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Remodeling of the binding site of nucleoside diphosphate kinase revealed by X-Ray structure and H/D exchange Alain Dautant, Julien Henri, Thomas E. Wales, Philippe Meyer, John R. Engen, and Florian Georgescauld Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01308 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 22, 2019
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Biochemistry
Remodeling of the binding site of nucleoside diphosphate kinase revealed by X-Ray structure and H/D exchange Alain Dautant,† Julien Henri,‡ Thomas E. Wales,§ Philippe Meyer,‡ John R. Engen,§ and Florian Georgescauld*,‡ † Université de Bordeaux, CNRS, Institut de Biochimie et Génétique Cellulaires, UMR5095, 146 rue Léo Saignat, 33077 Bordeaux, France § Department of Chemistry and Chemical Biology, Northeastern University, Boston, United States ‡ Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, UMR8226, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
IBGC, UMR 5095 CNRS Université de Bordeaux, Bordeaux, France IBPC, UMR 8226 CNRS Université Pierre et Marie Curie, Paris, France § Department of Chemistry and Chemical Biology, Northeastern University, Boston, United States * To whom correspondence should be addressed. Florian Georgescauld, Sorbonne Universités, UPMC Univ. Paris 06, CNRS, Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, UMR8226, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France. E-mail:
[email protected] † ‡
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ABSTRACT In order to be fully active and participate in the metabolism of phosphorylated nucleotides, most nucleoside diphosphate kinases (NDPK) have to assemble into stable hexamers. Here we studied the role played by six inter-subunit salt bridges R80-D93 in the stability of NDPK from the pathogen Mycobacterium tuberculosis (Mt). Mutating R80 into Ala or Asn abolished the salt bridges. Unexpectedly, compensatory stabilizing mechanisms appeared for R80A and R80N mutants and we studied them by biochemical and structural methods. R80A mutant crystallized into I222 space group unusual for NDPK and its hexameric structure revealed occurrence at the trimer interface of a stabilizing hydrophobic patch around the mutation. Functionally relevant, a trimer of the R80A hexamer showed a remodeling of the binding site. In this conformation, the cleft of active site is more open, and then active His117 is more accessible to substrates. HDX-MS analysis of WT, R80A and R80N mutants showed that the remodeled region of the protein is highly solvent accessible indicating that equilibrium between open and closed conformation is possible. We propose that such equilibrium occurs in vivo and explains how bulky substrates access the catalytic His117.
INTRODUCTION Nucleoside diphosphate kinases (NDPKs) are metabolic enzymes encoded by NME genes, also called NM23. They are present in all kingdoms of life and in certain viruses. NDPKs are responsible for the transfer of the γ-phosphate from nucleoside triphosphates (NTP) to nucleoside diphosphates (NDP) and, in this way, they maintain the equilibrium between the pools of phosphorylated nucleosides NTP/NDP [1]. The γ-phosphate transfer reaction takes place in two steps according to a ping-pong mechanism, with transient phosphorylation of the enzyme on a 100% conserved histidine residue [2, 3]. NDPK recognizes with low specificity all tri- and di-phosphate nucleosides [4]. As expected from their intracellular concentrations, it has been shown that the major phosphate donor in the cell is the ATP while the major acceptor is the GDP [1]. In mammals, by efficiently supplying with GTP the dynamin superfamily through direct interaction, it has recently been shown that NDPK plays an important role in membrane remodeling and trafficking events [5, 6]. Moreover, direct interaction between NDPK and different compounds or regulatory proteins from the cytoskeleton has been observed, indicating a clear role in nucleotide channeling and cell motility [1, 6]. Secondary enzymatic activities conserved from bacteria to humans, have been established for NDPK. It can act as protein histidine kinase [7], bind to single stranded DNA and exhibit 3’-5’ exonuclease activity, playing a potential role in DNA repair [8]. Studies on drosophila, zebrafish and mice show a role of NDPK in early, larval and embryo development [9]. In mammals, ten different NDPK isoforms exist. Isoforms 1 to 7 were characterized and present at least one of the enzymatic activities, while the function of the isoforms 8 to 10 is less understood [1, 8]. Isoform 1, also known as NM23-A, is the most abundant one and was the first human metastasis suppressor to be reported [10-12]. The 2 ACS Paragon Plus Environment
