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Article Cite This: ACS Omega 2017, 2, 8445−8452
Phosphorylation-Dependent Effects on the Structural Flexibility of Phosphoglucosamine Mutase from Bacillus anthracis Kyle M. Stiers,‡ Jia Xu,‡ Yingying Lee,† Zachary R. Addison, Steven R. Van Doren, and Lesa J. Beamer* Biochemistry Department, University of Missouri, 117 Schweitzer Hall, Columbia, Missouri 65211, United States S Supporting Information *
ABSTRACT: Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme in the peptidoglycan biosynthetic pathway, catalyzing the reversible conversion between glucosamine 1- and 6-phosphate. Previous structural studies of PNGM from the pathogen Bacillus anthracis revealed its dimeric assembly and highlighted the rotational mobility of its C-terminal domain. Recent studies of two other enzymes in the same superfamily have demonstrated the long-range effects on the conformational flexibility associated with phosphorylation of the conserved, active site phosphoserine involved in phosphoryl transfer. Building on this work, we use a combination of experimental and computational studies to show that the active, phosphorylated version of B. anthracis PNGM has decreased flexibility relative to its inactive, dephosphorylated state. Limited proteolysis reveals an enhanced and accelerated cleavage of the dephosphorylated enzyme. 15 N transverse relaxation-optimized NMR spectra corroborate a conformational adjustment with broadening and shifts of peaks relative to the phospho-enzyme. Electrostatic calculations indicate that residues in the mobile, C-terminal domain are linked to the phosphoserine by lines of attraction that are absent in the dephosphorylated enzyme. Phosphorylation-dependent changes in protein flexibility appear linked with the conformational change and enzyme mechanism in PNGM, establishing this as a conserved theme in multiple subgroups of the diverse α-D-phosphohexomutase superfamily.
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INTRODUCTION Phosphoglucosamine mutases (PNGMs) comprise a distinct subgroup within the large enzyme superfamily known as the α1 D-phosphohexomutases. These enzymes participate in carbohydrate metabolism in organisms ranging from archaea to humans, catalyzing the reversible conversion of 1- to 6phosphosugars via a bisphosphorylated sugar intermediate. PNGM is an essential enzyme in Gram-negative bacteria1 due to its role in converting glucosamine 6-phosphate to glucosamine 1-phosphate in peptidoglycan biosynthesis.2 As such, it is an established target for antimicrobial design.2,3 Enzymes from other subgroups of the superfamily share a common catalytic mechanism, but show varying preferences for the sugar moiety of their substrates, including glucose (e.g., preferred by phosphoglucomutases), mannose, and N-acetyl glucosamine.1 The multistep catalytic reaction of PNGM comprises two consecutive phosphoryl transfer reactions: first, from a phosphoserine in the active site to substrate, forming a bisphosphorylated sugar intermediate; and second, from the intermediate back to enzyme, forming the product and regenerating the active state of the enzyme (Figure 1A). The reaction requires an unusual reorientation of the intermediate in the midst of the catalytic cycle to enable the second phosphoryl transfer (see arrow in Figure 1A). In addition to this conserved mechanism, structural studies of PNGM from © 2017 American Chemical Society
Bacillus anthracis (BaPNGM) and Francisella tularensis (FtPNGM) have shown that these enzymes share the fourdomain architecture common to all α-D-phosphohexomutases (Figure 1B), as well as the conformational mobility of their Cterminal domains (Figure 1C). Indeed, to date, proteins in the PNGM subgroup have been found to exhibit the greatest conformational mobility in the superfamily, with a rigid-body rotational variability of the C-terminal domain of up to 50° in structural comparisons.4,5 Both PNGMs of known structure also share a side-by-side dimeric arrangement, which appears to be unique to this subgroup of the superfamily.6 Recent studies of the related enzyme, phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, established a connection between enzyme mechanism and protein flexibility. PMM/PGM is also a member of the α-D-phosphohexomutase superfamily and shares 27% sequence identity with BaPNGM. In PMM/PGM, the phosphorylation state of the conserved, catalytic phosphoserine residue was shown to produce long-range effects on the flexibility of the enzyme, as assessed by detailed biophysical studies including hydrogen−deuterium exchange (HDX) mass Received: October 5, 2017 Accepted: November 16, 2017 Published: November 29, 2017 8445
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Figure 1. (A) Schematic of the reversible conversion of glucosamine 1-phosphate to glucosamine 6-phosphate catalyzed by BaPNGM. On left, the phosphoryl group of Ser100 (yellow) is donated to the substrate to form the bisphosphorylated intermediate (middle), which then reorients in the active site. On right, the original phosphoryl group from the substrate (blue) is transferred back to enzyme, creating the product and regenerating the active form of the enzyme. (B) The crystal structure of the BaPNGM dimer (protein data bank (PDB) entry 3PDK), showing the two protomers in blue and beige. Ser100 is highlighted in green; the metal-binding loop is in red. (C) A superposition of the two protomers from the dimer in (B), showing the rotational variability of the C-terminal domain (pink arrow).
