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Solution NMR structure of GATase subunit and structural basis of interaction between GATase and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii Rustam Ali, Sanjeev Kumar, Hemalatha Balaram, and Siddhartha Peddibhotla Sarma Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi400472e • Publication Date (Web): 31 May 2013 Downloaded from http://pubs.acs.org on June 7, 2013

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Biochemistry

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Solution NMR structure of GATase subunit and structural basis of interaction between GATase

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and ATPPase subunits in a two-subunit-type GMPS

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from Methanocaldococcus jannaschii

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Rustam Ali1, Sanjeev Kumar2, Hemalatha Balaram2 and Siddhartha P. Sarma1*

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1

Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka-560012, INDIA.

2

Molecular Biology and Genetics Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore-560064, Karnataka, INDIA.

Address Correspondence to Siddhartha P Sarma 207 Molecular Biophysics Unit Indian Institute of Science Bangalore – 560012 Karnataka, INDIA E-mail: [email protected] Tel: 918022932839 Fax: 918023600535

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The authors would like to thank the Department of Science and Technology and the Department

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of Biotechnology, Government of India for the NMR and Mass spectrometric facilities at the

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Indian Institute of Science. HB acknowledges Department of Biotechnology and Department of

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Science and Technology, Government of India for funding.

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Rustam Ali is grateful to the Indian Institute of Science for a doctoral fellowship. Sanjeev Kumar

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was a recipient of CSIR Junior Research and Senior Research fellowships.

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Keywords:

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de-novo purine nucleotide biosynthesis; solution NMR; fast chemical exchange; intermediate

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chemical exchange; glutamine amidotransferase; guanosine monophosphate synthetase; protein –

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ligand interaction; protein – protein interaction; amination; ammonia channeling.

34 35

1

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ATP, Adenosine Triphosphate; ATPPase, ATP pyrophosphatase; DSS, Sodium 4,4-Dimethyl-4-

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Silapentane Sulfonate; ESI-MS, Electrospray Ionization Mass Spectrometry; GAT, Glutamine

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dependent

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Amidotransferase; GMP, Guanosine monophosphate; GMPS, Guanosine monophosphate

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Synthetase;

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Imidazoleglycerolphosphate synthase; MALDI - TOF, Matrix Assisted Laser Desorption

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Ionization – Time of Flight; Mj GATase, Methanocaldococcus jannaschii GATase; Mj GMPS,

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Methanocaldococcus jannaschii GMPS; NAD, Nicotinamide adenine dinucleotide; NMR,

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Nuclear magnetic resonance; NOE, Nuclear Overhauser Effect; NOESY, Nuclear Overhauser

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effect spectroscopy; Ph, P. horikoshii; XMP, Xanthosine monophosphate; rmsd, root mean

46

square deviation.

Abbreviations:

amidotransferase;

HSQC,

GDH,

Glutamate

Heteronuclear

Single

dehydrogenase;

Quantum

47 48 49 50 51 52 53 54 55 56


GATase,

Coherence;

Glutamine

IGPS,

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Abstract:

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Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been

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determined using high-resolution nuclear magnetic resonance methods. Gel filtration

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chromatography and

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present in solution as a 21 kDa (188 residues) monomer. The ensemble of twenty lowest energy

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structures showed an rmsd of 0.35±0.06 Å for backbone and 0.8±0.06 Å for all heavy atoms.

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Furthermore, 99.4 % backbone dihedral angles are present in allowed region of the

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Ramachandran map, indicating the stereochemical quality of the structure. The tertiary structure

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of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices.

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The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. NMR

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chemical shift perturbation and changes in line width were monitored to identify residues on

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GATase that were responsible for interaction with magnesium and the ATPPase subunit

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respectively. These interaction studies showed that a common surface exists for the metal-ion

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binding as well as for the protein–protein interaction. The dissociation constant for the GATase-

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Mg2+ interaction has been found to be ~1 mM, which implies that interaction is very weak and

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falls in fast chemical exchange regime. The GATase-ATPPase interaction on the other hand

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falls in intermediate chemical exchange regime on NMR time scale. The implication of this

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interaction on the regulation of the GATase activity of holo GMPS is discussed.

The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the

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N backbone relaxation studies have shown that Mj GATase subunit is

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Protein–protein interactions are at the core of most of the physiological process (1-3).

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Enzymes have been evolved to carry out a chemical reaction in an efficient and concerted

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manner. Guanosine monophosphate synthetase (4), presents the special example of a coupled

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enzyme catalyzed reaction where the component reactions are carried out by two distinct

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domains/subunits.GMPS1 a class I glutamine amidotransferase (5, 6), is an important enzyme

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that catalyzes the final step of GMP biosynthesis. Two reactions are catalyzed by the GMPS

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enzyme; 1) the hydrolysis of glutamine to yield ammonia and 2) amination of XMP to yield

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GMP (7, 8). The overall reaction is ATP dependent and requires Mg2+. The enzyme thus contains

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two independent catalytic sites that could either be housed in individual domains in a single

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polypeptide chain, or in separate subunits, and are known as the GATase and ATPPase domains

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or subunits respectively. In eukaryotes, bacteria and some archaea GMPSs are encoded for by a

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single gene (two-domain-type). In most other archaeal species, e.g., M. jannaschii, the GATase

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and ATPPase functions are present on individual polypeptides (two-subunit-type) that are coded

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for by separate genes. GATase catalyzes the hydrolysis of glutamine and ATPPase uses the

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ammonia generated to convert XMP to yield GMP. GMP formation occurs via a nucleophilic

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attack by the released ammonia on the adenyl-XMP intermediate (9-11).

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A PDB query for GMPS gives eleven hits. All structures have been solved by X-ray

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crystallography. These structures belong to GMPS from P. falciparum, H. sapiens, T.

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thermophilus, C. burnetii, P. horikoshii and E. coli. Except for P. horikoshii all GMPSs are two-

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domain-type. An analysis of different crystal structures from PDB reveals that the core structural

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elements are highly similar in GMPS. The GATase domain/subunit contains α/β structures. The

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core of the structure is formed by mixed β-sheet mainly constructed from parallel β-strands. The

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catalytic triad is conserved across these enzymes. The catalytic triad of GATase appears similar 4 ACS Paragon Plus Environment

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Biochemistry

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to those in serine proteases, however, with serine being replaced by cysteine and aspartate by

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glutamate in GATase.

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The ATPPase domain/subunit of GMPS has a typical dinucleotide binding site which

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resembles dehydrogenases. This fold is made up of five stranded sheets sandwiched between α-

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helices. ATPPase has a signature nucleotide-binding motif or P-loop that is highly conserved in

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all the nucleotide binding proteins (12). X-ray crystal structures of single-chain GMPSs have

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shown that the glutamine and XMP binding sites are separated by a distance of 10-40 Å. Thus,

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the ammonia released at glutamine binding site has to be channeled to the acceptor or ATPPase

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site (12-14). Ammonia channeling is the hallmark of a large group of proteins known generally

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as glutamine dependent amidotransferases, of which the GMPSs form one sub-group (7, 12, 14,

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15). This ammonia channeling and cross talk between two domains have been a major thrust of

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investigation in glutamine amidotransferases. Most of the studies have been carried out on two-

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domain-type GMPS (12, 16-21), while only one study has been reported from two-subunit-type

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GMPS (22).

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The GMPS from Methanocaldococcous jannaschii is a two-subunit-type GMPS. The

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glutamine amidotransferase (GATase) subunit of M. jannaschii is a monomer of molecular

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weight of 21 kDa, whereas the ATPPase subunit is a dimer with each protomer having a

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molecular weight of 34 kDa. There are several open questions with regard to the functioning of

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this enzyme. Unlike regular enzymes GATs are bi-enzymes and therefore function through the

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formation of protein-protein complexes that is facilitated by ligand binding to the synthase

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domain. Further, their activities are tightly coordinated leading to maximization of overall

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catalytic efficiency. Interestingly, different GATs seem to adopt different molecular mechanisms

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and thus the molecular mechanism that co-ordinate the various events warrants investigation. 5 ACS Paragon Plus Environment

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Recent studies have shown that GATase subunit interacts with ATPPase only when the

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latter is fully liganded and furthermore, the glutaminase activity is tightly regulated by

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interaction between GATase and ATPPase subunits (22). In the case of M. jannaschii GMPS,

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the ATPPase subunit alone is capable of catalyzing the hydrolysis of ATP. On the other hand the

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GATase does not show glutaminase activity in the absence of fully liganded ATPPase subunit.

