Structural insights into substrate selectivity and activity of bacterial

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Structural insights into substrate selectivity and activity of bacterial polyphosphate kinases Boguslaw P. Nocek, Anna N. Khusnutdinova, Milosz Ruszkowski, Robert Flick, Malgorzata Burda, Khorcheska Batyrova, Greg Brown, Artur Mucha, Andrzej Joachimiak, Lukasz Berlicki, and Alexander F. Yakunin ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b03151 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 12, 2018

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ACS Catalysis

Structural Insights into Substrate Selectivity and Activity of Bacterial Polyphosphate Kinases

Boguslaw P. Noceka, Anna N. Khusnutdinovab, Milosz Ruszkowskic, Robert Flickb, Malgorzata Burdad, Khorcheska Batyrovab, Greg Brownb, Artur Muchad, Andrzej Joachimiaka, Lukasz Berlickid, and Alexander F. Yakuninb*

a

Midwest Center for Structural Genomics and Structural Biology Center, Department of

Biosciences, Argonne National Laboratory, Argonne, IL 60439, U.S.A. b

Department of Chemical Engineering and Applied Chemistry, University of Toronto,

Toronto, ON, M5S 3E5, Canada c Synchrotron

Radiation Research Section of MCL, National Cancer Institute, Argonne,

IL 60439, USA d

Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of

Science and Technology, 50-370 Wroclaw, Poland

*corresponding

author: [email protected]

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Abstract Polyphosphate (polyP) kinases are widely conserved enzymes with importance in basic bacterial metabolism and virulence in many pathogens. However, the molecular mechanisms of their substrate specificity and catalysis remain unknown. Here, we present the results of comprehensive biochemical and structural studies of three polyP kinases from different bacteria, which belong to different clusters of the PPK2 class III family. Purified PPK2 proteins catalyzed polyP-dependent phosphorylation of AMP, ADP, GMP, and GDP to corresponding nucleoside di- and triphosphates. Crystal structures of these proteins in complex with substrates, products, Mg2+, and inhibitors revealed the binding sites for the nucleotide and polyP substrates overlapping at the Walker A and B loops. The Walker A loop is involved in the binding of polyP and the Mg2+ ion, whereas the Walker B loop coordinates the nucleotide phosphate groups. Structure-based site-directed mutagenesis of CHU0107 from Cytophaga hutchinsonii demonstrated the critical role of several conserved residues from the PPK2 core and lid domains, involved in the coordination of both substrates and two Mg2+ ions. Additionally, a two-times higher activity was observed following deletion of the C-terminal tail in the CHU0107 mutant protein L285Stop. Crystal structures of PPK2 in complex with three aryl phosphonate inhibitors indicated the presence of at least two binding pockets for inhibitors located close to the Walker A loop and the catalytic residues Lys81 and Arg208. Our findings provide a molecular framework for understanding the molecular mechanisms of PPK2 kinases and have implications for future drug design and biocatalytic applications. Keywords: inorganic polyphosphate, polyphosphate kinase, crystal structure, site-directed mutagenesis, ATP regeneration


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1. Introduction Inorganic polyphosphates (polyP) are polymers of orthophosphate (Pi) residues linked by phosphoanhydride (P-O-P) bonds. Arthur Kornberg called polyP a “bioenergy fossil” that originated in the pre-biotic world which became a primary energy source for the first organisms on Earth 1. PolyP is ubiquitous and present in all species tested to date, from bacteria to higher eukaryotes 2-5. It has numerous biological functions that include important roles in basic metabolism, maintenance of cellular structure, structural stabilization of nucleic acids, proteins and protein complexes (a chaperone), sensing/responding to environmental changes, stress response and survival, as well as a general source of ATP and Pi 2, 5-12. In addition to a crucial role of polyP in basic metabolism, there are numerous findings indicating its importance in bacterial pathogenesis, including biofilm formation, motility, sporulation, surface attachment, stationary phase and stress survival, dormancy and other features linked to virulence in several important bacterial pathogens, e.g. Mycobacterium tuberculosis, Neisseria, Campylobacter, Francisella, and Pseudomonas 2, 5, 13-21. The importance of polyP and associated enzymes to the virulence and survival of a wide spectrum of pathogens has led to the proposal that polyP-associated enzymes represent potential therapeutic targets for anti-bacterial chemotherapy 17, 22-23. The major enzymes associated with polyP metabolism include the two different families of polyP kinases (PPKs): PPK1 and PPK2. PPK1 enzymes catalyze reversible polymerization of the terminal phosphate of ATP into a polyP chain, whereas PPK2 kinases preferentially catalyze the polyP-driven synthesis of ATP or GTP 1, 24-26. A large number of studies have demonstrated the importance of PKK2 enzymes for virulence in a


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wide spectrum of important bacterial pathogens, suggesting that these enzymes represent potential therapeutic targets for anti-bacterial chemotherapy 2, 6, 17. PPK2 kinases have also attracted significant interest in biocatalysis due to their potential for the development of polyP-dependent regenerating systems for ATP and other nucleotides for various biocatalytic applications 27-28. PPK2 enzymes belong to the large group of P-loop kinases, which share a conserved globular fold with a parallel β-sheet surrounded by α-helices and the lid module29. P-loop kinases are characterized by the presence of two conserved sequence motifs: Walker A (binds the β- and γ-phosphates of ATP) and Walker B (with the conserved carboxylate residue coordinating a metal ion bound to the nucleotide phosphate groups) 29,30. Most of the biochemically characterized PPK2s predominantly catalyze the polyP-driven phosphorylation of ADP, GDP, or AMP 24, 31-36, but the Corynebacterium glutamicum PPK2B (NCg12620) was more active in the polyP-forming direction 37 (Table S1). A recent phylogenetic analysis of 209 PPK2 sequences revealed the presence of three classes of these enzymes (I, II, and III) with classes I and II catalyzing the phosphorylation of either ADP or AMP, respectively, whereas class III was active against both substrates 38. The crystal structures of SMc02148 (class I) from Sinorhizobium meliloti and PA3455 (class II) from P. aeruginosa revealed the presence of a 3-layer α/β/α sandwich fold with an α-helical lid similar to the structures of microbial thymidylate and adenylate kinases 24. SMc02148 contains a single PPK2 domain, whereas PA3455 has two fused PPK2 domains, the catalytically inactive N-terminal and active C-terminal domains24. The structure of SMc02148 showed the presence of four PPK2 monomers assembled as a D2 tetramer, whereas the asymmetric unit of PA3455