in the structural flexibility of BaPNGM correspond to key states in its multistep reaction, and may have implications for many proteins regulated by post-translational modifications.
spectrometry7 and HDX NMR.8 These effects were also apparent in the solution using small-angle X-ray scattering (SAXS) and from traditional biochemical methods such as limited proteolysis.7,8 These results, which suggested a connection between increased enzyme flexibility and the unusual reorientation of the reaction intermediate (Figure 1A), prompted the question of whether proteins from other subgroups of this important superfamily, including oligomeric enzymes, would share this structural response to phosphorylation. Here, we characterize the phosphorylated and dephosphorylated states of BaPNGM with multiple, complementary methods. Unique features of this enzyme relative to the previously studied PMM/PGM include its differing substrate specificity, dimeric quaternary structure, and the significantly greater rotational mobility of its C-terminal domain.4,5 We demonstrate that phosphorylation of the active site serine correlates with a reduction in structural flexibility, as shown by the relative decreases in its susceptibility to proteolysis, binding to 8-anilinonaphthalene-1-sulfonic acid (ANS), and rotational correlation time in solution. Electrostatic calculations indicate an attraction between catalytic loops in the phospho-enzyme and the C-terminal domain, suggesting a role for electrostatic attraction in decreasing the structural mobility. Consistent with the conserved mechanism of the enzyme superfamily, changes
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RESULTS Biochemical Investigations of Protein Flexibility. The recombinant BaPNGM purifies with Ser100 in its dephosphorylated state.9,10 Samples phosphorylated at Ser100 were prepared as in Experimental Procedures. Phosphorylation status was verified by electrospray ionization mass spectrometry (ESIMS) (Figure S1) prior to use. Accessibility of phospho- and dephospho-BaPNGM to proteolytic cleavage was assessed by a time course of digestion with proteinase K, followed by visualization on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 2A). Relative to the phospho-enzyme, dephospho-BaPNGM shows a significant increase in susceptibility to proteolysis. This includes many more sites of cleavage as well as much more rapid degradation (Figure 2A, right panel). After 240 min, no intact dephosphoenzyme remains. The interaction of phospho- and dephospho-BaPNGM with the fluorescent dye ANS, a measure of accessibility of hydrophobic patches, was also assessed (Figure 2B). The dephospho-enzyme shows a modest but noticeable increase in fluorescence emission, consistent with enhanced association 8446
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Figure 2. Effects of phosphorylation on BaPNGM flexibility and susceptibility to proteolysis. (A) SDS-PAGE showing limited proteolysis of phospho (left) and dephospho-BaPNGM (right). Phospho/dephospho samples were 95 and 0% phosphorylated, respectively, according to ESI-MS. Length of digestion with proteinase K is given in minute. Zero incubation time indicates untreated (control) samples. (B) Binding of ANS to phospho (black) and dephospho-BaPNGM (blue).