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Our interest lies in understanding the structural basis for the interaction between the GATase and

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ATPPase subunits and the role of this important interaction in catalyzing the formation of GMP.

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To date, there is no structural information available for the two-subunit-type GMPS holoenzyme.

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Also, poorly understood are the conformational changes in GATase subunit upon interaction

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with liganded ATPPase that lead to its activation. In order to gain a better understanding of the

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mechanistic principles, we initiated structural studies of M. jannaschii GMPS. As a first step we

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have determined the solution structure of the Mj GATase subunit by high-resolution multinuclear

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NMR spectroscopy. We have also studied the interaction of GATase with the co-factor Mg2+ and

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interaction between Mj GATase and ATPPase subunit.

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Materials and Methods

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Over-expression and purification

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The expression and purification of the Mj GATase subunit of M. jannaschii has been

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described earlier (23). Isotopically enriched (13C,

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prepared using

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respectively (24). Gene for ATPPase subunit from M. jannaschii genomic DNA was cloned into

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pET3atr using NdeI and BamH1restriction sites, and subcloned into cassette III of pST39

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expression vector using Sac I and Kpn restriction sites. Plasmids carrying gene for ATPPase

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C6-glucose and

15

15

N or

15

N) samples of Mj GATase were

NH4Cl or 15NH4Cl as the sole source of carbon and nitrogen


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Biochemistry

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subunit were transformed into Rosetta (DE3) pLysS E. coli over-expression strain. Mj ATPPase

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subunit was over-expressed and purified using the same protocol as described for Mj GATase

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subunit.

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Chromatographic studies

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Gel filtration studies were carried out using a Superdex-200 column (10 mm x 300 mm)

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attached to an AKTA Basic HPLC system. The column was equilibrated with 50 mM phosphate

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buffer, pH 7.0. Beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum

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albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) were used for

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molecular weight calibration. The protein samples were eluted at a flow rate of 0.5 ml/min.

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Gel filtration co-chromatography

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The column was pre-equilibrated with buffer (20 mM Tris, pH 7.4)alone or with buffer

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containing appropriate ligands. The concentration of ATP and XMP in the running buffer was

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100 µM each. The concentrations of Mj ATPPase and GATase when used alone or in

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combination were 10 µM each. The concentrations of ATP and XMP used for pre-incubation

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with the enzyme were 3 mM and 0.2 mM, respectively. Equimolar Mj GATase and ATPPase

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were pre-incubated on ice with substrates under four conditions: A) 20 mM MgCl2, B) 20 mM

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MgCl2 + 3 mM ATP, C) 20 mM MgCl2 + 200 µM XMP and D) 20 mM MgCl2 + 3 mM ATP +

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200 µM XMP. These samples were chromatographed. Fractions were collected and analyzed by

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SDS-PAGE.

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Metal affinity co-chromatography

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An N-terminal hexa-histidine tagged Mj GATase was generated in pET21b. This clone was

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verified by DNA sequencing and protein expressed in Rossetta (DE3) pLysS and purified using

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Ni-NTA agarose beads. The protein was passed through a desalting column (Sephacryl-200) and

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concentrated before further use. Mj GATase affinity beads were generated by incubating Ni-

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NTA agarose with 10 µM purified Mj GATase followed by buffer wash to remove unbound

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enzyme. Mj ATPPase (10 µM) was incubated for 15 minutes on ice with 2 mM ATP, 0.2 mM

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XMP in 20 mM Tris buffer, pH 7.4, containing 20 mM MgCl2. The pre-incubated Mj ATPPase

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was mixed with the beads and incubated at 4 ºC for 30 min. The beads were then washed with

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buffer A (20 mM Tris HCl, pH 7.4) and subsequently with buffer B (20 mM Tris HCl, pH 7.4

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containing 3 mM ATP, 0.2 mM XMP, 20 mM MgCl2). The protein was eluted from the column,

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using buffer A solution containing 500 mM imidazole. The column fractions were analyzed by

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SDS-PAGE.

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Biochemical Assay

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Measurement of GATase activity

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Mj GATase activity was monitored by a coupled enzyme assay; wherein glutamate

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formation was coupled to the reduction of NAD+ to NADH by glutamate dehydrogenase and the

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reaction was followed by spectrophotometry. The concentration of NADH was estimated from

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absorbance measurements at 340 nm using a molar extinction co-efficient value of 6220 M-1 cm-1

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(25). The reaction mixture consisted of 100 µM GATase and 100 µM glutamine in 100 mM Tris

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HCl, pH 7.4 and was incubated at either 22 ºC for 30 min or 70 ºC for 15 min. The reaction was

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quenched by boiling for 5 minutes followed by centrifugation at13000 g. The supernatant (100 8 ACS Paragon Plus Environment

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µl) was mixed with 0.5 mM NAD+ and glutamate dehydrogenase (1.8 Units) in a buffer

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containing 50 mM KCl and 1 mM EDTA. The reaction mixture was incubated for 1 hour at 37

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0

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mutually excluded.

C. Separate control reactions were also carried out wherein GATase and glutamine were

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GATase activity in the presence of ligand bound ATPPase

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The Mj GATase activity was monitored by an assay described in previous section.

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Concentrations of enzymes and ligands used in the assays were 2 µM GATase, 2 µM ATPPase,

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0.2 mM XMP, 2 mM ATP and 5 mM glutamine. Reactions were carried out in a buffer

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containing 100 mM Tris HCl, pH 7.4, 20 mM MgCl2 at 70 ºC for15 min and quenched by

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boiling for 5 minutes.

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Stoichiometry of GATase-ATPPase complex

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Stoichiometry of the GATase-ATPPase complex was determined by monitoring the

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formation of GMP. The reaction mixture contained fixed concentration of 2 µM ATPPase, 3 mM

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ATP, 0.2 mM XMP, 20 mM glutamine and varying concentrations of GATase (0-16 µM). The

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reaction was carried out in 90 mM Tris HCl, pH 7.0, containing 20 mM MgCl2 at 70 ºC. The

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reactions were initiated with 2 µM Mj ATPPase and varied concentrations of GATase.

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Formation of GMP was monitored spectrophotometrically, by measuring absorbance at 290 nm

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and the concentration was estimated using a molar extinction co-efficient value of 1500 M-1cm-1

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(26).

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NMR spectroscopy

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Sample preparation

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Samples for NMR spectroscopy were prepared in 20 mM potassium phosphate buffer pH

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7.0, containing 2 mM DTT, 0.1 mM EDTA, 1 mM PMSF and 0.01% sodium azide. Three

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dimensional

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GATase that were 0.70 mM in 90% H2O/10%D2O or 100% D2O respectively. Nitrogen-15

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relaxation experiments were recorded using samples of Mj GATase (0.4 mM) in 90% H2O/10%

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D2O.

15

N-edited and

13

C-edited NMR experiments were recorded using samples of Mj

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Data acquisition

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NMR data were acquired either on an Agilent 600 MHz spectrometer equipped with a 5

230

mm triple resonance pulsed field gradient (TRPFG) probe or on a Bruker Avance 700 MHz

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spectrometers equipped with 5 mm TXI triple resonance PFG (z-axis) probe. The sample

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temperature was maintained at 30 ºC during all experiments. Chemical shifts were referenced to

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external DSS. Two dimensional 1H-15N HSQC spectra (27) were acquired at 700 MHz using

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proton spectral widths of 14006 Hz in the acquired dimension and 2129 Hz in the indirectly

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detected dimension. Water suppression was achieved by using a pulse program that incorporated

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the WATERGATE (28) solvent suppression scheme. The STATES-TPPI mode of quadrature

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detection was used for frequency selection in the indirectly detected dimension. The 1H-13C

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HSQC spectra were acquired at 700 MHz in sensitivity-enhanced mode. Proton and carbon

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spectral widths of 14006 Hz and 10914 Hz were recorded in the directly detected and indirectly

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detected dimensions respectively. Frequency discrimination in the indirectly detected dimension

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was achieved via coherence selection using pulsed field gradients. Water suppression was 10 ACS Paragon Plus Environment

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achieved via coherence selection and by the use of using trim pulses. Three-dimensional

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edited 1H-1H NOESY spectra (mixing time, τm= 150 ms and 200 ms) and

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NOESY (29) spectra (mixing time, τm = 150 ms) were acquired at 700 MHz using proton

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spectral widths of 14005.60 Hz and 14005.60 Hz in F3 and F1 dimensions, and nitrogen and

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carbon spectral width of 2128.65 Hz and 4225 Hz, respectively in the F2 dimension. Data were

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acquired in phase sensitive mode, using the States-TPPI (15N-edited NOESY) or Echo-AntiEcho

248

method (13C-edited NOESY) of quadrature detection for the indirectly detected dimensions.