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Biochemistry
molecular mechanism by which NM23-A inhibits in vivo the metastasis spreading is largely unknown and has been linked with the role of NDPK in the cell motility. However, metastasis suppression function is dependent on NDPK capacity to perform its enzymatic activities [1]: nucleoside diphosphate kinase [13], histidine kinase [7, 14-16] and 3’-5’ exonuclease [8, 17, 18]. In order to be functional, NDPK monomers have to auto-assemble into hexameric or tetrameric complexes, sub-oligomeric species like monomers or dimers presenting at most 1 to 2 % of the total activity [19]. Hexamers are formed in eukaryotes, archaea, some viruses and Gram-positive bacteria [20], while two different types of tetramers are present in Gram-negative bacteria [21]. In terms of subunits composition of complexes, NDPK hexamers as well as tetramers are homo-oligomeric or hetero-oligomeric [19]. Interestingly, hetero-oligomers are formed in vivo as well as in vitro, either from different cellular isoforms, either after infection from a mix of host and pathogen monomers, a potential way to have more interactions with other proteins and hence more cellular functions [19, 22]. All NDPK structures deposited at the PDB concern homo-oligomers. Figure 1 A shows one monomer. The ferredoxin-fold is conserved with two additional specific elements: the helix hairpin formed by αA and α2 helices linking β1 and β2 strands and the Kpn-loop (named from the Killer of prune lethal mutation in Drosophila [23]) inserted between α3 and α3’ helices [4, 21]. All structures show that monomers are in the same conformation inside the hexamers or tetramers, in agreement with the idea that active sites are identical and independent. Complete genome sequencing of Mycobacterium tuberculosis (Mt), the causative agent of tuberculosis, showed that it contains only one gene coding for NDPK (UniProtKB A5U5E1) and its crystal structure showed it forms hexamers (Pdb Id 1k44; Figure 1) [24]. In patients infected by tuberculosis, the bacterium is phagocytosed by macrophages, but achieves to escape degradation by preventing the fusion of the phagosome with the lysosome [25]. The escape mechanism responsible for an enhanced intracellular survival within macrophages is not fully understood but requires several thermostable proteins [26], including NDPK [25, 27]. In this context, we previously studied the stabilizing mechanisms allowing Mt-NDPK to present a 15 to 20°C higher Tm when compared to NDPK from other species [20]. We showed that this unusually high stability is partially based on the presence of six inter-subunit ionic bridges R80-D93 (Figure 1 B). When the bridges were abolished by the point mutation D93N, the mutant was still hexameric and enzymatically active, but its Tm dropped by 28°C [20]. Equilibrium analysis of WT and D93N mutant by hydrogen deuterium exchange mass spectrometry (HDX-MS) allowed us to show that the bridge R80-D93 was not only important in the hexamer stability but also influenced its conformational dynamics [28]. We have continued our research to understand the role of ionic bonds on Mt-NDPK stability and conformational dynamics by mutating R80, the other amino acid involved in the ionic bridge formation (Figure 1 B). Mutants R80A and R80N (R80A/N) were produced and their properties 3 ACS Paragon Plus Environment
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studied by enzymology, fluorescence, circular dichroism (CD) and size-exclusion chromatography (SEC). The structure of mutant R80A which crystallized into an unusual space group for NDPKs was solved. We noticed important conformational remodeling which has not been stated so far. It mainly concerned peripheral helices αA and α2 which are involved in nucleotide binding and in its locking inside the active site. In the current structure, the catalytic site is remodeled and becomes accessible for substrates bigger than a nucleotide. To show that such conformational change exists in solution, we performed at equilibrium HDX-MS measurements for WT, R80A and R80N in presence and absence of nucleotides. The HDX data which deciphered Mt-NDPK dynamics in solution was in perfect agreement with the structural remodeling observed in the crystal structure. We propose that the highlighted conformational change explains how bulky substrates access the catalytic His117.