(expected molecular mass of 102 kDa) and previous SAXS studies.4 The apparent τc of the dephospho- and phosphoenzymes suggests that despite the differences in TROSY spectra, the effective radius of gyration for each dimer is similar in the solution. Comparison of Electrostatics. Previous structural comparisons have shown that the C-terminal domain of PNGM has significant rotational mobility.4 The phosphorylation-dependent differences in the solution behavior of BaPNGM raised the question of whether phosphorylation modulates interactions with the mobile, C-terminal domain. Precedent for this notion comes from the electrostatic calculations of P. aeruginosa PMM/PGM that indicated a long-range attraction between the positively charged residues in its C-terminal domain and the negative charge in the vicinity of the catalytic phosphoserine.8 A similar arrangement for BaPNGM seemed possible, but could not be assumed due to multiple differences between the proteins, including their low overall sequence identities (27%). Moreover, the C-terminal domain of BaPNGM is smaller (79 vs 93 residues in PMM/PGM), has a different topology, and the rotational freedom of its C-terminal domain observed in the crystal structure is much greater in BaPNGM than in PMM/ PGM (∼30 vs 10°).5 Models for phospho- and dephospho-BaPNGM were prepared as described in Experimental Procedures. Partial charges were calculated for the atoms around the divalent metal-binding site and Ser100, both with and without phosphorylation. Electrostatic field lines for structures of both phosphorylated and dephosphorylated BaPNGM were calcu-
with ANS. Increased binding of ANS has been associated with increased structural fluctuations of proteins, such as in molten globule states.11 NMR Spectra and Hydrodynamics. To assess the potential structural differences between phospho- and dephospho-BaPNGM in a solution, 2H/15N-labeled samples were prepared for NMR studies. An overlay of their transverse relaxation-optimized spectra (TROSY) showing 1H−15N correlations in the backbone is in Figure 3A. Overall, both samples show a good peak dispersion in most regions, indicating a well-ordered tertiary structure. Multiple differences between the two samples, however, are also apparent. Dephosphorylation not only shifts amide peaks but also causes a minor reduction in the number of peaks (Figure 3A). This decrease in the number of TROSY peaks of the dephosphoenzyme is suggestive of line broadening by exchange between conformational substates in msec, i.e., an increase in flexibility on a slow time scale. The overall hydrodynamic behavior of the phosphorylated and dephosphorylated forms of 2H/15N-labeled BaPNGM was compared in terms of their 15N NMR transverse crosscorrelated relaxation rate constants, η xy (Figure 3B). Phosphorylated BaPNGM has ηxy of 62 ± 4 s−1; the relaxation of the dephospho-enzyme is similar with ηxy of 72.5 ± 13 s−1 at 308 K (Figure 3B). These relaxation rates were interpreted using the TRACT approach,12 suggesting rotational correlation times (τc) at 308 K near 43 ± 3 ns for phospho-PNGM and 51 ± 9 ns for dephospho-PNGM. These time constants for the tumbling in solution are consistent with a dimer of PNGM 8447
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animation of conformational change in D4 observed between the protomers of the BaPNGM dimer.) To probe the potential impact of D4 mobility on the electrostatic environment of the active site, residues connected by the attractive field lines were examined in different conformers of the enzyme, from B. anthracis or F. tularensis. For simplicity, we compare the interresidue distances between the Oγ of phosphoserine 100, and one of the key arginines on the face of D4 (Arg419 in BaPNGM, Arg417 in FtPNGM) (Figure 5A). These interresidue distances vary significantly by conformer, ranging from 9.4 to 19.9 Å. As electrostatic effects are well known to be distance-dependent, the orientation of D4 likely has a significant impact on the electrostatic environment of the active site, diminishing the electrostatic attraction between this domain and the phosphoserine as D4 is rotated away. Thus, phosphorylation state, D4 orientation, and electrostatics appear to be intimately connected in these enzymes. The orientation of D4 is also relevant to enzyme mechanism, which requires the protein to cycle between a “closed” state, suitable for high-affinity interactions with bound substrate and phosphoryl transfer, and an “open” state that would permit initial interactions with substrate, release of product, and is also required to enable the reorientation of the bisphosphorylated reaction intermediate (Figure 1A). In particular, the closed conformer of D4 positions Arg419 appropriately for interactions with phosphate group of bound substrate (Figure 5B), a highly conserved role for this residue in the enzyme superfamily.9 Given that the phosphorylated state of the enzyme is its active form (Figure 1A), the observed electrostatic environment would thus seem to favor the formation of closed conformers relevant to catalysis. In the dephospho-enzyme, the lack of electrostatic interactions between Ser100 and D4, along with the concomitant increase in protein flexibility, would favor the open enzyme conformers, which could facilitate the reorientation of the intermediate required in the midst of the catalytic cycle (Figure 1A).
Figure 3. (A) Effects of phosphorylation on the NMR fingerprint, 15N TROSY spectra (phospho-PNGM: blue contours, dephospho-PNGM: red). Conditions were 400 μM enzyme, 800 MHz 1H Larmor precession frequency, 308 K, and pH 7.4. (B) 15N NMR transverse cross-correlated relaxation. The relaxation rate constant ηxy4 was obtained by exponential fitting to one-dimensional (1D) NMR spectra integrated over 8.5−10 ppm. Conditions were 400 μM enzyme, 800 MHz, 308 K, and pH 7.4.