13

N-

C-edited 1H-1H

249 250

15

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T1, T2 measurement

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Spin–lattice relaxation rate, T1, and spin–spin relaxation, T2, were measured by recording

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two dimensional 1H-15N HSQC spectra on an Agilent 600 MHz NMR spectrometer equipped

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with a 5 mm triple resonance pulsed field gradient (TRPFG) probe (30, 31). The sample

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concentration used for T1 and T2 measurements was 0.4 mM. The data used for T1 measurement

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were acquired with 2 sec recycle delays and relaxation delays of 10, 50, 100, 250, 400, 550, 750,

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900 and 1200 ms. T2 data were acquired with 2 sec recycle delays and relaxation delays of 10,

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30, 50, 70, 90, 110, 130, 150 and 170 ms. T1 and T2 values were determined by fitting peak

259

heights using the nonlinear least-squares routine, using Analysis (32).

N Backbone Dynamics

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Calculation of Rotational correlation time (τc)

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Rotational correlation time (τc) was measured using the following formula (30, 33)

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τc = 1/4πνN (6×T1/T2− 7)1/2 where νN is the 15N resonance frequency (in Hz).

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(1)

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Hydrogen – deuterium exchange

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A series of 2D 1H-15N HSQC spectra at an interval of 45 minutes over a period of 24 hours,

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were acquired in 100% D2O. Lyophilized samples of Mj GATase subunit were dissolved in D2O

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just before data acquisition.

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Data processing and analysis

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All 2D and 3D NMR data were processed by using NMRPipe/NMRDraw processing

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software (34) on an Intel PC workstation running Suse Linux 11.3. The directly and indirectly

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detected time domain data of 2D and 3D spectra were processed by applying a 90° phase-shifted

274

squared sinebell or a Gaussian filter with a line-broadening parameter of 10 Hz as weighting

275

functions. Data sets were zero-filled prior to Fourier transformation. Processed data were

276

analyzed using the ANALYSIS module in CCPN.

277 278

Structure determination

279

Distance and dihedral angle restraints

280

Inter-proton distance restraints were calculated from the intensities of unambiguously

281

assigned NOE correlations in the 3D 15N-edited NOESY-HSQC and

13

282

spectra. On the basis of integrated intensities, NOEs were classified as strong, medium, weak and

283

very weak with upper distance bounds of 2.8, 3.5, 5.0 and 6.0 Å respectively. All distance

284

restraints employed a lower bound of 1.80 Å. Hydrogen bond restraints were generated for

285

residues that were slow to exchange in H/D exchange experiments. The upper distance bound

286

employed for the hydrogen bonds were set to 2.2 Å for HN···O and 3.2 Å for N···O pairs.

287

Backbone dihedral angles phi (φ) and psi (ψ) were predicted using the program TALOS (35)

C-edited NOESY-HSQC

+


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Biochemistry

α

13

α

288

using the observed HN, 1H ,

289

allowed during structure calculation.

C ,

13

β

C chemical shifts. An angular variation of ± 30º was

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Solution structure evaluation

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Three-dimensional structures were calculated using torsion angle dynamics protocol in the

293

program CYANA-3.0 (36, 37). Experimentally derived distances from NOEs, hydrogen-bond

294

distances and dihedral angle restraints were used as input for the structure calculation. A hundred

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random conformers were subjected to 20000 steps of annealing. An ensemble of twenty lowest

296

energy conformers out of hundred calculated conformers were selected on the basis of target

297

function and which had no upper distance violations greater than 0.2 Ǻ and no dihedral angle

298

violations greater than 5°. The conformer with minimum energy/target function was chosen as a

299

representative conformer. The structures were analyzed using MOLMOL (38). The quality of

300

structure was further assessed by Protein structure validation suit (PSVS) (39). Structure

301

alignment was performed using either DALI (40) or PyMOL (41).

302 303

Interaction studies

304

Interaction with the cofactorMg2+

305

Perturbations in chemical shift of residues in Mj GATase, as observed in 1H-15N HSQC

306

spectra, upon interaction with Mg2+, were followed by NMR titration studies.

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GATase (50 µM) was titrated with varying concentration of MgCl2 (0.5 mM–20 mM). Chemical

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shift perturbation as a function of MgCl2 concentration in HSQC spectrum was used to calculate

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dissociation constant (Kd). Weighted chemical shift change per residue (∆) was calculated by the

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equation (42); 13 ACS Paragon Plus Environment

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N-enriched Mj

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∆ = [(δH)2 + (0.1δN)2]1/2

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(2)

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where δH and δN are changes in chemical shifts of 1HN and

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constant for the interaction was determined by plotting the ∆ as a function of increasing MgCl2

314

concentration. The resulting curve was fitted into the following equation

315

15

N, respectively. Dissociation

∆ = ∆max([L]T + [P]T + Kd - {([L]T + ([P])T + Kd)2 - 4[L]T[P]T}1/2)

(3)

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where ∆ is the observed chemical shift change at a given total ligand concentration, [L]T,

317

(relative to the resonance frequency in absence of MgCl2), ∆max is the change in chemical shift at

318

saturation and [P]T is the total protein concentration (43-47).

319 320

Interaction with ATPPase

321

A method, similar to the one described in the previous section was used to study the

322

interaction between Mj GATase and Mj ATPPase subunits. Nitrogen-15 labeled Mj GATase (50

323

µM) was titrated with varying concentration of unlabeled Mj ATPPase (20 µM to 160 µM), in

324

presence of saturating concentration of all substrates viz; MgCl2 (20 mM), ATP (8 mM) and

325

XMP (2 mM). All the experiments were carried out in a 40 mM potassium-phosphate buffer, pH

326

7.0, containing 50 mM NaCl. The control experiment was recorded on 15N enriched Mj GATase

327

(50 µM) sample in presence 20 mM MgCl2, 8 mM ATP and 2 mM XMP. To this, varying

328

concentration of unlabeled Mj ATPPase (20 µM to 160 µM) was added. Each titration

329

experiment was started on a freshly prepared sample to avoid a dilution effect upon addition of

330

ligand. All samples were prepared from the same stock solutions. Data were recorded by placing

331

the sample in a 5 mm, deuterium susceptibility matched Shigemi tube (Pennsylvania, USA). The

332

total sample volume used in a Shigemi tube was kept constant at 300 µl. The concentration of

333

stock solutions of Mj GATase and Mj ATPPase were 0.43 mM and 0.53 mM respectively. The 14 ACS Paragon Plus Environment

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Biochemistry

334

Mj GATase and Mj ATPPase protein concentrations were measured on a NanoDrop 1000

335

spectrophotometer using molar extinction coefficient values of 15930 M-1cm-1 and 22190 M-

336

1

337

intensity (I0). The decrease in intensity due to line broadening/chemical exchange contribution to

338

the relaxation, arising from interaction between two subunits was determined by measuring peak

339

height. Change in intensity (∆I) upon ligand binding was calculated as

cm-1 respectively. The intensity of a resonance in the control experiment was taken as reference

∆I = I0 – I

340

(4)

341

where I0 is the peak intensity in absence of ligands in control experiment and I represents peak

342

intensity in presence of ligand, analogous to the approach used by (48). The data was normalized

343

with respect to reference intensity (I0). The intensity of a resonance that disappeared on titration

344

with ATPPase subunit was taken as zero. The calculated ∆Inormalized was plotted against residue

345

number. A cut off of 0.8 was set for the residue interacting directly with Mj ATPPase subunit

346

while a cut off ≥ 0.75 but less than 0.8 was set for the residues those interacting indirectly. The

347

residues involved in interaction with Mj ATPPase subunit were mapped onto the calculated

348

solution NMR structure of Mj GATase.