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contained a dimer with the four PPK2 domains arranged like a pseudotetramer. Sitedirected mutagenesis identified nine conserved residues required for activity of both proteins, which include the residues from both Walker A and B motifs and the lid 24. Recently, the crystal structures of PPK2s from Francisella tularensis (FTT1564, class I) and Meiothermus ruber (MrH2468, class III) in complex with nucleotides and/or polyP were determined, revealing that the folds and active sites are similar to those of SMc02148 and PA3455 16, 39. These structures provided insight into the molecular basis of substrate binding and nucleotide specificity of PPK2 enzymes. However, our understanding of PPK2 catalyzed phosphorylation of nucleotides is still limited, including the mechanistic details for activity, the role of Mg2+ in catalysis, and potential for PPK2 inhibitor development. Here, we have performed detailed biochemical, structural, and mutational studies of three class III PPK2 enzymes from Cytophaga hutchinsonii (CHU0107), Arthrobacter aurescens (AAur2811), and Deinococcus radiodurans (DR0132), as well as the class I PPK2 from S. meliloti (SMc02148). These enzymes predominantly catalyze the poly-P dependent phosphorylation of both adenosine and guanosine mono- and diphosphates to the corresponding di- and triphosphates. Ten crystal structures of four PPK2s from different phylogenetic groups in complex with substrates, products, Mg2+, and inhibitors revealed the binding sites for these ligands. Site-directed mutagenesis identified the PPK2 residues critical for activity and demonstrated two times greater activity in the CHU0107 protein with a removed Cterminal extension. Our results provide a molecular framework toward understanding the activity of PPK2 kinases and allow for the development of PPK2 inhibitors and more efficient ATP-regenerating systems.


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2. Materials and Methods 2.1. Gene cloning, protein purification, and mutagenesis. The genes encoding four selected PPK2 proteins (CHU0107, AAur2811, DR0132, and SMc02148) were amplified by PCR from corresponding genomic DNA and cloned into a pMCSG19 plasmid as described previously 40. The proteins were expressed as a fusion with a dual N-terminal 6His/maltose-binding protein (MBP) tag and affinity purified as described 40. The oligomeric state of purified proteins was determined using size-exclusion chromatography on a Superdex 200 10/300 column (GE Healthcare). Site-directed mutagenesis of CHU0107 was performed using a protocol based on the QuickChange site-directed mutagenesis kit (Stratagene) as described previously 41. The presence of mutations was verified by DNA sequencing, and mutant proteins were overexpressed and purified in the same manner as the wild-type CHU0107. 2.2. Enzymatic assays. PolyP-dependent phosphorylation of AMP and GMP was measured spectrophotometrically using a continuous enzyme-coupled assay with pyruvate kinase and lactate dehydrogenases in a reaction mixture (0.2 ml) containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, nucleoside monophosphate (0.01-5 mM), 0.5 mM polyP (polyP12-13; Sigma), 5 mM phosphoenolpyruvate, 0.3 mM NADH, 2 units of pyruvate kinase (from rabbit muscle, Sigma), 5 units of L-lactate dehydrogenase (Sigma), and 0.02-4 μg of purified PPK2 42. In addition, an HPLC-based protocol was used for the analysis of polyP-dependent (and independent) PPK2 activity with nucleoside mono- and diphosphates (1 mM ADP, 2 mM AMP, 5 mM GDP; 0.5 - 40 μg protein), as well as the reverse PPK2 reaction (polyP-dependent dephosphorylation of ATP) and adenylate kinase reaction (10-80 μg protein/assay). Enzymatic reactions (0.2 ml; 50 mM Tris-HCl,


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pH 8.0, 10 mM MgCl2, 1 mM polyP) were carried out at 30 °C (15 min), filtered through 10 kDa cut-off spin filters, and reaction products were analyzed using reverse phase chromatography on a Varian ProStar HPLC system equipped with an AXXI-CHROM ODS C18 column (5 μm particles; 4.6 × 250 mm; Cole Scientific) 43. The dependence of PPK2 activity on divalent metal cations was determined using HPLC (50 mM Tris-HCl, pH 8.0, 5 mM ADP, 0.5 mM polyP, 2 mM metal ions, and 5 μg protein). For determination of kinetic parameters (Km and kcat), enzymatic assays were performed using a range of substrate concentrations, and kinetic parameters were calculated as previously described 41. 2.3. Protein crystallization. Selenomethionine-labeled or native PPK2 proteins (CHU0107, AAur2811, DR0132, and SMc02148) were purified using metal chelate affinity chromatography as described previously 44. Purified proteins were dialyzed against a uniform crystallization buffer containing 20 mM HEPES-K (pH 7.5), 150 mM NaCl, and 1 mM TCEP and concentrated to ~ 20 mg/ml. Crystallization was performed by the sitting-drop vapor-diffusion method at 18°C using a robotic Mosquito system (TTP LabTech). Three hundred commercially available conditions were used for the initial screening and the most promising hits were further optimized. Crystals of AAur2811 were grown in 0.1 M phosphate citrate and 0.4 M PEG 300. Purified DR0132 was incubated with 1 mM MgCl2, 10 mM sodium triphosphate and 20 mM ADP for 20 minutes on ice and then mixed at 1:1 ratio with the crystallization solution containing 0.2 M CaCl2, 0.1 M Bis-Tris (pH 6.5), and 45% methylenepentanediol (MPD). For CHU0107 complexes with different ligands, purified protein was mixed with AMP, ADP, guanosine 5'-tetraphosphate (G4P), or inhibitors (final concentration: 10 mM for each),


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and crystals were grown in solutions containing 1.1 M ammonium tartrate (pH 7.0). SMc02148 was crystallized in the presence of 0.1 M Tris-HCl (pH 7.5), 0.12 M Naformate, 0.12 M Li-sulfate, 0.4 M NDSB 211, and 10 mM ADP. 2.4. Data collection, structure determination, and refinement. The data sets for each crystal were collected on CCD detector at the 19ID or 19BM beamlines of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory. The experimental set-up involved taking two test diffraction images 90° apart and collecting data near the selenium white line (λ1 = 0.9795 Å). All diffraction data were collected at 100 K and processed using the HKL3000 45 or XDS 46 programs. The structures have been determined using single anomalous dispersion (SAD) or molecular replacement methods. Manual correction of the models was carried on in COOT 47, and the models were refined with REFMAC 48 of the CCP4 49 and phenix.refine 50. The quality of structural models was assessed with Molprobity 51. The data collection and final refinement statistics are presented in Table S2. The atomic coordinates have been deposited in the Protein Data Bank with accession codes 6ANG (CHU0107-AMP), 6AN9 (CHU0107-ADP), 6ANH (CHU0107-G4P), 6AU0 (CHU0107-inh9), 6B18 (CHU0107inh24), 6AQN (CHU0107-inh25), 6AQE (DR0132-ATP-Mg), 3RHF (AAur2811-citratephosphate), and 6DZG (SMc02148-ADP/AMP). 2.5. Sequence and structural analyses. Phylogenetic analysis of the PPK2 protein family was performed using 12,273 PPK2 sequences (IPR022488, InterPro database) and the approximately-maximum-likelihood algorithm of FastTree 2.1.5 built in Geneious 8.1.8. Multiple sequence alignments were constructed using the MAFFT-online server (https://mafft.cbrc.jp/alignment/server/). The PPK2 protein surface conservation was


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calculated using ConSurf-2016 based on 500 sequences from UNIREF90 extracted from the list of 2,215 unique HMMER homologues with sequence identities between 40% and 95% 52-54. The PPK2 surface electrostatic potential was calculated using the PDB2PQR and APBS servers 55-56.