MATERIALS AND METHODS Reagents Chemicals of the highest purity grade were bought from Sigma. Solutions of urea were freshly prepared for each experiment. Mutagenesis and Protein Purification The gene mutation R80A was introduced using a TransformerTM site-directed mutagenesis kit (Clonetech) and the mutation was confirmed by nucleotide sequencing. The individual expression of WT, R80A, R80N and D93N Mt-NDPK recombinant proteins was realized using a pET24 vector (Novagen) in the BL21-derived host strain BL21-CodonPlusH(DE3)-RIL (Stratagene). The 2YT culture medium contained 16 g/L bacto tryptone, 10 g/L bacto yeast extract, 5 g/L sodium chloride, in the presence of 80 mg/mL of kanamicyn. The expression was induced with 1 mM IPTG for 6 h at 37°C, once the optical density reached 0.5–0.7 units. As previously described, the purification steps were carried out at 4°C [20, 28]. After harvesting, the E. coli cells were sonicated and centrifuged in order to recuperate the soluble fraction containing Mt-NDPKs. The DNase-treated bacterial extract was loaded onto a Q-Sepharose column equilibrated in 100 mM Tris-HCl, pH 7.4. The enzyme was eluted at 0.5–0.6 M NaCl, in a linear gradient of 0–0.8 M NaCl in the same buffer. Active fractions were precipitated with 80% saturated ammonium sulfate and further purified by salting-out chromatography on a Sepharose 6B column equilibrated with 80% ammonium sulfate, 100 mM TrisHCl, pH 7.4. The protein was eluted by a linear gradient from 80% to 20% ammonium sulfate in the same buffer. The active fractions were pooled, dialyzed against 100 mM Tris-HCl, pH 7.4, and further purified on a Source 15Q column, under the conditions described for the Q-Sepharose chromatography. The enzymes were precipitated by dialysis against a saturated solution of ammonium sulfate, recovered by centrifugation and further purified by size exclusion chromatography on a Sephacryl S-200 column equilibrated with 0.2 M sodium phosphate buffer, pH 7.0. This step allowed cleaning up the sample, by eliminating aggregated and dissociated protein. The enzymes were 4 ACS Paragon Plus Environment
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Biochemistry
essentially pure as ascertained by polyacrylamide gel electrophoresis in the presence of SDS. The concentrations of WT and mutant Mt-NDPKs were determined from the optical density at 280 nm using an extinction coefficient of 0.48 for 1 mg/mL, which was calculated from the amino acid composition. The molecular weight of proteins was checked by mass spectrometry. Size exclusion chromatograms (SEC) were performed on SuperoseTM 12, 10/300 GL column equilibrated with 20 mM Tris-HCl, pH 7.0, 100 mM NaCl to insure that all proteins were hexameric. Molecular markers: cytochrome c, 12.4 kDa; myoglobin, 17 kDa; carbonic anhydrase, 29 kDa; ovalbumin, 44 kDa; bovine serum albumin (BSA), 68 kDa; aldolase, 158 kDa and beta amylase, 200 kDa; (GE Healthcare Life Sciences) were used. Thermal denaturation and chemical unfolding experiments Thermal denaturation experiments were performed by incubating 10 μg/mL of NDPK in a water bath and increasing the temperature at the rate of 1°C/min. At different time points (corresponding to different temperatures) aliquots were taken and the enzyme activity was measured according to the NDPK enzymatic assay. The chemical unfolding experiments were performed as previously described [20, 28]. A 10 μg/mL final concentration of native or unfolded Mt-NDPK was incubated for 16 h in 0–8 M urea or 0–5 M GuHCl and 20 mM phosphate buffer (pH 7.0) at 25°C. Fluorescence intensities of the single tryptophan residue, Trp132, were measured at 335 nm with excitation at 295 nm. NDPK enzymatic assay Enzymatic activity of NDPK was measured using a coupled assay which involves the three independent reactions shown below [20]. Enzymes performing the catalysis of each reaction are indicated in parentheses. NDPK catalyzed the transfer of the γ-phosphate from ATP to 8-bromoinosine 5′-diphosphate (8-BrIDP). Reaction took place into 0.8 mL of enzyme mix containing 1 mM ATP, 0.2 mM 8-BrIDP, 1 mM phosphoenolpyruvate (PEP), 0.1 mM NADH, 20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 100 mM KCl, 1 mg/mL BSA, 2 units/mL pyruvate kinase and lactate dehydrogenase. NADH disappearance was measured at 340 nm and 25°C, using a PerkinElmer spectrophotometer. ATP