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DISCUSSION Enzymes in the PNGM subgroup are perhaps best known for their roles in bacterial pathogenesis.13−16 Overall, however, these proteins have not been well-characterized biochemically or structurally, with only a few kinetic reports to date13,16−20 and just two representative structures in the PDB, one of which is published.9 As members of the ubiquitous α-D-phosphohexomutase superfamily, they are expected to share key mechanistic and structural features that have been characterized well in other subgroups of the superfamily.1 Here, we pursued the intriguing connection between phosphorylation and protein flexibility, first established in P. aeruginosa PMM/PGM.7,8 Recent biochemical studies have revealed similar patterns in human phosphoglucomutase 1 (PGM1).21 As is the case for P. aeruginosa PMM/PGM, human PGM1 has distinct differences relative to BaPNGM, including a longer sequence length (562 residues), a monomeric state in a solution, and limited sequence identity (only 25%). To determine whether BaPNGM undergoes analogous phosphorylation-dependent changes in flexibility, several experimental approaches were utilized, including several from previous studies of these related proteins. In particular, limited proteolysis demonstrates a significant increase in the structural flexibility of dephospho-BaPNGM, relative to its phosphorylated state (Figure 2). These effects appear to be manifested across the structure of the enzyme, as is apparent from the
lated as described in Experimental Procedures. An overview of the long-range (>10 Å) lines of attraction for the BaPNGM dimer are highlighted in Figure 4A, showing extensive interactions between the C-terminal domain 4 (D4) and the rest of the protein. Overall, many of the interdomain electrostatic interactions revealed by the field lines are similar between the phospho- and dephospho-enzyme structures. However, within the active site, striking differences are observed in the region spanning the vicinity of the phosphoserine and D4. Here, converging field lines (those colored in a gradient from red to blue) are obvious in phoshopho-enzyme (Figure 4B), where they connect a region of negative charge near the phosphoserine and metal-binding loop to a positively charged patch in D4 that includes two arginine residues (410 and 419). In contrast, in the dephosphoenzyme, the number of attractive field lines between these two regions is markedly diminished (Figure 4C). Relationships between Electrostatics, Conformational Variability, and Enzyme Mechanism. As noted above, the crystal structures of BaPNGM and the related FtPNGM reveal a dramatic rotational mobility of D4 as a rigid body, with variability seen between protomers within a dimer and when comparing the two dimers.4 (Supporting file S1 shows an 8448
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Figure 4. Electrostatic calculations for the phospho and dephospho states of BaPNGM. (A) An overview of the BaPNGM dimer showing numerous, long-range field lines between D4 and the rest of the protein. Chains A and B (PDB entry 3PDK) are shown in slate blue and olive, respectively. Dimer is in similar orientation as in Figure 1B. (B) A close-up view of the active site cleft of the phospho-enzyme (chain A from PDB entry 3PDK; P-Ser modeled as in Experimental Procedures), showing extensive short-range field lines between residues in D4 and the vicinity of P-Ser100 and metal-binding site (residues 240−244 shown as stick model). (C) A view of the active site for dephospho-enzyme, similar in orientation to (B), showing the diminished attractive field lines between D4 and the same loops.