3. Results and Discussion 3.1. Comprehensive phylogenetic analysis of the PPK2 family of polyP kinases. The vast majority of identified PPK2 sequences (PPK2 domain, InterPro IPR022488) are present in bacterial genomes (12,702), with just 162 PPK2 genes found in archaea, 103 in metagenomes, and 6 in eukaryotes. After removing redundant and incomplete sequences, the phylogenetic analysis of over 7,000 PPK2 sequences revealed the presence of the three major clades corresponding to the recently proposed 38 PPK2 classes I (6,717 sequences), II (2,511 sequences), and III (3,716 sequences) (Figure 1A). Phylogenetic analysis of PPK2 sequences within these classes identified at least nine clusters in class I, five clusters in class II, and three clusters in class III (Figures 1B and S1). Sequence alignment of PPK2 proteins from the three classes revealed the conservation of the Walker A (GxDxxGK) and B (DR) motifs, as well as several charged and aromatic residues in the lid domain (overall sequence identity 25-45%) (Figure S2). Interestingly, approximately half of the class III PPK2 sequences showed conservative substitutions of the Walker-A Gly to Ala (Ala75 in CHU0107) and Walker-B Asp to Asn (Asn132 in CHU0107). In the class III phylogenetic tree, PPK2 proteins with the Walker B-Asp motif are associated with cluster III-3, whereas the Walker B-Asn containing proteins are


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found in clusters III-1 and III-2 (Figure 1B). For the class II enzymes, one- and twodomain PPK2 proteins were found in all five clusters (Figure S1B).

Figure 1. Phylogenetic analysis of the PPK2 family. (A), Unrooted phylogenetic tree of PPK2 proteins (IPR022488) showing three monophyletic groups: I, II, and III. (B), Unrooted phylogenetic tree of the class III PPK2 proteins (3,050 sequences) showing three major monophyletic groups (1, 2, 3). The class III PPK2 proteins characterized in


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this work (CHU0107, DR0132, AAur2811) are indicated by black spheres, whereas blue spheres indicate PPK2 proteins with experimentally confirmed polyP kinase activity. Most biochemically characterized PPK2 enzymes belong to class I (PA0141, SMc02148, and FTT1564), with only two proteins characterized from class II (PA3455, A. johnsonii PAP) and one from class III (Mrub2488 from M. ruber strain DSM 1279) (Table S1).

3.2. Biochemical characterization of class III PPK2 proteins CHU0107, DR0132, and AAur2811. In this work, we have crystallized class III PPK2 proteins from Arthrobacter (Paenarthrobacter) aurescens (AAur2811), Cytophaga hutchinsonii (CHU0107), and Deinococcus radiodurans (DR0132) (Tables S1, S2). These PPK2 proteins belong to different class III clusters: III-1 (CHU0107), III-2 (DR0132), and III-3 (AAur2811) (Figure 1B). They share low sequence similarity to each other (24.8 to 42 % sequence identity) with the conservation of eight residues found to be essential for the activity of SMc02148 and PA3455 (Figure S2). In contrast to AAur2811, the sequences of both CHU0107 and DR0132 show replacement of the Walker B Asp by Asn (Figure S2). The three purified class III PPK2 proteins catalyzed polyphosphate- and Mg2+dependent phosphorylation of AMP to ADP and ADP to ATP (Table 1). In contrast to SMc02148 (class I) and PA3455 (class II) 24, class III PPK2s can use a broader range of divalent metal cations, and showed significant activity in the presence of Mg2+, Mn2+, Co2+, Ca2+ and Ni2+ (Table 1, Figure S3). In general, they showed higher affinity (lower Km) for ADP, but higher activity toward AMP, especially DR0132 which exhibited very low activity with ADP (kcat of 94.5 s-1 for AMP and 0.4 s-1 for ADP) (Table 1). Both CHU0107 (cluster-1) and AAur2811 (cluster-3) exhibited comparable catalytic efficiency


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(kcat/Km) with AMP and ADP, whereas the catalytic efficiency of DR0132 (cluster-2) with AMP was two orders of magnitude higher than with ADP (kcat/Km 3.5 × 105 and 0.1 × 103 s-1M-1, respectively) (Table 1). With GMP and GDP as substrates, CHU0107 and AAur2811 showed lower activity compared to adenosine nucleotides, while DR0132 was only active against GMP (Table 1). In addition, AAur2811 and DR0132 had higher affinities to polyP compared to CHU0107 (Table 1). In the absence of polyP, both CHU0107 and AAur2811 catalyzed detectable dephosphorylation of ATP to ADP, but this activity was at least three orders of magnitude lower than polyP-driven phosphorylation of AMP or ADP (Table S3).

Table 1. Kinetic Parameters of Purified PPK2 Enzymes with Various Substrates. Proteins and variable substrates CHU0107 AMP dAMP ADP dADP GMP GDP polyP ATP Mg2+

Other substrates

Measured Products

Km, mM

kcat, s-1

kcat/Km, s-1 M-1

polyP polyP polyP polyP polyP polyP ADP polyP ADP, polyP

ADP dADP ATP dATP GDP GTP ATP ADP ATP

0.62 ± 0.10 1.69 ± 0.61 0.35 ± 0.06 0.53 ± 0.12 3.15 ± 0.64 2.74 ± 0.55 0.29 ± 0.07 2.71 ± 0.71 0.57 ± 0.05

16.83 ± 0.91 2.77 ± 0.54 9.39 ± 0.52 0.58 ± 0.05 6.91 ± 0.52 3.86 ± 0.35 29.63 ± 4.70 0.29 ± 0.05 9.11 ± 0.21

2.7 × 104 1.6 × 103 2.7 × 104 1.1 × 103 2.2 × 103 1.4 × 103 1.0 × 105 0.1 × 103 1.6 × 104

AAur2811 AMP dAMP ADP dADP GMP GDP polyP ATP Mg2+

polyP polyP polyP polyP polyP polyP ADP polyP ADP, polyP

ADP dADP ATP dATP GDP GTP ATP ADP ATP

0.83 ± 0.11 1.03 ± 0.34 0.17 ± 0.03 0.11 ± 0.04 17.70 ± 2.51 39.20 ± 9.61 0.011 ± 0.001 8.64 ± 0.72 2.84 ± 0.03

21.24 ± 0.83 0.60 ± 0.08 4.12 ± 0.12 0.55 ± 0.07 0.97 ± 0.07 2.12 ± 0.37 5.53 ± 0.09 3.32 ± 0.22 5.04 ± 0.18

2.6 × 104 0.6 × 103 2.4 × 104 5.0 × 103 0.1 × 103 0.1 × 103 5.0 × 105 0.4 × 103 1.8 × 103