+ 8-BrIDP → ADP
+ 8BrITP
(NDPK)
ADP
+
→ ATP
+ pyruvate
(pyruvate kinase)
→ lactate
+ NAD+
(lactate dehydrogenase)
PEP
Pyruvate + NADH
Crystallization and X-Ray diffraction data collection
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Recombinant Mt-NDPK-R80A was purified by Superdex 200 size-exclusion chromatography in buffer 100 mM NaCl, 20 mM Tris-HCl, pH 7.9. Collected peak fractions were pooled and concentrated to 12.1 mg/mL by ultrafiltration. Sparse-matrix screening of candidate crystallization conditions was set up on TTP Labtech IQ plates with mixes of 50 nL protein and 50 nL commercial precipitant solutions (Qiagen) and incubated at 20°C. Monocrystalline 100 µm x 50 µm platelets of R80A Mt-NDPK grown for 5 weeks in condition JCSG IV 58 (0.8 M ammonium sulfate, 0.1 M HEPES-NaOH, pH 7.0) were cryo-protected by addition of 25% glycerol and flash-frozen in liquid nitrogen. 2000 images of 0.1° rotation increment each were collected on ID30A-3 MASSIF microfocus beamline at the European Synchrotron Radiation Facility (Grenoble, France) at 12.81 keV. Diffraction data were recorded on a Eiger X 4M detector. The data were indexed and integrated with IMOSFLM [29] and SCALA [30] in orthorhombic space group I222 with unit cell parameters a=113.1 Å, b=121.7 Å, c=150.71 Å. The dataset was complete to >99% at a resolution of 2.2 Å. The structure was solved by molecular replacement method with MOLREP using the highest resolution available Mt-NDPK structure (Protein Data Bank code 4ane) as a search model [31]. The unit cell contained six molecules in the asymmetric unit resulting in a solvent content of 59% (Matthews’ coefficient (Vm) of 2.99 Å3 Da-1). HDX-MS Stock solutions of 4.0 μM native Mt-NDPKs (WT and R80A/N) were prepared in 20 mM Tris-HCl, pH 7.0, 100 mM NaCl and H2O. Deuterium exchange was initiated by 20-fold dilution of the MtNDPK stock solution into 100 mM NaCl, 20 mM Tris-HCl, pD 7.0, and 99.9% D2O at 10°C. At different incubation times, a 100 μL aliquot was removed from the exchange reaction mixture (from 10 s to 120 min) and labeling was quenched at pH to 2.5 by adding 10 μL solution of (1% formic acid, 5 M GuHCl), at 0°C. 100 μL of each acid-quenched sample was immediately injected into an HDX Waters nanoACQUITY Ultra Performance Liquid Chromatography (UPLC) [32]. The sample passed through a Poroszyme-immobilized pepsin cartridge (Applied Biosystems) accommodated within the HDX manager at a flow rate of 100 μL min–1 and 15°C. Peptic peptides eluting from the pepsin column were trapped and desalted for 3 min at a rate of 100 μL min–1. Peptides were separated in 6 min with a 5 to 35% acetonitrile/water gradient in 0.1% formic acid, at a rate of 100 μL min–1 with a 1.0 mm × 50.0 mm ACQUITY UPLC C18 HSS T3 column (Waters) containing 1.8 μm particles (back pressure 13,000 psi). All chromatographic elements were held at 0°C for the entire time of the measurements. The calculated average amount of back exchange was of 31% on the basis of analysis of fully deuterated peptides of NDPK in GuDCl. Since all comparison experiments were performed under identical experimental conditions, there was no correction for back exchange [33]. The error in the determination of the deuterium levels was ±0.20 Da in this experimental setup, consistent with previously obtained values [34, 35]. Mass spectra were recorded in HDMSE mode using a Waters Synapt G2 Si instrument with a standard ESI source (Waters Corp., Milford, MA) over an m/z range of 50–2000. Mass accuracy was ensured by calibration with Glu-fibrinogen peptide and was