supported by the crystallographic studies and normal mode analyses of BaPNGM,4 as well as the conservation of D4 motion across the entire α-D-phosphohexomutase enzyme superfamily.6 This makes its potential modulation by phosphorylation an attractive notion. The possibility of phosphorylation affecting more localized protein dynamics would require a more detailed investigation. We note that both phosphorylated (FtPNGM) and dephosphorylated (BaPNGM) forms of these proteins crystallize, and show no obvious structural differences related to phosphorylation (e.g., disordered regions, etc.). Thus, phosphorylation or lack thereof does not appear to produce structural differences observable by crystallography, despite the differences observable in their solution behavior. Indeed, for P. aeruginosa PMM/PGM, where both dephospho- and phosphoenzyme crystal structures are available at high resolution, no significant structural differences can be discerned.7 This serves as an interesting, and perhaps cautionary, example of how solution studies can add unexpected insights into proteins of known structure. As noted above, rotation of D4 in BaPNGM is likely necessary at several points in the multistep reaction, including binding of substrate, release of product, and to permit rotation
more rapid proteolytic digestion and increased number of cleavage sites in the dephospho-enzyme. NMR also suggests differences between phospho- and dephospho-BaPNGM in a solution, based on changes in the 15N TROSY spectrum (Figure 3). Cross-correlated 15N relaxation data might be consistent with a subtle slowing of the rotational diffusion of dephospho-BaPNGM, but the differences are within experimental error. The molecular features of BaPNGM responsible for its observed phosphorylation-dependent behavior were also considered. In principle, these could include many factors, such as changes in interdomain mobility, differences in the time scales of conformational changes, increased “breathing motions” reflecting some structural destabilization, or even order-to-disorder transitions. Evidence for the former is suggested by the electrostatic calculations of the active site of BaPNGM, which show attraction between a negatively charged region that includes the catalytic phosphoserine residue and positively charged residues in D4. In contrast, in the dephosphorylated version of the active site serine, this electrostatic attraction is absent, consistent with increased relative mobility of D4, and potentially other regions of the protein as well. The functional importance of D4 mobility is 8449
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effects of post-translational modifications on protein structure, with the unique addition of its mechanistic implications.
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EXPERIMENTAL PROCEDURES Protein Preparation. Expression and purification of BaPNGM for the biochemical studies was done as previously described.27 For NMR studies, 2H/15N-labeled BaPNGM was expressed using the protocol in ref 28, with several minor modifications: initial small-scale cultures were grown in 70% D2O and then transferred into 99% D2O for final growth (250 mL). Cultures were harvested when the bacterial growth reached a plateau at an optical density at 600 nm of ∼6.0. Purification of the labeled protein was done as referenced above for the unlabeled sample, yielding 65 mg of pure, labeled protein from 500 mL of bacterial culture. Purified proteins were dialyzed into 50 mM 3-(N-morpholino)propane sulfonic acid (MOPS) pH 7.4 and 1 mM MgCl2, and concentrated to ∼10 mg/mL. Samples were either used immediately or flash frozen in liquid nitrogen for storage at 193 K. Phosphorylation and Assessment by Mass Spectrometry. The phosphorylation of the active site phosphoserine (Ser100) of BaPNGM was assessed before and after treatment with glucose 1,6-bisphosphate using electrospray ionization mass spectrometry (ESI-MS). Protein at a concentration of 430 μM was incubated with a 10-fold molar excess of glucose 1,6bisphosphate for 16 h at 4 °C. Excess glucose 1,6-bisphosphate was removed by extensive dialysis against 50 mM MOPS, pH 7.4, and 1 mM MgCl2. For mass spectrometric studies, 10 μL protein samples at 1 pmol/μL in 1% formic acid were analyzed by NanoLCNanospray QTOF (Agilent 6520) in positive ion mode with a Zorbax C8 trap column. Data were examined using the Qual software provided with the instrument. The mass error between samples is 0.11 Da (2.1 ppm), and the quantification error is 2%. Percent phosphorylation was calculated by normalizing the sum of the dephosphorylated and phosphorylated peak heights to 1.0. In all of the samples, no more than one phosphorylated residue was observed. Assays of Accessibility. Limited proteolysis of BaPNGM in its phospho and dephospho states was done as follows. Protein samples at 1.5 mg/mL were incubated with Tritirachium album proteinase K (Fisher Scientific) in 50 mM MOPS, pH 7.4 at a 500:1 (w/w) ratio. Digestion was conducted at room temperature, aliquots removed at various time points, and the reaction terminated by the addition of phenylmethylsulfonyl fluoride (final concentration: 3 mM). The reaction mixtures were subjected to SDS-PAGE using 14% polyacrylamide gels. ANS-binding of phospho- and dephospho-enzyme samples was assessed as follows. ANS at 0.5 mM was incubated with protein samples at 12 μM in 50 mM MOPS, pH 7.4, 1 mM MgCl2 for 1 h at 25 °C. Data were collected using a BioTek Synergy Mx Microplate reader. The excitation wavelength was 365 nm, and the emission spectra were recorded from 400 to 560 nm. Fluorescence intensities of samples were corrected for ANS emission spectra in buffer. TROSY Spectra and Rotational Correlation Times by NMR. 2H/15N-labeled PNGM was concentrated to approximately 360 μM in 50 mM MOPS (pH 7.4), 1 mM MgCl2, and 7% D2O. Two-dimensional 15N band-selective excitation shorttransient traverse relaxation-optimized NMR spectroscopy29 and 1D cross-correlation relaxation spectra30 were collected at 35 °C on a Bruker Avance III 800 MHz spectrometer with TCI
Figure 5. Close-up view of varying conformers of D4 in PNGM. (A) Inter-residues distances shown on a superposition of the two protomers of BaPNGM (chains A in blue; B in tan from PDB entry 3PDK) with chain A (green) of FtPNGM (PDB entry 313W). Distances indicated are between Og of Ser100 and the NH2 of Arg419 in BaPNGM, or the corresponding atoms in FtPNGM. (B) The same superposition as in (A) with a model of bound ligand (glucose 1phosphate in yellow) based on the enzyme−ligand complex of P. aeruginosa PMM/PGM (PDB entry 1P5D); for more details see also ref 20. Note how the closed conformer of chain B (tan) positions Arg419 for interaction with the phosphate group of ligand.