349 350

H/D exchange studies

351

In an effort to identify interface residues in the Mj GATase-ATPPase complex, H/D

352

exchange NMR experiments were also carried out. Sample for control experiment was prepared

353

by lyophillization of 50 µM Mj GATase. Equivalent amounts of powdered MgCl2, ATP and

354

XMP were added and the mixture was dissolved in D2O, just prior to data acquisition. The final

355

concentrations of MgCl2, ATP and XMP in the solution were 20 mM, 8 mM and 2 mM

356

respectively. Similarly, Mj GATase (50 µM) and ATPPase (50 µM) were co-lyophillized and 15 ACS Paragon Plus Environment

Biochemistry

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357

subsequently equivalent amount of powdered form of MgCl2, ATP and XMP were added. D2O

358

was added just before data acquisition. 1H-15N HSQC spectra were acquired as described above.

Page 16 of 66

359 360

Reaction assay

361

All reaction assays were carried out in 20 mM phosphate (pH 7.0) buffer at 303 K. The

362

reaction mixture contained 5 µM Mj GATase, 5 µM Mj ATPPase, 20 mM MgCl2, 2 mM ATP,

363

240 µM XMP and 20 mM Glutamine. The substrates and co-factors were mixed. The reaction

364

was initiated by the addition of enzymes and this reaction mixture was transferred into a NMR

365

tube. Time course of the reaction was followed by recording one-dimensional proton NMR

366

spectra. GMP formation was tracked by monitoring the H8, 6NH2 and H1' resonances of GMP,

367

which are known to occur at 8.3 ppm, 6.3 ppm and 5.8 ppm respectively. Chemical shifts were

368

referenced to internal DSS. In a similar manner, formation of AMP was monitored by tracking

369

H8 resonance of AMP at 8.55 ppm. GATase and glutamine were excluded from the reaction

370

mixture, where only AMP formation was monitored. Spectra were recorded at intervals of 10

371

minutes, processed and analysed.

372 373

Results

374

Solution properties of Mj GATase

375

Figure 1 shows the gel filtration chromatography elution profile when the Mj GATase

376

subunit is passed through a Superdex-200 gel filtration column. Comparison of the elution

377

volume (Ve) of Mj GATase with those of proteins of known molecular weight showed that Mj

378

GATase behaves as a monomer in solution with a molecular weight of ~ 21 kDa. In light of this,


Page 17 of 66

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Biochemistry

379

the protocols followed for structure determination were those that were generally applicable for

380

monomeric proteins.

381 382

NMR spectroscopy

383

Sequence specific assignment and secondary structure

384

Figure 2 shows the sequence specifically assigned 1H-15N HSQC of the GATase subunit of

385

Mj GMPS. The sequential connectivity assignments obtained from analysis of the three-

386

dimensional 15N-edited NOESY-HSQC spectrum for residues 35 to 40 are shown in figure 3. A

387

summary of the sequential and short range NOEs between backbone atoms and side chain and

388

backbone atoms is shown in Figure S1 (Supplementary Information). The Mj GATase subunit

389

consists of eleven β-strands and five α-helices. Assignment of sequential, short and medium

390

range NOEs provided corroborative evidence for the secondary structure determined from 1H

391

and

392

sequence number and its comparison with the secondary structure of GATase subunit from P.

393

horikoshii, has been listed in table S1 (Supplementary information) (23).

13

α

C ,

13

β

C secondary chemical shifts. Distribution of secondary structure as a function of

394 395

Three dimensional Structure

396

The three dimensional structure of Mj GATase was determined using NMR derived

397

distance, dihedral and hydrogen bond restraints. The number and type of restraints used for

398

structure calculation are listed in table 1. The table also shows the structural parameters derived

399

for the twenty lowest energy structures. Figure 4a shows the superposition of twenty lowest

400

energy conformers of Mj GATase subunit when superimposed on their backbone (N, C, and C )

α


Biochemistry

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Page 18 of 66

α

401

atoms. The rmsd, when superposed on the backbone (N,C and C ) atoms and on all heavy atoms,

402

with respect to their mean coordinate position, were 0.35±0.06 Å and 0.8±0.06 Å respectively.

403

An analysis of the backbone dihedral angles in the ensemble of calculated structures shows that

404

99.4 % backbone dihedral angles are present in allowed region of the Ramachandran map (49,

405

50). The low rmsd values for backbone and heavy atoms of all residues (1-188), and the high

406

percentage of residues occupying the allowed regions in the Ramachandran map, indicate that

407

the quality of calculated structure is good. Figure 4b shows the lowest energy conformer of Mj

408

GATase in ribbon representation. The core of the GATase structure is a mixedβ-sheet formed by

409

sevenβ-strands. This mixed β-sheet is made up almost entirely of parallel β-strands, except the

410

β-strand XI, which is anti-parallel in orientation to both strands IX and X. This arrangement

411

makes the mixed β-sheet appear as though it is made up of one β-sheet consisting of five parallel

412

strands and one β-sheet made up of two anti-parallel strands. The first and last β-strands of the

413

core β-sheet are twisted. The core β-sheet is flanked on both sides by five α-helices. The

414

catalytic Cys76, is the only residue with backbone dihedral angles that is present in the

415

disallowed region of the Ramachandaran map. This residue is present at the end of a β-strand

416

and beginning of an α-helix. A similar structural feature has been also observed in other α/β

417

hydrolases (51) (peroxidases, esterases etc.), where the nucleophilic residue is present in a tight

418

α/β turn called “nucleophilic elbow”. Presence of this nucleophilic elbow forces the catalytic

419

residue to populate the disallowed region in the Ramachandaran map.

420 421 422


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Biochemistry

423

Comparison withGATase of Pyrococcus horikoshii

424

The GATase subunit of Mj GMPS and that of Ph GMPS share a 56 % sequence identity.

425

The distribution of secondary structural elements as a function of sequence in Mj GATase is

426

similar to that observed in the X-ray structure of the GATase subunit of the Ph GMP synthetase

427

(52) (PDB ID 2D7J). Figure 5 shows a backbone superposition of these two structures.

428

Significant differences are found only for residues in β-strands V, VI, VII and VIII, which are

429

shorter in length in the solution structures. Additionally, residues 68–70, which are present in a

430

310-helix in the crystal structure, exist in a coil-like conformation in the solution structure. These

431

differences could be attributed to the local dynamics. The Mj and PhGATase are structurally

432

similar to the GATase in two-domain-type GMPS.

433 434

15

435

Figure 6 shows the measured15N T1 and T2 relaxation times for Mj GATase, as a function

N backbone dynamics

15

436

of residue number. The average values of

N T1 and T2 for GATase polypeptide backbone

437

nitrogen atoms are 0.650 ± 0.03 and 0.089 ± 0.003s, respectively. The residues in α-helices and

438

β-sheets exhibit a tight distribution of T1 and T2 values. This is an indication of well- structured

439

regions and corroborates well with the calculated solution structure. On the other hand residues

440

in loop regions show wider distribution of T1. The residues present in loops showed a lower than

441

average value of T2, e.g., G51and G52.

442

Using the average values of T1 and T2, the average global rotational correlational time τc,

443

was found to be ~8.0 ns. The observed value is lower than what one could expect for a 21 kDa

444

molecule (i.e., 10.5 ns).

445 19 ACS Paragon Plus Environment

Biochemistry

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446

Page 20 of 66

Biochemical studies

447

Substrate liganded ATPPase is required for Mj GATase activity

448

Mj GATase, in absence of ligand bound ATPPase subunit is inactive. In the presence of

449

substrate liganded ATPPase, viz., Mg2+, ATP and XMP, Mj GATase shows glutaminase activity.

450

A quantitative estimate of this glutaminase activity is shown in Figure 7a. Figure 7b shows the

451

one-dimensional NMR spectra of the conversion of XMP to GMP as a function of time. The

452

resonance lines at δ 5.9 ppm and 5.92 ppm and those at 8.12 ppm and 8.18 ppm correspond to

453

the H1' and H8 protons of XMP and GMP respectively. The resonance line at 6.35 ppm

454

corresponds to that of amino protons of GMP. The decrease in intensity of resonance lines of

455

XMP with the concomitant increase in intensity for resonance lines of GMP is unequivocal proof

456

for the conversion of XMP to GMP. Similar changes in intensity, of resonance lines of ATP and

457

AMP can also be observed. The decrease in intensity of the resonance lines of glutamine is not

458

apparent due to the large stoichiometric excess of this substrate in the reaction mixture. A similar

459

NMR study of GATase alone shows no discernable decrease in the amount of glutamine as a

460

function of time (data not shown). Mj ATPPase, on the other hand is catalytically active even in

461

the absence of GATase subunit. Figure 8 shows that the concentration of XMP (δ 5.90, 8.12)

462

remains unchanged, while that of AMP (δ 8.55 ppm) increases as a function of time.