DR0132


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AMP dAMP ADP GMP GDP polyP a ND, not detected.

polyP polyP polyP polyP polyP AMP

ADP dADP ATP GDP GTP ADP

0.27 ± 0.03 94.47 ± 2.80 0.71 ± 0.39 0.06 ± 0.01 0.20 ± 0.05 0.40 ± 0.02 2.88 ± 0.63 0.80 ± 0.09 a ND ND 0.013 ± 0.002 132.01 ± 5.11

3.5 × 105 0.1 × 103 2.0 × 103 0.3 × 103 ND 10.2 × 106

PolyP synthase activity of PPK2s (analyzed based on the polyP-dependent transformation of ATP to ADP) was found to be at least three orders of magnitude lower than polyP-dependent phosphorylation of nucleotides (Table 1). With pyrimidine nucleoside mono- and diphosphates as substrates, no significant polyP-dependent phosphorylation activity was observed (data not shown). This is in contrast to the class III PPK2 kinase Mrub2488 from M. ruber, which was found to be active with CMP, CDP, UMP, and UDP 38. With deoxyribonucleotides as substrates, purified CHU0107, AAur2811, and DR0132 showed substantial activity against dAMP and dADP producing dADP and dATP, respectively (Table 1). This suggests that these PPK2 proteins might also contribute to the polyP-dependent synthesis of DNA precursors, consistent with the role of polyP and PPKs in DNA replication and bacterial chromosome structure 57. Thus, while PPK2 proteins from different phylogenetic clusters are active against both adenosine and guanosine mono- and diphosphates, they exhibit different substrate preferences and affinities. Phosphonates and their derivatives have been widely used as isosteric mimics of phosphates in the design of inhibitors for enzymes catalyzing phosphate transfer 58-60. In this work, we screened purified CHU0107 (class III) and PA3455 (class II) against a set of 24 mixed aryl, alkyl, and heterocyclic phosphonate compounds (Table S4) using both HPLC and spectrophotometric enzyme assays. These screens revealed strong inhibition


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of PPK2 enzymes by at least six compounds including #3, #7, #9, #11, #21, and #24 which represent chlorinated or substituted aryl phosphonates or bisphosphonates (Figure S4). For CHU0107 and PA3455, these compounds were found to have IC50s in the range of 0.1 – 0.4 mM (data not shown), and were used for co-crystallization with purified PPK2 proteins.

3.3. Overall fold of CHU0107, DR0132, and AAur2811. Eight crystal structures for CHU0107, AAur2811, and DR0132 were determined to 1.80 – 2.65 Å resolution using single anomalous dispersion (SAD) or molecular replacement methods (Table S2). The overall structures of the CHU0107, DR0132, and AAur2811 monomers revealed a 3layer α/β/α sandwich fold (Figures 2, S5, S6), similar to that of the class I (SMc02148) and class II (PA3455) PPK2s 24.


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Figure 2. Overall crystal structure of CHU0107. Two views each of the CHU0107 monomer (A), dimer (B), and tetramer (C) related by 90° rotations. (A), The protein core domain is colored gray with the Walker A and B motifs indicated by violet and green colors, respectively, whereas the lid domain is colored teal. Secondary structure elements are labelled, and the position of the active site is indicated by the bound ADP (shown as balls-and-sticks with red oxygens and khaki carbons). The protein subunits are colored differentially, and shown as ribbon diagrams (B) or by surface presentation (C). Like class II PPK2s, the class III enzymes CHU0107, AAur2811, and DR0132 have a 5-stranded parallel β-sheet (β2-β3-β1-β4-β5) flanked by four (α1, α3, α4 and α17) or eight (α6, α7, η8, α9, η10, η11, α12 and α15) α-helices and covered by the lid with three α-helices (α13, α14 and α15) (Figures 2, S5, S6). Below the lid, there is a large shallow cavity accommodating two loops with Walker motifs A (residues 75-81 in CHU0107) and B (residues 131-136 in CHU0107). In the class III PPK2 enzymes, the side chain of the Walker B Arg residue (Arg133 in CHU0107) is located close (3.2 Å) to the side chain of the conserved Glu (Glu137 in CHU0107), which is the class III PPK2 signature residue (Figures S2, S7). In contrast, the structure of class I PPK2 protein SMc02148 shows the signature Asn153 at this position, whereas Gly367 is present in the class II PA3455 (Figure S7). However, we found that site-directed mutagenesis of the CHU0107 Glu137 to Asn (as in class I PPK2s) or Gly (as in class II PPK2s) produced mutant proteins with greatly reduced activities in polyP-dependent phosphorylation of AMP, ADP, or GDP (Figure 3). These results are consistent with previous work on Mrub2488 from M. ruber, which suggested that the class III signature Glu residue alone does not determine PPK2 substrate specificity 38. In contrast to class I and II PPK2s16, 24, the structures of class III PPK2s revealed the presence of a C-terminal extension, which expands from the bottom of the


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core domain (Figures 2, S5, S6). In CHU0107, this extension (aa 285-305) includes the α18 helix connected by a long (eight residues) loop, stretched to the protein core (Figure 2).

Figure 3. Site-directed mutagenesis of CHU0107: polyP kinase activity of purified mutant proteins with AMP (A), ADP (B), or GDP (C) as substrates. Enzymatic activity was measured using an HPLC-based assay in a reaction mixture containing 1 mM polyP, nucleotide substrate (2 mM AMP, 1 mM ADP, or 5 mM GDP) 10 mM MgCl2, and purified protein (0.5-4.0 μg) as described in Materials and Methods. The truncated mutant protein L285Stop was prepared by replacement of Leu285 by a stop codon (TGA),


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producing a truncated CHU0107 protein (1-284 aa) without the C-terminal extension (Leu285-Asp305). The CHU0107 K81A, N132A, Y148A, and R204A mutant proteins were also produced, but found to be insoluble. Despite low sequence similarity, the overall folds of the CHU0107, DR0132 and AAur2811 monomers are very similar (rmsd 1.0 – 1.1 Å), except for the presence of two 310 helices (η10 and η11, residues 145-164) in CHU0107, whereas the other two structures contain a five-residue loop (Figure S8A). Surface conservation analysis of the three PPK2 proteins using the ConSurf web-server 52 demonstrated strong conservation of residues located in the predicted binding sites for the nucleotide and polyP substrates, as well as at the end of the C-terminal extension (Figures S9, S10). In addition, surface charge analysis of CHU0107 showed that positively charged residues dominate in the predicted polyP binding site and on the top of the lid domain, whereas the C-terminal extension contained more positively charged residues, which are likely to interact with the lid domain of another monomer (Figure S9). It is known that the degree of amino acid conservation in protein structures is strongly dependent on functional importance of protein residues 52. Therefore, high conservation of positively charged residues in the predicted polyP binding site is consistent with their importance for PPK2 activity. The structures of CHU0107, AAur2811, and DR0132 revealed the formation of antiparallel dimers through the creation of a common large β-sheet (although with a disrupted H-bonding between the subunits), and interactions between the helices α4 and η5 (Figures 2B, S5, S6). The formation of stable dimers was verified using the quaternary structure prediction server PISA. The dimeric oligomeric states of the MBP-tagged DR0132 and CHU0107 were confirmed using size-exclusion chromatography, which demonstrated that both proteins exist as dimers in solution (apparent Mw for the MBP-