of the reaction intermediate. This reorientation is a unique mechanistic feature of the enzyme superfamily, which is intimately linked to the reversibility of the reaction and the pivotal metabolic and biosynthetic roles of these enzymes. For P. aeruginosa PMM/PGM and rabbit PGM,22,23 this reorientation is known to occur “on enzyme” (i.e., without dissociation of the intermediate), which is an additional mechanistic challenge for the enzyme. The observed coupling between dephosphorylation and increased enzyme flexibility, which coincides with the catalytic step requiring reorientation of the intermediate, is a strong argument for the conservation of this mechanistic feature across this ubiquitous enzyme superfamily. Phosphorylation is just 1 of more than 400 types of posttranslational modifications identified in proteins to date.24 The functional effects of post-translational modification are well known and manifold. However, their structural effects are often assumed to be localized, operating via changes in the immediate environment of the phosphorylation site, or, on the other extreme, through induced, large-scale structural rearrangements. Some recent studies, e.g.,24−26 however, are providing hints of nuanced yet significant structural effects that remain largely unappreciated. This study adds to the growing list of 8450
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cryoprobe. The NMR spectra were processed using NMRpipe31 and viewed using SPARKY.32 Transverse crosscorrelated 15N NMR relaxation rate constants ηxy30 (relaxation involving dipole−dipole interactions with chemical-shift anisotropy) were measured for phosphorylated and dephosphorylated BaPNGM. Series of 1D versions of the spectra were collected to measure the average ηxy values for the amide spectral regions corresponding to the rigidly structured (not random coil) regions of PNGM using the relaxation periods of 0, 2, 4, 7, 10, 14, and 20 ms, each in triplicate. The TREND software package33 was used to integrate backbone amide proton signals from 8.5 to 10 ppm. These integrals were fitted to a single exponential decay to extract the relaxation rate constant ηxy.30 The rotational correlation time τc was estimated using eqs 3−6 in ref 12 which assume isotropic rotational diffusion. However, crystallographic coordinates of the PNGM dimer suggest some axial symmetry. Electrostatic Field Calculations. PDB entry 3PDK, which is dephosphorylated, was the starting point for the electrostatic calculations of the BaPNGM active site. The phosphorylated version of Ser100 in BaPNGM was modeled based on a superposition with the related P. aeruginosa PMM/PGM (PDB entry 1K35), which is phosphorylated on the corresponding serine. In addition, because the low pH of the crystallization buffer precluded the binding of divalent cations in the active site of BaPNGM (PDB entry 3PDK), the metal-binding loop (residues 240−244) for both phospho- and dephospho-enzyme was also modeled as in PDB entry 1K35,8 which has an identical amino acid sequence in the loop. Atomic charges and sizes around the metal-binding site of BaPNGM were parameterized using the Amber tools module MCPB/ MTKPP, which invokes Gaussian 2009.34,35 Other atomic sizes and charges were defined by the Amber ff99SB force field.35 The electrostatic potentials were then computed using the adaptive Poisson−Boltzmann solver36 at 310 K, with protein and solvent dielectric constants of 2.0 and 78.5, respectively, and ionic strength of 30 mM. Electrostatic field lines were visualized using visual molecular dynamics37 as previously described.38,39
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ACKNOWLEDGMENTS
We thank Brian Mooney and Bevery DaGue of the University of Missouri Charles W. Gehrke Proteomics Center for mass spectrometry and Andrew Muenks for assistance with figure preparation. Kyle Stiers was supported by National Institutes of Health Training Grant T32 GM008396-26 from NIGMS and predoctoral fellowship 17PRE33400210 from the American Heart Association. This work was supported by a grant from the National Science Foundation (MCB-1409898) to L.J.B. and S.V.D. The University of Missouri and National Institutes of Health Grant RR022341 generously supported acquisition of the 800 MHz NMR spectrometer.
