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The Solvation of the E. coli CheY Phosphorylation Site Mapped by XFMS

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Hamid, Maham, Khalid, Muhammad Farhan, Chaudhary, Safee Ullah, Khan, Shahid·2022·International Journal of Molecular Sciences
DOI 10.3390/ijms232112771PMC PMC9659070

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Abstract

The Escherichia coli CheY protein belongs to a large bacterial response regulator superfamily. X-ray hydroxy radical foot-printing with mass spectroscopy (XFMS) has shown that allosteric activation of CheY by its motor target triggers a concerted internalization of aromatic sidechains. We reanalyzed the XFMS data to compare polar versus non-polar CheY residue positions. The polar residues around and including the 57D phosphorylated site had an elevated hydroxy radical reactivity. Bioinformatic measures revealed that a water-mediated hydrogen bond network connected this ring of residues with the central 57D. These residues solvated 57D to energetically stabilize the apo-CheY fold. The abundance of these reactive residues was reduced upon activation. This result was supported by the bioinformatics and consistent with the previously reported activation-induced increase in core hydrophobicity. It further illustrated XFMS detection of structural waters. Direct contacts between the ring residues and the phosphorylation site would stabilize the aspartyl phosphate. In addition, we report that the ring residue, 18R, is a constant central node in the 57D solvation network and that 18R non-polar substitutions determine CheY diversity as assessed by its evolutionary trace in bacteria with well-studied chemotaxis. These results showcase the importance of structured water dynamics for phosphorylation-mediated signal transduction.