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tagged proteins are 138 kDa and 186 kDa ± 5 kDa, respectively). Analysis of the PPK2 crystal contacts using PISA also suggested that CHU0107, AAur2811, and DR0132 are likely to form homotetramers (dimers of dimers) through multiple interactions between residues located on the α6, α7 and α12 helices (Figures 2C, S5C, S6C). The tetrameric structures of the class III PPK2s are similar to those of the class I and II enzymes 16, 24. In PPK2 dimers, the C-terminal extensions of both monomers are placed on the top of the lid domain of another monomer acting like a clamp, fastening the dimer (Figures 2B, S5, S6). Using site-directed mutagenesis, we replaced Leu285 of CHU0107 with the TGA stop codon and found that the purified truncated protein (1-284 aa) exhibited twice as high polyP kinase activity compared to the wild type protein (Figure 3). This suggests that the C-terminal extension of the class III PPK2 proteins might limit access of substrates to the active site. Thus, the truncated class III PPK2 enzymes appear to be more efficient biocatalysts for applications in ATP regeneration.

3.4. Crystal structures of CHU0107 in complex with AMP, ADP, or guanosine 5'tetraphosphate. Purified CHU0107 was co-crystallized with AMP, ADP, or guanosine 5'-tetraphosphate (G4P), and the crystal structures revealed the presence of these substrates bound in the active site (Figure 4, Table S2). As predicted in our previous work 24 and recently shown by Parnell et al. 39, the nucleotide substrates were bound on the right side of the Walker B motif. A Michaelis complex structure of the CHU0107 bound with AMP revealed that the adenine base is bound in the anti configuration in the deep cleft between the side chains of Glu137 and Val104-Pro105 (Figure 4A, 4B).


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Figure 4. Crystal structures of CHU0107 (class III) and SMc02148 (class I) in complex with nucleotides. (A, B), CHU0107+AMP; (C, D), CHU0107+ADP; (E, F), SMc02148+ADP; (G, H), CHU0107+G4P (guanosine 5'-tetraphosphate). The left panels (A, C, E, G) present protein ribbon diagrams showing the position of ligand bound between the protein core domain (gray) and lid (teal) with green meshes representing OMIT Fo – Fc electron density maps (at 3 σ level) contouring the ligands. The right panels (B, D, F, H) present close-up stereo views of the protein ligand-binding sites indicated by red rectangles on the left panels. Model orientations are preserved in all panels. The Walker A and B motifs are indicated by violet and green colors, respectively,


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whereas ligand molecules are shown as ball-and-stick models with light blue carbons, and side chains of the core domain residues are shown as sticks with orange carbons. The exocyclic N6-amino group of adenine is H-bonded to the side chain of the semiconserved Asn138 (3.2-4.0 Å), and its N7 atom is close to the conserved Arg117 (3.2 Å) (Figure 4B). Ribose sits close to the Walker B Arg133 with its 2'-hydroxyl interacting with the side chain of the Class III-specific Glu137 (2.8 Å), whereas its ribofuranose oxygen interacts with the main chain amide of Val104 (3.3 Å). The α-phosphate group of AMP is not specifically bound to the protein, and is positioned near the Walker B Arg133 (3.9 Å). The structure of the CHU0107-AMP complex also suggests that this enzyme might be able to bind guanosine nucleotides, consistent with the observed polyP kinase activity against GMP and GDP (Table 1). The structure of the CHU0107-ADP complex demonstrated similar binding of the adenine base and ribose, with the two phosphates of ADP forming ionic interactions with the side chains of Lys103 (2.9 Å), Arg133 (2.8-2.9 Å), Lys81 (2.8 Å), and Asp82 (2.9 Å) (Figure 4C, 4D). In the structures of the CHU0107-AMP and ADP complexes, the αphosphates have similar positions regarding the catalytic Asp77 (Walker B), whereas the β-phosphate of ADP resides deeper in the active site (Figure 4A-D). This suggests that depending on the nature of the nucleotide substrate (AMP or ADP) bound in the ternary PPK2-nucleotide-polyP complex, the terminal phosphate of polyP can be positioned at one of two adjacent sub-sites separated by a distance of one phosphate group (2.5-3.0 Å). Recent structures of the class III PPK2 enzyme MrH2468 from M. ruber demonstrated similar binding of AMP and ADP in the active site 39. In contrast, the two structures of SMc02148 (class I) in complex with ADP or AMP revealed a different orientation of the


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adenine base, which is stacked between the side chains of Phe163 and Lys120 (Figures 4E, 4F, S11A, S11B). In addition, the ribose ring is inverted almost 180° with both hydroxyls oriented toward the active site bottom (close to the side chains of Arg111 and Asn153). Different binding of the nucleobase and ribose in the SMc02148 active site is probably due to the presence of Glu158 (precluding adenine binding deeper in the active site) and Met222 (pushing the ribose hydroxyls toward the active site bottom) (Figures 4E, 4F). Similar ADP binding in the active site was recently demonstrated in the structure of the FTT1564-ADP complex 39. Interestingly, the position of the ADP β-phosphate in the SMc02148-ADP complex matches the location of α-phosphates in the CHU0107AMP and –ADP complexes (Figure 4). Guanosine 5'-tetraphosphate (G4P) is a known inhibitor of several nucleotide active enzymes (e.g. guanylate cyclase, dinucleoside tetraphosphatase, phosphodiesterase I) 61-63, which can also be considered as a transition state mimic for PPK2 enzymes. The structure of the CHU0107-G4P complex revealed one molecule of G4P bound in the active site in a manner similar to that of ADP, but with the tetraphosphate moiety extending further into the polyP-binding site (Figures 4E, 4F). This structure serves as a model for the binding of GDP in the CHU0107 active site (Figure S11C), demonstrating similar coordination of ribose and α- and β-phosphates as in the ADP complex (Glu137, Lys103, Arg133, and Lys81) (Figure 4F). Furthermore, the γ- and δ-phosphates of G4P are coordinated by the side chain of the conserved Arg208 (3.0 Å), located in the lid domain, and part of the polyP binding site (Figure 4F). Thus, it appears that the lid domains of CHU0107 and other PPK2 proteins interact only with the reactive end of the polyP chain, which is bound close to the nucleotide substrate.