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ABBREVIATIONS ANS, 8-anilinonaphthalene-1-sulfonic acid BaPNGM, B. anthracis PNGM ESI-MS, electrospray ionization mass spectrometry HDX, hydrogen−deuterium exchange MOPS, 3-(N-morpholino)propane sulfonic acid PDB, protein data bank PGM, phosphoglucomutase PMM/PGM, phosphomannomutase/phosphoglucomutase PNGM, phosphoglucosamine mutase SAXS, small-angle X-ray scattering REFERENCES
(1) Stiers, K. M.; Muenks, A. G.; Beamer, L. J. Biology, mechanism, and structure of enzymes in the α-D-phosphohexomutase superfamily. Adv. Protein Chem. Struct. Biol. 2017, 109, 265−304. (2) Barreteau, H.; Kovac, A.; Boniface, A.; Sova, M.; Gobec, S.; Blanot, D. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol. Rev. 2008, 32, 168−207. (3) Gautam, A.; Vyas, R.; Tewari, R. Peptidoglycan biosynthesis machinery: A rich source of drug targets. Crit. Rev. Biotechnol. 2011, 31, 295−336. (4) Mehra-Chaudhary, R.; Mick, J.; Tanner, J. J.; Beamer, L. J. Quaternary structure, conformational variability and global motions of phosphoglucosamine mutase. FEBS J. 2011, 278, 3298−3307. (5) Mehra-Chaudhary, R.; Mick, J.; Tanner, J. J.; Henzl, M. T.; Beamer, L. J. Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens. Proteins 2011, 79, 1215−29. (6) Luebbering, E. K.; Mick, J.; Singh, R. K.; Tanner, J. J.; MehraChaudhary, R.; Beamer, L. J. Conservation of functionally important global motions in an enzyme superfamily across varying quaternary structures. J. Mol. Biol. 2012, 423, 831−846. (7) Lee, Y.; Villar, M. T.; Artigues, A.; Beamer, L. J. Promotion of enzyme flexibility by dephosphorylation and coupling to the catalytic mechanism of a phosphohexomutase. J. Biol. Chem. 2014, 289, 4674− 4682. (8) Xu, J.; Lee, Y.; Beamer, L. J.; Van Doren, S. R. Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase. Biophys. J. 2015, 108, 325−37. (9) Mehra-Chaudhary, R.; Mick, J.; Beamer, L. J. Crystal structure of Bacillus anthracis phosphoglucosamine mutase, an enzyme in the peptidoglycan biosynthetic pathway. J. Bacteriol. 2011, 193, 4081− 4087. (10) Lee, Y.; Furdui, C. M.; Beamer, L. J. Data on the phosphorylation state of the catalytic serine of enzymes in the a-Dphosphohexomutase superfamily. Data Brief 2017, 10, 398−405. (11) Ray, S. S.; Balaram, P. 1-Anilino-8-naphthalene-sulfonate (ANS) Binding to Proteins Investigated by Electrospray Ionization Mass Spectrometry: Correlation of Gas-Phase Dye Binding to Population of Molten Globule States in Solution. J. Phys. Chem. B 1999, 103, 7068− 7072.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01490. Animation of conformational change in D4 observed between the protomers of the BaPNGM dimer (MPG) Supporting Figure 1 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: 573-882-6072. Fax: 573884-4812. ORCID
Lesa J. Beamer: 0000-0001-5689-200X Present Address †
Catalent Pharma Solutions, 10245 Hickman Mills Drive, Kansas City, Missouri 64137, United States (Y.L.). Author Contributions ‡
K.M.S. and J.X. contributed equally to this manuscript.
Notes
The authors declare no competing financial interest. 8451
DOI: 10.1021/acsomega.7b01490 ACS Omega 2017, 2, 8445−8452
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DOI: 10.1021/acsomega.7b01490 ACS Omega 2017, 2, 8445−8452