463 464

Stoichiometry of GATase and ATPPase in the GMPS complex

465

Figure 9 shows the result of Mj ATPPase titration with varying concentration of Mj

466

GATase. A maximum GMP synthetase activity was observed at GATase-ATPPase ratio 1,

467

indicating the stoichiometry of functional of Mj ATPPase-GATase in a functional Mj GMPS is

468

1:1. 20 ACS Paragon Plus Environment

Page 21 of 66

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469

Biochemistry

Interaction studies

470

Interaction between GATase and cofactor Mg2+

471

Binding of Mg2+ to the Mj GATase subunit was studied by NMR spectroscopy. A

472

concentration dependent perturbation of chemical shifts was observed for several residues in the

473

protein. An overlay of the HSQC spectra of Mg2+ free GATase and of Mg2+ saturated15N-

474

labeledMj GATase is shown in figure S2 (Supplementary Information). Figure 10ashows the

475

chemical shift changes in the HSQC spectra for some of the selected residues that are affected by

476

Mg2+ binding. According to Cavanagh et. al., “The near continuous change in chemical shift as a

477

function of increasing Mg2+ concentration is a clear indication that the protein and metal are in

478

a fast chemical exchange regime on NMR time scale”(42). There were no detectable changes in

479

the spectrum at concentrations less than 400 µM and no further change in chemical shift at Mg2+

480

concentrations >10 mM, indicating that protein has reached saturation.

481

The absolute value of the deviation in chemical shift, when compared to the free protein

482

is shown in figure S3 (Supplementary Information). Residues that showed a weighted chemical

483

shift change (∆) greater than 0.05 ppm were considered for calculation of dissociation constant.

484

Twelve residues were showing ∆ greater than 0.05 ppm. Upon mapping these residues on the

485

solution structure of Mj GATase subunit, all residues showing major chemical shift perturbation

486

were present on one side/face of the structure, around the catalytic residue (Figure10b). Residues

487

close to active site, such as H16, G78, H79, S124, H125, D127 and V166 experience significant

488

chemical shift perturbation. Other residues viz; I5, G9, I28, G88 and A93 also experience

489

measurable perturbation. This could be attributed to changes in conformation induced upon Mg2+

490

binding.


Biochemistry

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Page 22 of 66

491

The binding curves for the Mg2+-Mj GATase interaction for some of the selected residues

492

are shown in figure 10c. The dissociation constant, Kd, was calculated using equation 2, and was

493

found to be ~ 1 mM. That the Kd is in the millimolar range indicates that the interaction between

494

GATase and Mg2+ is very weak, and falls in very fast exchange regime on NMR time scale. The

495

implications of Mg2+ binding to GATase are discussed below.

496 497

Interaction between Mj GATase and ATPPase

498

Gel filtration chromatography

499

The gel filtration studies (vide supra) have shown that Mj GATase exists in solution as a 15

500

monomer, with a molecular weight of ~ 21 kDa. Evidence for this also comes from the

N-

501

relaxation studies described above. The gel filtration studies also showed that the Mj ATPPase

502

subunit exists as a dimer of molecular weight of ~ 80 kDa. Figure 11a shows the chromatogram

503

of elution profile of Mj GATase and ATPPase in different conditions. Under the

504

chromatographic conditions used the complex should elute as a distinct peak ahead of ATPPase.

505

However, as this was absent, fractions of the peak corresponding to ATPPase were examined on

506

SDS-PAGE and a low level of GATase was seen in the ATPPase fractions (Figure 11b–11e). To

507

improve the ratio of ATPPase and GATase co-eluting, a pull down experiment was carried out.

508

This yielded greater recovery of the protein-protein complex (Figure 12).

509 510

NMR spectroscopic studies

511

The one dimensional proton NMR spectrum of the interaction of the Mj GATase and

512

ATPPase subunits is shown in figure 13a. An overall broadening of the resonances in Mj

513

GATase can be observed in the spectrum. Broadening of the resonance could arise due to 22 ACS Paragon Plus Environment

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Biochemistry

514

chemical exchange of the components of the complex or due to increase in the rotational

515

correlation time (τc) as a function of the molecular size of the complex. The former would

516

suggest that the complex is in intermediate exchange, while the later would indicate that the

517

complex is in slow exchange. In order to get further insight into the interaction between Mj

518

GATase and ATPPase subunits at atomic level, 1H-15N HSQC spectra of 15N-labeled Mj GATase

519

in presence of varying concentration of unlabeled Mj ATPPase and constant saturating

520

concentration of ATP, Mg+2 and XMP were acquired. Figure 13b shows the ovelay of two 1H-

521

15

522

bind to the Mj ATPPase subunit. However, upon addition of MgCl2, ATP and XMP, a significant

523

reduction in the intensity of the correlation peaks was observed for specific residues in Mj

524

GATase (Figure 13c). The change in intensities for some of the interacting residues with respect

525

to the intensities in control experiment has been shown in figures13d and S4 (Supplementary

526

Information). The molecular weight of the Mj GATase-ATPPAse complex is large (~112 kDa)

527

and therefore, the observed overall reduction in intensity could be attributed to the increased

528

molecular weight. The greater than average reduction in intensity observed for specific residues

529

in Mj GATase,as a function of increasing concentration of Mj ATPPase subunit in presence of

530

all substrates indicate that Mj GATase and ATPPase are in chemical exchange, between bound

531

and unbound forms, at a rate that is intermediate on the NMR time scale.

N HSQC spectra. In the absence of substrates and co-factors the Mj GATase subunit does not

532

A plot of normalized change in intensity, ∆Inormalized, as a function of residue number of Mj

533

GATase subunit is shown in figure 14a. In all, 37 residues showed significant decrease in

534

intensity (∆Inormalized > 0.80) upon Mj ATPPase binding. These residues were identified as those

535

that were directly involved in the interaction between Mj GATase and ATPPase subunits. In

536

addition to these, another set of 27 residues also showed significant decrease in intensity 23 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 66

537

(∆Inormalized ≥ 0.75 ≤ 0.80). The decrease in intensities of these residues is most likely due to

538

secondary effects. The residues of Mj GATase subunit that are involved in interaction and form

539

the interaction surface are listed in Table 2. When mapped ontothe structure of Mj GATase

540

structure, these residues were found to be concentrated in five regions of the structure (Figure

541

14b). The residues stretching from V12 to I22, formα-1 helix, while the other four stretches of

542

the residues are present in loopV, loopVII, loopXI, and loop XIV around catalytic triad (C76,

543

H163 and E165). Figure 14c shows the surface representation of Mj GATase subunit showing

544

residues involved in interaction. Residues involved in direct interaction with Mj ATPPase

545

subunit are shown in blue, while the residues involved in indirect interaction are shown in cyan

546

colored. Interestingly, the catalytic residues C76 and H163 were also showing change in

547

intensity greater than 0.80. We could not evaluate change in intensity of E165 because this cross

548

peak overlaps with V29. This clearly indicates that catalytic residues of Mj GATase also undergo

549

conformational change upon interaction with Mj ATPPase subunit. This probably explains why

550

Mj GATase subunit is inactive in the absence of the ATPPase subunit. Therefore we surmise that

551

this interaction brings about a conformational change, which is transmitted around the catalytic

552

site, leading to the activation of Mj GATase subunit.

553 554

Complex between GATase and ATPPase results from a weak interaction:

555

The stability of the Mj GATase-ATPPase complex was probed by H/D exchange NMR

556

experiments. Figure S5 shows an overlay of HSQC spectra of solutions containing Mj

557

GATaseand ATPPase in the absence (in red) and presence (in blue) of all substrates (induces

558

complexation), both of which were acquired in D2O. The residues in αI are slow to exchange in

559

the both cases, which is not surprising as the backbone amide protons are hydrogen bonded. 24 ACS Paragon Plus Environment

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Biochemistry

560

However, residues in loops exchange rapidly with D2O. Thus the residues in the loop regions are

561

not protected in the complex and it is safe to conclude that the interaction between Mj GATase

562

and ATPPase subunits is a transient one and does not result in a stable complex.