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The exocyclic O6 atom of guanine can potentially form a weak hydrogen bond with the Asn138 side chain Nδ (3.4 Å), whereas the N2 atom of guanine points towards Tyr148, but does not clash with its side chain nor enforce a non-preferred conformation (Figure 4F). This mode of the base binding is consistent with the significant activity of CHU0107 toward GMP and GDP (Table 1). In contrast, both AAur2811 and DR0132 have an Asp residue (Asp142 and Asp127, respectively) at the position equivalent to the CHU0107 Asn138 and exhibit negligible or low activity against both GMP and GDP (compared to CHU0107) (Table 1). We also found that the CHU0107 N138A mutant protein showed wild-type activities with AMP, ADP, or GDP (Figure 3). In contrast, replacement of Asn138 by Asp, His, Gln, or Glu had stronger negative effects on its activity toward GDP compared to that with AMP or ADP (Figure 3). In the class I PPK2 enzyme FTT1564 from F. tularensis, the base-coordinating residue is Asn122, whose side chain can potentially hydrogen bond with the guanine O6, consistent with comparable activities of FTT1564 against both ADP and GDP 16. Thus, PPK2 structures in combination with kinetic analysis revealed that significant activity of these enzymes against GMP and GDP correlates with the presence of an asparagine residue in the nucleobase binding pocket, which appears to represent a molecular determinant for PPK2 activity toward guanosine nucleotides.

3.5. Crystal structure of AAur2811 in complex with phosphates and citrates. The crystal structure of AAur2811 revealed a tetramer containing four phosphates, four citrates, and one polyethylene glycol (PEG) molecule bound in the active site on the left side of the Walker-A motif (Figures 5A, 5B). In the AAur2811 tetramer, there are at least


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four distinctive binding sites for phosphates, which are bound close to the side chains of conserved Lys40 (3.3-3.6 Å), Lys44 (2.9 Å), Lys85 (3.0 Å), Arg197 (2.8-3.5 Å), Lys203 (3.2 Å), Lys206 (2.8 Å), and Arg251 (2.6 Å) (Figure 5A).

Figure 5. Crystal structures of PPK2 proteins in complex with ATP, Mg2+, phosphate, and citrate. (A), Close-up view of AAur2811 showing four bound phosphates (P1, P2, P3, P4; shown as balls-and-sticks). (B), Close-up view of AAur2811 showing two bound citrates (CIT1 and CIT2; with pink-colored carbons), as well as one bound phosphate (P2). Panels A and B are composite images, created using different protein chains in the asymmetric unit. (C), Overall view of the DR0132 dimer in complex with four ATP molecules and Mg2+ (shown as balls-and-sticks). Ribbon diagrams are


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colored: violet (Walker A), green (Walker B), teal (the lid), and salmon (the second DR0132 monomer). (D), DR0132 residues involved in the binding of two ATP molecules (ATP1 and ATP2, with gray carbons) and two Mg2+ ions. (E), Close-up view of the DR0132 active site, showing two Mg2+ ions and the residues involved in the coordination of phosphates and catalysis. AAur2811 monomer-A contains two phosphates bound below the lid domain with one molecule (P1) positioned close to the Walker A motif (2.8-3.2 Å) and the side chain of conserved Lys85 (3.0 Å). The second phosphate (P2) is located just 4.7 Å from the P1 molecule and is coordinated by the side chains of the conserved Arg197 (2.8 Å), Lys203 (3.4 Å), and Lys206 (2.8-3.2 Å) residues from the lid domain (Figure 5A). The monomer-C structure revealed the presence of two other phosphate molecules (P3 and P4) bound at the other end of the large shallow cavity, and located 7.7-9.2 Å away from the P2 molecule (Figure 5A). The P3 and P4 molecules are positioned close to each other (2.3 Å) and to the side chains of the conserved Lys40 (3.6 Å), Lys44 (2.9 Å), and Arg251 (3.2 Å) residues. The shallow cavity on the left side of the Walker A motif accommodates a large number of positively charged residues (e.g. Lys40, Lys44, Lys246, Lys247, and Arg251 in AAur2811), and this positive surface shows a strong conservation in the structures of all PPK2 proteins (Figures S9, S10). Similar binding of four phosphate molecules was observed in the active site of the class III PPK2 protein MrH2468 from M. ruber H328 (PDB code 5LCD)39. This positively charged surface represents the polyP binding site of PPK2 proteins as shown by the structure of the class I PPK2 enzyme FTT1564 from F. tularensis in complex with polyP (PDB code 5LL0) 39. This is also supported by our results of site-directed mutagenesis of CHU0107, which revealed a critical role of Lys86, Lys258, and Arg262 for polyP-dependent phosphorylation of AMP, ADP, and GDP (Figure 3). In the FTT1564-polyP complex, the


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terminal phosphate of polyP bound in the active site corresponds to the P1 phosphate of the AAur2811-phosphate complex (Figure 5A) and likely represents the polyP binding position for ADP phosphorylation. The distal end of the polyP binding site in AAur2811 shows no obvious barriers, which could limit the chain length of the bound polyP (Figures 5A, 5B). This is consistent with the ability of PPK2 enzymes to use polyP substrates with a broad range of chain lengths (3-700 Pi groups; 30-50 residues are optimal) (Table S3) 31.

3.6. Crystal structure of DR0132 in complex with ATP and Mg2+. Purified DR0132 was co-crystallized with ADP, tripolyphosphate, and MgCl2, and the crystal structure was solved to 1.80 Å resolution (Table S2). Surprisingly, the structure revealed the presence of two molecules of ATP bound in one active site (Figures 5C, S12), probably due to the ability of DR0132 (and PA3455 24) to phosphorylate ADP using tripolyphosphate (polyP3) as a phosphodonor (Table S3). The first ATP molecule (ATP1) is bound in the nucleotide binding pocket with the exocyclic adenine N6 amino group hydrogen bonded to the Asp127 side chain (2.8 Å; Asn138 in CHU0107). The second ATP molecule (ATP2) is bound near the dimer interface with the adenine base sandwiched between the side chains of Tyr51 and Asn82 of the second monomer (Figures 5C, 5D, S12). The triphosphate parts of both ATP molecules are positioned parallel to each other, and are oriented toward the bottom of the active site. The γ-phosphates of the two ATP molecules are located near the side chain of the Walker A Asp66 (2.8-3.0 Å), and Lys70 on the bottom (2.7-2.9 Å) (Figure 5E). The Walker B Arg122 interacts with the γ-phosphate of ATP1 (2.9 Å) and β-phosphate of ATP2 (3.0 Å). In addition, the α-phosphate oxygens of