563 564

Discussion

565

The biosynthesis of GMP in most of the archaea is catalyzed by a two-subunit-type

566

GMPS enzyme. On the other hand, higher organisms including eubacteria possess a multi-

567

domain GMPS, which probably arose through gene fusion. While much is known of the structure

568

and kinetic and enzymatic properties of the two-domain-type GMPS, the two-subunit-type is less

569

well characterized. Here we have determined the three-dimensional solution NMR structure of

570

the GATase subunit of a two-subunit-type GMPS from M. jannaschii. The structure shows that

571

the molecule is compact and exhibits an α + β fold. The calculated average global correlation

572

time for the Mj GATase subunit was almost 2.5 ns smaller than the expected average global

573

correlation time. This strongly indicates that the molecule is rigid, tightly packed and tumbles

574

isotropically in solution. H / D exchange studies showed that almost 40 percent of the residues

575

Mj GATase residues are either involved in hydrogen bonding in secondary structure or are

576

buried inside the protein core.

577

During the course of our studies, we had observed that optimal activity of GMPS was

578

achieved only under conditions in which the Mg2+ was present at a concentration that was

579

significantly higher than that essential for charge stabilization of ATP. Similar observations

580

have also been made in the case of the two-domain-type GMPS from P. falciparum (16) and

581

human GMPS (19). Our titration data, for the first time, showed that magnesium interacts with

582

Mj GATase subunit in a site-specific manner. The residues that are perturbed in chemical shift 25 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

583

due to interaction with Mg2+, lie on one face of the molecule (figure 10c) and form a ring around

584

the catalytic site. Figure 15 shows the electrostatic surface potential for Mj GATase. The

585

interaction between Mg2+ and Mj GATase is not a case of a non-specific electrostatically driven

586

interaction. This is supported by the fact that several other negatively charged regions, which are

587

present on the surface of Mj GATase are unperturbed in the presence of Mg2+. For instance, the

588

surface of helix II is highly negatively charged (4 out of 6 residues are negatively charged).

589

However, no change in chemical shift for the residues in helix II was observed upon titration

590

with Mg2+, indicating that the metal–protein interaction is a highly specific one. The high value

591

of the dissociation constant indicates that this is a weak interaction. In the case of human and Pf

592

GMPS, the site of Mg2+ binding is as yet unknown.

Page 26 of 66

593

The mechanism by which ammonia is channeled from the GATase to the ATPPase is

594

complex and is poorly understood. Structural studies of two-domain-type GMPS do not show a

595

clear conduit for the passage of ammonia from one domain to the other. The structural basis for

596

subunit association in two-subunit-type GMPS’s is not known. Using high-resolution NMR

597

spectroscopic methods, we have also identified the residues on Mj GATase that are responsible

598

for interaction with the ATPPase subunit. There are eight residues on Mj GATase that are

599

common to the interaction with Mg2+ and ATPPase. These residues showed chemical shift

600

perturbation upon Mg2+ binding as well as reduction in intensity upon interaction with ATPPase

601

subunit. Though the exact role of Mg2+ is not known, the fact that a significant number of

602

residues are involved in Mg2+ binding and that these residues are also involved in interaction

603

with ATPPase, indicates that Mg2+ binding must play an important role. Since Mg2+ can bind to

604

GATase subunit independently, one may postulate that Mg2+ binding may play an important role

605

in charge stabilization of the GATase-ATPPase complex. 26 ACS Paragon Plus Environment

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Biochemistry

606

It is important to note that the Mj GATase-ATPPase interaction takes place only

607

in the presence of substrates. The Mj GATase-ATPPase complex undergoes exchange that is

608

intermediate on the NMR time scale. Evidence for this comes from the fact that correlation peaks

609

of 15N-labeled Mj GATase subunit showed neither chemical shift perturbation nor appearance of

610

new correlation peaks with increasing concentration of ATPPase subunit. This rules out the

611

possibility of a fast chemical exchange regime or a slow exchange chemical regime, respectively.

612

Instead, a significant line broadening effect was observed as function of increasing concentration

613

of ATPPase. This could be attributed to the intermediate chemical exchange between two

614

subunits in Mj GATase-ATPPase complex, on NMR time scale. The intermediate chemical

615

exchange regime is characterized by complete broadening of resonances at approximately half of

616

the saturating concentration of ligand and then reappears, probably at slightly different chemical

617

shifts, at much higher concentration of ligand (42). Given the fact that the apparent molecular

618

weight of Mj GATase increases from 21 kDa to 112 kDa, upon complexation with the ATPPase

619

subunit, it is expected that there will be some contribution to the observed line width from the

620

increased rotational correlation time.

621

Most of the studies on GMPS have shown that glutaminase activity is regulated

622

by interaction between GATase and ATPPase subunits. However a recent study on IGPS (53, 54)

623

from Thermotoga maritima, a two subunit-type-amidotransferase has shown that indeed GAT is

624

independently active and absence of constitutive activity is due to the presence of a plug in the

625

channel in the GAT domain. Removal of plug results in constitutive activity. We therefore

626

checked in Mj GATase for such an independent activity using high equimolar quantities of

627

enzyme and glutamine. No measurable activity was observed when GATase and glutamine were

628

incubated at either 22ºC or 70 ºC. It should be noted that under our assay condition, glutamate 27 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

629

concentrations as low as 5 µM can be reliably measured. Thus it is safe to conclude that Mj

630

GATase has no activity and that its activity seems to be tightly regulated. Our NMR studies have

631

shown that catalytic residues C76 and H163 also undergo conformational change upon Mj

632

GATase-ATPPase interaction. The role of catalytic cysteine in GATase domain has been known

633

to be very important (20, 21, 55, 56). This is strong indication that there are conformational

634

changes at catalytic site upon interaction with ATPPase and that these changes could lead to

635

activation of the Mj GATase subunit.

Page 28 of 66

636

Hydrogen-deuterium exchange data provides valuable information about the

637

physical environment of exchangeable protons, i.e., whether they are present in regions of the

638

molecule that are solvent inaccessible and / or are strongly hydrogen bonded. Residues that are

639

solvent exposed often become protected from solvent when they are part of an intermolecular

640

interface. The rate at which these protons undergo solvent exchange is a measure of the strength

641

of association, particularly when the interaction is between large molecules. The rates of

642

exchange of solvent exposed residues in Mj GATase in the presence and absence of ATPPase

643

were nearly identical. This once suggests that the complex is not in slow exchange, but rather

644

exchanges on a time scale that is intermediate to fast.

645

The residues involved in the interaction with ATPPase are those that are present

646

in helix I and in loops V, VII, XI and XIV. Except for helix I, the residues in the loops are

647

present around the catalytic site. The residues in helix I are V12, H13, R14, V15, H16, R17, S18,

648

L19, K20, Y21, and I22. A survey of protein–protein interaction surfaces has shown that Trp,

649

Arg and Tyr predominate and these are considered as “hot spots”. In addition, His, Ile, Lys, Leu,

650

and Met are also found in high proportion. Helix I is well represented by these “hot spot”

651

residues. It has been shown that protein-protein interaction surfaces are generally uneven and 28 ACS Paragon Plus Environment

Page 29 of 66

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

652

consist of rigid and non-rigid structural elements. Furthermore, ~ 65 % of the core interface is

653

formed by rigid residues (57-59). In our case, a tight distribution of T1 and T2 values for residues,

654

V12 to I22, indicates that this helix is rigid. Additionally, the preponderance of positively

655

charged residues in helix I suggests that electrostatic interactions may play a very important role

656

steering complex formation, which could be stabilized by salt bridges (60).

657

In conclusion, we have determined the solution NMR structure of Mj GATase

658

subunit and have shown its interaction with MgCl2. Most importantly, we have identified the

659

specific residues on Mj GATase that are responsible for interaction with the ATPPase subunit.

660

The interaction between these subunits in a two-subunit-type GMPS has not been established

661

earlier.

662

The interaction between Mj GATase and Mg2+ falls in the fast chemical exchange regime

663

on the NMR time scale. The interaction between Mj GATase and ATPPase subunits fall in

664

intermediate chemical exchange regime on NMR time scale. This implies that ammonia

665

channeling occurs at a rate that is fast compared to the exchange rate of the protein-protein

666

complex.

667 668

Accession numbers

669

The structural coordinates for the twenty low energy structures of Mj GATase have been

670

deposited in the Protein Data Bank, PDB ID 2LXN. The chemical shift assignments for Mj

671

GATase have been deposited in the Biological Magnetic Resonance Bank, BMRB ID 17935.

672

673


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

674

Supporting Information available

675

Figures providing corroborative evidence for the data shown in the manuscript are included as

676

Supporting information. This material is available free of cost via the internet at

677

http://pubs.acs.org.

678


Page 30 of 66

Page 31 of 66

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Biochemistry

679

References:

680

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687 688

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Jones, S., and Thornton, J. M. (1996) Principles of protein-protein interactions., Proc Natl Acad Sci USA.93, 13-20.

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Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning,

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Zalkin, H., and Smith, J. L. (1998) Enzymes using glutamine as an amide donor, Adv Enzymol Relat Areas Mol Biol.72, 87-144.

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Fukuyama, T. T. (1966) Formation of an adenyl xanthosine monophosphate intermediate

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von der Saal, W., Anderson, P. M., and Villafranca, J. J. (1985) Mechanistic

701

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intermediate by positional isotope exchange experiments., J. Biol Chem.260, 5343-5350.

703

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von der Saal, W., Crysler, C. S., and Villafranca, J. J. (1985) Positional isotope exchange

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Tesmer, J. J., Klem, T. J., Deras, M. L., Davisson, V. J., and Smith, J. L. (1996) The

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crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural

708

paradigm for two enzyme families, Nat Struct Biol.3, 74-86.

709

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ammonia-channeling glutamine amidotransferases, Curr Opin Struct Biol.17, 653-664.

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Bhat, J. Y., Shastri, B. G., and Balaram, H. (2008) Kinetic and biochemical characterization of Plasmodium falciparum GMP synthetase, Biochem J.409, 263-273.

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Huang, X., Holden, H. M., and Raushel, F. M. (2001) Channeling of substrates and intermediates in enzyme-catalyzed reactions, Annu Rev Biochem.70, 149-180.

714 715

Raushel, F. M., Thoden, J. B., and Holden, H. M. (2003) Enzymes with molecular tunnels, Acc Chem Res.36, 539-548.

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Mouilleron, S., and Golinelli-Pimpaneau, B. (2007) Conformational changes in

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Bhat, J. Y., Venkatachala, R., and Balaram, H. (2011) SUbstrate-induced conformational

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changes in Plasmodium falciparum guanosine monophosphate synthetase, FEBS J.278,

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Nakamura, J., and Lou, L. (1995) Biochemical characterization of human GMP

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Nakamura, J., Straub, K., Wu, J., and Lou, L. (1995) The glutamine hydrolysis function

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of human GMP synthetase. Identification of an essential active site cysteine, J Biol

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Zalkin, H., and Truitt, C. D. (1977) Characterization of the glutamine site of Escherichia

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Maruoka, S., Horita, S., Lee, W. C., Nagata, K., and Tanokura, M. (2010) Crystal

731

structure of the ATPPase subunit and its substrate-dependent association with the

732

GATase subunit: a novel regulatory mechanism for a two-subunit-type GMP synthetase

733

from Pyrococcus horikoshii OT3, J Mol Biol.395, 417-429.

734

23.

Ali, R., Kumar, S., Balaram, H., and Sarma, S. P. (2012) 1H, 13C, 15N assignment and

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secondary structure determination of glutamine amido transferase subunit of gaunosine

736

monophosphate synthetase from Methanocaldococcus jannaschii., Biomol NMR

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nuclear magnetic resonance., Methods Enzymol.177, 44-73.

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Mori, S., Abeygunawardana, C., Johnson, M. O., and van Zijl, P. C. (1995) Improved

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fast HSQC (FHSQC) detection scheme that avoids water saturation, J Magn. Reson.

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Biochemistry

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Studied by 15N Inverse Detected Heteronuclear NMR Spectroscopy: Application to

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775

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predicting protein backbone torsion angles from NMR chemical shifts, Journal of

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Conformational analysis of protein and nucleic acid fragments with a new grid search

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algorithm FOUND., Journal of biomolecular NMR12, 543-548.

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analysis of macromolecular structures, J Mol Graph.14, 51-55, 29-32.

784 785

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786 787

Koradi, R., Billeter, M., and Wuthrich, K. (1996) MOLMOL: a program for display and

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Academic Press, Amsterdam.

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Demonstration of protein-protein interaction specificity by NMR chemical shift mapping,

797

Protein Sci.6, 2624-2627.

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perturbation mapping of DNA binding by a zinc-finger domain from the yeast

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transcription factor ADR1, Protein Sci.6, 1835-1848.

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816

Crystal structure of glutamine amidotransferase from Pyrococus horikoshii OT3, Proc.

817

Japan Acad.81, 459-462.

818

53.

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819

glutaminase variants provide insight into the activation mechanism of anthranilate

820

synthase., Biochemistry51, 2812-2818.

821

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822

Catalysis uncoupling in a glutamine amidotransferase bienzyme by unblocking the

823

glutaminase active site, Chem Biol.19, 1589-1599.

824

55.

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825

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826

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827

56.

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828 829

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57.

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830

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831

J Mol Biol.365, 663-679.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

832

58.

Reichmann, D., Rahat, O., Albeck, S., Meged, R., Dym, O., and Schreiber, G. (2005) The

833

modular architecture of protein-protein binding interfaces, Proceedings of the National

834

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835

59.

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836

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837

334.

838 839

60.

Sheinerman, F. B., Norel, R., and Honig, B. (2000) Electrostatic aspects of proteinprotein interactions, Curr Opin Struct Biol.10, 153-159.

840

841

842 843 844 845 846 847


Page 38 of 66

Page 39 of 66

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

848 849

Table 1

850

NOE-based distance restraints Total Intra-residue [|i-j| = 0] Sequential [|i-j| = 1] Medium range [|i-j| ≤ 4 Long range [|i-j|] ≥ 5 NOE restraints per residueb Hydrogen bond restraints Dihedral angle restraints Total number of restraintsb Total number of restraints per residueb Total number of structures calculated Number of structures used Restraint violatonsa,c Dihedral angle > 5° Distnace > 0.2 Ǻ Van der Waals Ramachandaran Plot (Procheck) Most favoured regions (%) Additionally allowed regions (%) Generously allowed regions (%) Total allowed regions (%) Disallowed regions (%) Ramachandaran Plot (Richardson’s lab) Most favoured regions (%) Allowed regions (%) Total allowed regions (%) Disallowed regions (%) rmsd from mean structure coordinate (Å) Backbonea Average heavy atoma a Analysed for residues 1 to 188

851

b

There are 183 residues with conformationally restricting restraints

852

c

Calculated for all restraints for the given residues, using average r-6

1781 670 526 200 385 9.73 110 364 2255 12.32 100 20 0 0 3 81.6 17.7 0.1 99.4 0.6 92.5 7.0 99.5 0.5 0.3 0.8

853 854 855 39 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

856 857

Table 2 Directly interacting residues

V12, H13, H16, R17, S18, L19, K20, Y21, I22, V24, N31 (side chain), L49, G51, G52, I55, N67 (side chain), I75, C76, L77, H79, A121, W122, A123, S124, H125, H141, C145, A149, V160, F162, H163, V166, H168, T169, E174, L176 and N178(side chain)

Indirectly interacting residues

V3, I4, D6, G23, I28, K57, I82, V90, G91, R92, A93, A95, E96, E97, Y98, A99, K102, V103, Y104, D106, D127, E128, V129, K130, Q146, E165, N173 (side chain)

858 859 860

861

862

863

864

865

866

867

868


Page 40 of 66

Page 41 of 66

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

869

870

Figure Legends

871

Figure 1:

872

and ATPPase (dimer) on a Superdex-200 column. Inset shows the molecular weight calibration

873

curve obtained using β-amylase, ADH (alcohol dehydrogenase), BSA (bovine serum albumin),

874

carbonic anhydrase and cytochrome c as the molecular weight markers.

875

Figure 2:

876

GATase subunit.

877

Figure 3:

878

HSQC spectrum of Mj GATase subunit. Strong sequential backbone HN-HN and Hα-HN NOE

879

correlations for the residues 35 to 40 indicate that these residues constitute an α-helix. Side-chain

880

to backbone NOE correlations have been also shown in figure.

881

Figure 4:

882

have been superposed on the backbone (N, Cα, and C’ atoms).

Gel filtration chromatogram showing the elution profile of Mj GATase (monomer)

Sequence specifically assigned two-dimensional 1H-15N HSQC spectrum of Mj

Two-dimensional strip-plots from the three-dimensional

15

N-edited1H-1H NOESY-

(a) Ensemble of 20 low-energy conformers of Mj GATase subunit. The structures

(b) A ribbon representation of lowest energy conformer of Mj GATase subunit.