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ATP2 are bound to the side chains of Arg182 (3.0 Å) and Lys191 (2.7 Å) from the lid domain (Figures 5D, 5E). In some way, the two ATP molecules bound in the DR0132 active site resemble the two nucleotide substrates (2 ADP, AMP+ATP, or AMP+ADP) bound in the active site of adenylate kinases 64-66. Both PPK2s and adenylate kinases belong to P-loop kinases, and exhibit significant structural similarity to each other, suggesting that these enzymes have a common evolutionary origin 29. This prompted us to determine if PPK2 enzymes can catalyze the adenylate kinase reaction, the reversible transformation of two ADP molecules to AMP and ATP. Both HPLC-based and enzyme-coupled assays revealed the presence of detectable metal-dependent adenylate kinase activity in CHU0107, AAur2811, and DR0132, as well as in the class I (SMc02148) and class II (PA3455) PPK2 proteins (Figure S13, Table S3). Analysis of kinetic parameters revealed that adenylate kinase activity of purified PPK2 proteins was three orders of magnitude lower than the polyP-dependent phosphorylation of ADP (Table S3). These results suggest that adenylate kinase activity of PPK2s might represent an ancestral activity of these enzymes that evolved from a common P-loop kinase ancestor in order to catalyze the polyPdependent phosphorylation of nucleoside mono- and diphosphates. Although, from an evolutionary point of view PPK2 proteins are closer to thymidylate kinases than to adenylate kinases 29, our experiments showed no detectable thymidylate kinase activity in purified PPK2 proteins. The DR0132-ATP structure also revealed the presence of two Mg2+ ions bound in the active site close to both triphosphate chains, with a 5.5 Å distance between the two cations (Figures 5D, 5E). This is close to the 5.2 Å distance between the Mg2+ and Mn2+


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ions bound in the active site of the E. coli PEP carboxykinase (PCK), for which a general model of a binuclear metal cluster has been postulated 67-68. According to this model, one of the metal ions (Mg2+ in PCK) is bound to the nucleotide β- and γ-phosphates acting as a co-substrate with the nucleotide, whereas the second metal ion (Mn2+ in PCK) acts as a bridge between the two substrates 67. A similar model with two Mg2+ ions bound in the active site has also been proposed for the E. coli adenylate kinase and Pyrococcus furiosus UMP kinase 69-70. In the DR0132-ATP structure, the Mg1 ion appears to be a cosubstrate ion, which interacts with the ATP1 β-phosphate (2.0 Å) and is additionally coordinated by the side chain of conserved Asp196 (2.0 Å; Asp222 in CHU0107 and Asp211 in AAur2811) (Figure 5E). The Mg2 ion is bound close to the side chain of the Walker A Asp66 (2.1 Å; Asp77 in CHU0107 and Asp81 in AAur2811) and positioned between the triphosphate moieties of two ATP molecules (2.0-2.1 Å to both γ- and βphosphates), suggesting that it functions as a bridge, reducing electrostatic repulsion between the negatively charged phosphate groups (Figure 5E). Both Mg2+-coordinating Asp residues are conserved in all PPK2 enzymes (Figure S2), and their critical role in the activity of CHU0107 (Asp66 and Asp196), as well as SMc02148 (Class I, Asp93 and Asp223) and PA3455 (Class II, Asp307 and Asp437) was confirmed using site-directed mutagenesis (Figure 3) 24. Thus, our results suggest that PPK2 enzymes might also employ a binuclear metal center for catalysis.

3.7. The active site channel and potential catalytic mechanism of PPK2s. Crystal structures of PPK2s in complex with different substrates demonstrated that their active site is covered by a lid domain, which provides several positively charged residues


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for the coordination of the reactive polyP chain end, as well as an Asp residue coordinating one of the Mg2+ ions (Mg1) (Figures 4, 5). Cross section analysis of PPK2 structures from different classes revealed the presence of an internal substrate channel, connecting the polyP and nucleotide binding sites near the catalytic Asp (Walker A): Asp93 in SMc02148 (class I), Asp307 in PA3455 (class II), and Asp77 in CHU0107 (class III) (Figure 6). Examination of the channel surface charge indicated that positively charged residues prevail at the polyP binding site and in the central part of the channel in all PPK2s, as well as in the nucleotide site of SMc02148 (Figure 6). The surface of the nucleotide binding sites of PA3455 and CHU0107 (and other class III PPK2s) exhibited a reduced positive charge and showed higher similarity to each other (than to SMc02148), in line with their preference for AMP as substrate (Figures 6B, 6C, Table 1). As shown in Figure 6, the characterized PPK2 proteins from three classes displayed similar broad openings at the polyP entry side, with notable differences in nucleotide entrance sides. With the exception of DR0132, the class III PPK2s (CHU0107, AAur2811, and MrH2468) showed a wider entrance at the nucleotide binding sites compared to SMc02148 and PA3455, consistent with a broader nucleotide range of the class III enzymes (Figure 6).


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Figure 6. Active site channels of PPK2 proteins from different classes: a crosssection analysis. PPK2 structures from different classes are shown in surface representation with electrostatic potential: (A), SMc02148 (class I); (B), PA3455 (class II); (C), CHU0107 (class III); (D), AAur2811 (class III); (E), DR0132 (class III); (F), MrH2468 (class III). The protein dimer structures were superimposed and clipped using the same clipping planes. The position of the lid domain is indicated, whereas the white labels designate the location of the catalytic (Walker A) aspartates and binding sites for polyP and nucleotides. The narrow nucleotide entrance in the DR0132 structure is in line with a high preference of this enzyme for AMP compared to ADP (at least 200 times higher kcat/Km), which makes it biochemically similar to PA3455. Thus, analysis of the substrate channels for


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available PPK2 structures suggests that the broader nucleotide range of the class III PPK2s correlates with the wider nucleotide entrance sites of these proteins. Overall, the structure of DR0132 in complex with two ATP and Mg2+ ions might represent a catalytically competent conformation of PPK2 enzymes, in which the reactive groups of two substrate molecules are positioned in close proximity, favouring catalysis, whereas the CHU0107-G4P structure might represent a transition state with the covalently linked substrates. We propose that PPK2 enzymes orient the terminal phosphates of both substrates (nucleotide and polyP) optimally for a nucleophilic attack of the nucleotide α- (in AMP) or β- (in ADP) phosphate oxygen on the terminal polyP phosphorus atom (Figure 7). This is accomplished through coordination of phosphate groups by a bridging Mg2+ ion (Mg2 in DR0132) and several positively charged residues involved in phosphate binding, orientation, transfer, and charge compensation (Lys81, Arg133, Arg208, Lys214, and Lys217 in CHU0107; Lys70, Arg122, Arg182, Lys188, and Lys191 in DR0132). All these residues, as well as the two conserved Asp residues involved in Mg2+ coordination (Asp77 and Asp222), have been shown to be critical for activity in CHU0107 (Figures 3 and 5). The structures of CHU0107+AMP and CHU0107+ADP (section 3.4) suggest that the terminal phosphate of polyP can be located at one of the two adjacent sub-sites separated by a length of one phosphate group. Crystal structures of PPK2s also suggest that in the transition state, the transferred phosphoryl group of polyP is stabilized by the side chains of two conserved positively charged residues: Arg from the lid domain (Arg182 in DR0132 and Arg208 in CHU0107), and Lys from the bottom of the active site (Lys70 in DR0132 and Lys81 in CHU0107), with


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the phosphoryl group likely remaining attached to these side chains over the whole transfer path (Figure 7).