883

884

Figure 5:

An overlay of solution NMR representative structure of Mj GATase, shown in green

885

and X-ray structure of Ph GATase(PDB ID 2D7J), shown in blue. The shortening of β-sheets

886

observed in solution structure, have been circumscribed byred elipses. Residues 68-70 and 51, 52

887

are shown in red and magenta colors respectively. The 310 helix observed in X-ray structure of


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 66

888

Ph GATase has been circumscribed by a red circle. The catalytic residues Cys, His and Glu have

889

been shown in stick representation.

890

Figure 6:

891

values of T1(top panel) and T2(bottom panel) have been plotted as function of residue number.

892

Shown in inset in lower panel is the T2 value of C-terminus E188. The secondary structural

893

elements have been shown as cylinder (α-helix) and arrow (β-sheet).

894

Figure 7:

895

GATase + ATPPase + Q; 4 GATase + ATPPase + Mg2+ + Q + ATP; 5, GATase + ATPPase +

896

Mg2+ + Q + XMP; 6, GATase + ATPPase + Mg2+ + Q + ATP + XMP. See the text for details.

15

N T1, T2 relaxation times, measured on an Agilent 600 NMR spectrometer. The

(a) Measurement of Mj GATase activity: 1, GATase + Q; 2, ATPPase + Q; 3,

(b) GMP sythetase activity monitored by NMR. The formation of GMP is

897 898

accompanied by a concomitant decrease in the XMP concentration. See text for details.

899

Figure 8:

900

intensity of the AMP peak as a function of time is visible as the reaction progresses. Note that

901

AMP formation results from the XMP-dependent cleavage of ATP. AMP build up can be seen

902

clearly by tracking the H8 resonance of AMP.

903

Figure 9:

904

and ATPPase. Maximum activity is observed for a 1:1 ratio of GATase:ATPPase.

905

Figure 10: (a) Change in chemical shift observed in various 1H-15N HSQC spectra of

906

labeled MjA GATase subunit, upon titration with varying of MgCl2 concentration. The change in

907

chemical shift has been shown with respect to unbound protein.

Progress of the reaction independently catalyzed by Mj ATPPase subunit. Increase in

Measurement of Mj GMP synthetase activity for different stoichiometries of GATase


15

N-

Page 43 of 66

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Biochemistry

908

(b) Cartoon-surface representation of the Mg2+ binding sites on Mj GATase structure.

909

The catalytic residues are shown in red (stick representation). The residues which bind with

910

Mg2+ and are present around the catalytic site, have been shown in blue. The residues shown in

911

cyan are those showing significant chemical perturbation due to conformational changes upon

912

Mg2+ binding.

913

(c) Fitting of titration curve for some of the well resolved Mj GATase residues.

914

Figure 11: Ligand mediated association of GATase with ATPPase. (a) Elution profile of Mj

915

GATase (A) and ATPPase (B) on an analytical size-exclusion Superdex 200 column under

916

different conditions viz., 2 mM MgCl ; 2 mM MgCl + 100 µM ATP; 2 mM MgCl + 100 µM 2

917

2

2

XMP; 2 mM MgCl + 100 µM ATP + 100 µM XMP (b to e) SDS-PAGE of the the protein 2

918

fractions collected under different conditions indicated in panel by analytical gelfiltration.

919

Protein bands were developed by silver staining of the gel.

920

Figure 12: Interaction between Mj GATase and ATPPase subunits. Lane1, GATase flow through

921

(cont); lane2, GATase flow through (exp); lane 3, GATase wash (cont); lane 4, GATase wash

922

(exp); lane5, GATase + ATPPase flow through (cont.); lane 6, GATase + ATPPase flow through

923

(exp); lane 7, GATase + ATPPase wash (cont); lane 8, GATase + ATPPase wash (exp); lane 9,

924

Elute (cont); lane10 Elute (exp); lane 11 Elute fraction 2(cont); lane 12 Elute fraction 2 (exp);

925

lane 13 beads (cont); lane 14 beads (exp); M is the molecular weight marker. Note (exp) refers

926

to experiment done in presence of ligands, while (cont.) is that in the absence of ligands.

927

Experimental details are as described in methods.


Biochemistry

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Page 44 of 66

928

Figure 13: (a) Overlay of proton one dimensional spectra of GATase, ATPPase and GATase,

929

ATPPase in presence of all substrates. The observed line broadening has been highlighted in red

930

rectangles.

931

(b) Overlay of two-dimensional 1H-15N spectra of

15

N-labeled Mj GATase subunit.

932

The red and blue colored spectra represent the spectra of GATase in absence and presence of

933

ATPPase subunit, respectively. Both of the spectra were acquired in absence of substrates.

934

(c) Overlay of two-dimensional 1H-15N spectra of

15

N-labeled Mj GATase subunit.

935

The spectrum shown in red represents the GATase spectrum in presence of unbound ATPPase

936

subunit. The blue spectrum represents the GATase spectrum in presence of ligand bound

937

ATPPase. Shown in inset is decrease in intensity upon GATase-ATPPase interaction.

938

(d) Decrease in intensity of 1H-15N correlation peaks of 15N-labeled GATase subunit

939

as a function of ATPPase concentration. All spectra were acquired in presence of the constant

940

saturating concentration of substrates. See text for details.

941

Figure 14: (a) Plot of normalized change in intensity as a function of residue number of Mj

942

GATase subunit. The residues showing ∆Inormalized> 0.80 and ∆Inormalized ≥0.75≤ 0.80 are shown in

943

blue and cyan, respectively. The catalytic residues are shown in red.

944

(b) Cartoon representation of Mj GATase subunit showing ATPPase interaction sites.

945

The catalytic residues have been shown in red (stick representation). The residues involved in

946

direct interaction are shown in blue while those involved in indirect interaction are shown in

947

cyan. The positively charged α-helix is highlighted in red elipse.


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Biochemistry

(c) Surface representation of Mj GATase subunit, showing the interaction surface

948 949

between two subunits.

950

Figure 15: Surface potential representation of Mj GATase structure. Large negatively charge

951

patches can be seen on both sides of the Mj GATase structure.

952

Figure 1

953 954 955 956 957 958 45 ACS Paragon Plus Environment

Biochemistry

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959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975


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976

Biochemistry

Figure 2

977 978 979 980 981 982


Biochemistry

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983

Figure 3

984 985 986 48 ACS Paragon Plus Environment

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987

Biochemistry

Figure 4a

988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010


Biochemistry

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1011

Figure 4b

1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034


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1035

Biochemistry

Figure 5

1036 1037 1038 1039 1040 1041 1042 1043 1044


Biochemistry

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1045

Figure 6

1046 1047 1048 1049


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1050

Biochemistry

Figure 7a

1051 1052 1053 1054 1055 1056 1057 1058 1059

Figure 7b

1060 1061 53 ACS Paragon Plus Environment

Biochemistry

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1062

Figure 8

1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 54 ACS Paragon Plus Environment

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1073

Biochemistry

Figure 9

1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 55 ACS Paragon Plus Environment

Biochemistry

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1084

Figure 10a

1085 1086 1087 1088 1089 1090 1091 1092 1093


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1094

Biochemistry

Figure 10b

1095

1096 1097

Figure 10c

1098 1099 1100 1101


Biochemistry

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1102

Figure 11 (a)

1103 1104 1105 1106 1107 1108 58 ACS Paragon Plus Environment

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1109

Biochemistry

Figure 12

1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 59 ACS Paragon Plus Environment

Biochemistry

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1125

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Figure 13a

1126 1127 1128

Figure 13b

Figure 13c

1129


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1130

Biochemistry

Figure 13d

1131

1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145


Biochemistry

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1146

Figure 14a

1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169


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1170

Biochemistry

Figure 14b

1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185


Biochemistry

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1186

Figure 14c

1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 64 ACS Paragon Plus Environment

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1200

Biochemistry

Figure 15

1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215


Biochemistry

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1216

For Table of Contents Use Only

1217

Solution NMR structure of GATase subunit and structural basis of interaction between GATase

1218

and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii

1219

Rustam Ali, Sanjeev Kumar, Hemalatha Balaram and Siddhartha P. Sarma

1220

1221

1222

1223

1224

1225

1226

1227

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1229

1230

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