Figure 7. Proposed catalytic mechanism of PPK2s: polyP-dependent phosphorylation of ADP to ATP. (A), Ternary enzyme-substrate complex (PPK2+polyPn+ADP); (B), Transition state intermediate; (C), Enzyme-product complex (PPK2+polyPn-1+ATP). PolyP and ADP are bound in the active site in close proximity to each other with two Mg2+ ions coordinated by conserved aspartates (Asp77 and Asp222 in CHU0107). In addition, the transferred terminal phosphoryl group of polyP is stabilized by the side chains of two conserved positively charged residues (Arg208 and Lys81 in CHU0107). The reaction is initiated by nucleophilic attack of the β-phosphate


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oxygen of ADP on the terminal phosphorus of polyP, producing a cross-linked intermediate (B). The residue numbers for the three PPK2 proteins studied in this work are shown: CHU0107 (C), AAur2811 (A), and DR0132 (D). In CHU0107, the critical role of the lid domain Arg208 was confirmed using site-directed mutagenesis (the K81A mutant protein was found to be insoluble) (Figure 3). These two residues are highly conserved in all PPK2 proteins (Figure S2), as well as in adenylate kinases (Lys13 and Arg156 in the E. coli adenylate kinase).

3.8. Crystal structures of CHU0107 in complex with aryl phosphonate inhibitors. We have also determined co-crystal structures for CHU0107 in complex with aryl phosphonate inhibitors #9, #24, and #25 to 2.1 – 2.3 Å resolution (Figure 8). These structures revealed that the inhibitors were bound in the active site below the lid, between the polyP and nucleotide binding sites and near the Walker A loop (Figure 8). All three inhibitors bind in positions overlapping with the positions of P1 and P2 phosphates in the AAur2811 structure and utilize the interactions responsible for polyP recognition. The overall fold of the inhibitor-bound CHU0107 structures was similar to those determined with substrates (AMP or ADP) except for a minor change in the lid domain (Phe218 – Asp222) of the CHU0107-24 complex due to the accommodation of the second aromatic ring (Figure S8). In the CHU0107-9 complex, the 3,5-dichlorobenzyl ring is stacked between the Arg208 side chain (interplanar distance to the guanidinium group ≈ 3.3 Å) and the Walker A main chain (3.7 Å) (Figures 8A-C). One chloride group is positioned near the backbone amide of Phe205 (3.5 Å), whereas the second chloride is positioned close to the Asp222 side (3.2 Å), which is involved in the coordination of Mg1 ion in DR0132.


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Figure 8. Crystal structures of CHU0107 in complex with the aryl phosphonate inhibitors #9, #24, and #25. Panels A, D, and G show the overall view of the CHU0107 subunit (ribbon diagrams) with the inhibitor molecule bound near the Walker A (violet) and B (green) motifs. The core and lid domains are colored gray and teal, respectively, whereas the inhibitor molecules are shown as balls-and-sticks. Panels B, E, and H represent close-up views of the inhibitor binding sites indicated by red rectangles in panels A, D, and G. Panels C, F, and I show clipped surface representations of the same binding sites (shown in B, E, and H, respectively) with the OMIT Fo – Fc electron density maps (green meshes) contouring the inhibitors at 3 σ level and inhibitor chemical structures shown on the right (#9, #24, and #25). Similarly, the bisphosphonate moiety of inhibitor-9 is sandwiched between the Walker A main chain (2.9 Å to the Gly80 backbone amide) and Arg208 side chain (2.7 – 3.1 Å), which is proposed to coordinate the transferred phosphoryl group during catalysis (Figure 8A-C). Therefore, binding of inhibitor-9 in the CHU0107 active site will interfere with


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the binding of the polyP terminal phosphate and phosphoryl transfer to the nucleotide substrate. In contrast to the CHU0107-9 complex, the inhibitor molecule in the CHU0107-24 complex is wrapped around the side chain of the catalytic Arg208 (3.1 – 3.5 Å) with the bisphosphonate moiety fixed by ionic interactions with the side chains of Lys217 (3.3 Å) and Arg208 (3.1 Å) (Figure 8D-F). The second benzyl ring of inhibitor-24 occupies a hydrophobic pocket formed by the side chains of Phe205, Ile209, Phe218 and Leu223. In addition to inhibitor-24, the active site of CHU0107 contains a phosphate group bound similarly to the P1 phosphate in the AAur2811 structure and positioned between Arg208 (2.6 Å), Walker A loop (3.1 Å), and Lys81 (4.9 Å), resulting in a shift of the position of the bisphosphonate of inhibitor-24 compared to that of inhibitor-9. Inhibitor-25 was found to be bound in a position similar to that of inhibitor-9 with the aromatic ring stacked between the Arg208 side chain and the Walker A backbone, whereas its monophosphonate moiety interacts with the backbone amides of Ala78 (2.9 Å), Gly80 (2.7 Å) and Asp82 (2.7 Å) within the Walker A motif (Figure 8G-I). Thus, the structures of CHU0107-inhibitor complexes revealed at least two binding pockets for aryl phosphonate inhibitors. The first pocket (for monoaryl inhibitors-9 and -25) is located between the Walker A backbone and Arg208 side chain, which are involved in the binding of polyP and coordination of the transferred phosphoryl group during catalysis. The second pocket is indicated by the diaryl bisphosphonate inhibitor-24 stacked between the side chains of catalytic Arg208 and Lys217, with the second aromatic ring bound in the hydrophobic cleft between Phe205 and Phe218. The


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information regarding these binding pockets can be used for future inhibitor design by extending the aryl phosphonate fragments into the nucleotide binding site.

4. Conclusion. We describe here the crystal structures of bacterial PPK2 proteins in complex with substrates, products, Mg2+ ions, and inhibitors. We have identified the binding sites for both nucleotide and polyP substrates and confirmed the critical role of active site residues using site-directed mutagenesis. The crystal structures of PPK2s also suggested a molecular basis for substrate preference and activity against adenosine and guanosine mono- and diphosphates. Based on these results, we proposed the potential catalytic mechanism for PPK2 enzymes, which involves two Mg2+ ions. Since the identified active site residues are conserved in all PPK2 proteins, the proposed catalytic mechanism is also applicable for the class I and II PPK2 kinases. The structures of CHU0107 in complex with three aryl phosphonate inhibitors revealed the presence of two binding pockets in the active site, which can be used for inhibitor development. Our results contribute to the understanding of the PPK2 catalytic mechanism, and provide important information for the design of specific PPK2 inhibitors and engineering of highly active PPK2 kinases for biocatalytic applications.

SUPPORTING INFORMATION Supporting Information includes 13 figures and four tables and can be found with this article online at https://pubs.acs.org/

ACKNOWLEDGMENTS


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This work was supported by the NSERC Strategic Network grant IBN (A.F.Y.) and the NIH grants GM094585 and GM115586 (A.J.). This project was also supported in part by the Intramural Research Program of the NCI Center for Cancer Research. Diffraction data were collected at the Structural Biology Center at the Advanced Photon Source, operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357.

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Structural insights into substrate selectivity and activity of bacterial

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