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Figure A1 Screenshot of Mass Hunter GUI.
Figure A1 Screenshot of Mass Hunter GUI.CC BY · PubMed Central
Figure A2 Examples of oxidized peaks (Po’s) with ΣAA > k for 2 overlapping peptides (ΣPeptOxoverlap = 2). (a) k = 1, ΣAA = 2. (b) k = 2, ΣAA = 3. For ΣPeptOxoverlap = 3 or >, the equations have extra terms, but the logic is the same. Overlapping peptides do not occur for unoxidized peaks (Pu’s).
Figure A2 Examples of oxidized peaks (Po’s) with ΣAA > k for 2 overlapping peptides (ΣPeptOxoverlap = 2). (a) k = 1, ΣAA = 2. (b) k = 2, ΣAA = 3. For ΣPeptOxoverlap = 3 or >, the equations have extra terms, but the logic is the same. Overlapping peptides do not occur for unoxidized peaks (Pu’s).CC BY · PubMed Central
Figure 1 XFMS dose-response curves for residues around the 57D phosphorylation site. Oxidation rates were computed from the slopes of single exponential fits, weighted by reciprocal of the standard error, for all ten residues, including 57D. The CheYA rates (black dashed lines) were lower than those for CheYI (grey dashed lines) in all cases, except for 13D/K and 59N. The intrinsic reactivity to hydroxy radical oxidation is greater for K (2.2) than D (0.42).
Figure 1 XFMS dose-response curves for residues around the 57D phosphorylation site. Oxidation rates were computed from the slopes of single exponential fits, weighted by reciprocal of the standard error, for all ten residues, including 57D. The CheYA rates (black dashed lines) were lower than those for CheYI (grey dashed lines) in all cases, except for 13D/K and 59N. The intrinsic reactivity to hydroxy radical oxidation is greater for K (2.2) than D (0.42).CC BY · PubMed Central
Figure 2 PF differences between residue types and activated states. (a). The solvent accessibility of non-polar residues (magenta symbols) varies linearly with SASA. The least-square best fit linear regression (thick dashed line) and 95% confidence limits (thin dashed lines) are shown. Most polar residues (white symbols (N)) follow the best-fit relation, but a significant fraction (yellow symbols (L)) have anomalously high solvent accessibility (low Log (PF)) A = CheYA, I = CheYI. (b). The activation-dependent change in polar residue Log (PF) values. There is a marked positive Log (PF) (Δ(CheYA−CheYI)) increase for the 34E, 35E, 37E, 38D residue quadruplet ringing 57D, modest increases for 12D, 18R, 57D, 62N, and no significant change for 13D/K, 59N. The Δ(CheYA−CheYI) Log (PF) for 75D outside the 57D solvation network has a negative value.
Figure 2 PF differences between residue types and activated states. (a). The solvent accessibility of non-polar residues (magenta symbols) varies linearly with SASA. The least-square best fit linear regression (thick dashed line) and 95% confidence limits (thin dashed lines) are shown. Most polar residues (white symbols (N)) follow the best-fit relation, but a significant fraction (yellow symbols (L)) have anomalously high solvent accessibility (low Log (PF)) A = CheYA, I = CheYI. (b). The activation-dependent change in polar residue Log (PF) values. There is a marked positive Log (PF) (Δ(CheYA−CheYI)) increase for the 34E, 35E, 37E, 38D residue quadruplet ringing 57D, modest increases for 12D, 18R, 57D, 62N, and no significant change for 13D/K, 59N. The Δ(CheYA−CheYI) Log (PF) for 75D outside the 57D solvation network has a negative value.CC BY · PubMed Central
Figure 3 Activation-dependent change in fold energetics. The Frustratometer2 algorithm computes residue contact energies relative to a decoy contact distribution with a coarse-grained AWSEM force-field. The PDB structures with superimposed high (red lines) and low (green lines) contact energies relative to the decoy distributions are shown. (a) Apo-CheY. High-energy contacts radiate out from 57D (red sidechain) to surrounding residues that include residue positions detected by XFMS (stick sidechains) with anomalous low (yellow) or comparable (white) Log (PF) values to non-polar residues (see text). 106Y sidechain (salmon). (b) Mg2+ bound apo-CheY. Magnesium ion (cyan sphere). (c) 13DK,106YW CheY. Activated by bound FliMN (not shown). XFMS residue positions in (b,c) as in (a). There is a reduction in the high energy contacts of 13D/K, 106YW CheY 57D with surrounding residue positions relative to the apo-CheY forms. Waters (red dots).
Figure 3 Activation-dependent change in fold energetics. The Frustratometer2 algorithm computes residue contact energies relative to a decoy contact distribution with a coarse-grained AWSEM force-field. The PDB structures with superimposed high (red lines) and low (green lines) contact energies relative to the decoy distributions are shown. (a) Apo-CheY. High-energy contacts radiate out from 57D (red sidechain) to surrounding residues that include residue positions detected by XFMS (stick sidechains) with anomalous low (yellow) or comparable (white) Log (PF) values to non-polar residues (see text). 106Y sidechain (salmon). (b) Mg2+ bound apo-CheY. Magnesium ion (cyan sphere). (c) 13DK,106YW CheY. Activated by bound FliMN (not shown). XFMS residue positions in (b,c) as in (a). There is a reduction in the high energy contacts of 13D/K, 106YW CheY 57D with surrounding residue positions relative to the apo-CheY forms. Waters (red dots).CC BY · PubMed Central
Figure 4 Changes in the Bridge2 CheY hydrogen-bonded protein-water network upon activation. Bars = Color-coded Cbc values (Blue (Low) -> Red (High)). (a–c). Inactive apo-CheY. (a) The Cbc nodes mapped onto the 3D structure. Residues detected by XFMS (stick sidechains). Water (dotted spheres). Mg2+ (cyan spheres), BeF3 (magenta sticks). (b) The 2D map of the network. Local nodes (i) 109K -> 57D (circled [19]) (ii) 18R. (iii) 94N -> 106Y. (c) The Cbc nodes targeted by XFMS detected residues (low (yellow), normal (white)). (d–f). Activated CheY13DK106YW in complex with FliMN. (d) The Cbc nodes mapped onto the 3D structure. Residues detected by XFMS and waters (dotted spheres) as in (a). (e) The 2D map of the network. Local nodes (i) 109K -> 57D (circled [19]) (ii) 18R. (iii) 37E -> 41D, 38D -> 62N, 75D (f) The Cbc nodes targeted by XFMS detected residues (anomalous (yellow), comparable (white)). Bridge2 residue nomenclature, where type precedes position, is different from nomenclature used elsewhere in the text.
Figure 4 Changes in the Bridge2 CheY hydrogen-bonded protein-water network upon activation. Bars = Color-coded Cbc values (Blue (Low) -> Red (High)). (a–c). Inactive apo-CheY. (a) The Cbc nodes mapped onto the 3D structure. Residues detected by XFMS (stick sidechains). Water (dotted spheres). Mg2+ (cyan spheres), BeF3 (magenta sticks). (b) The 2D map of the network. Local nodes (i) 109K -> 57D (circled [19]) (ii) 18R. (iii) 94N -> 106Y. (c) The Cbc nodes targeted by XFMS detected residues (low (yellow), normal (white)). (d–f). Activated CheY13DK106YW in complex with FliMN. (d) The Cbc nodes mapped onto the 3D structure. Residues detected by XFMS and waters (dotted spheres) as in (a). (e) The 2D map of the network. Local nodes (i) 109K -> 57D (circled [19]) (ii) 18R. (iii) 37E -> 41D, 38D -> 62N, 75D (f) The Cbc nodes targeted by XFMS detected residues (anomalous (yellow), comparable (white)). Bridge2 residue nomenclature, where type precedes position, is different from nomenclature used elsewhere in the text.CC BY · PubMed Central
Figure 5 The evolutionary importance of ALPF 18R in CheY evolution. (a) The phylogenetic tree for E. coli CheY. Based on 443 sequences used by EV-Trace. Scale bar = 2.0. (b) Top (10%) residue positions. The E. coli CheY residue positions were ranked by rv.ET score. Asterisks denote residues known previously to be important for chemotaxis. (c). Residue conservation of the top rv.ET ranked positions. From an MSA of 56 sequences from 12 distinct species. (d) The 3D CheY structure color-coded by ev.ET score. The residues oxidized by XFMS are labelled (stick sidechains). Spheres mark the residues 12D and 18R (spheres) important for CheY evolution under selection for chemotaxis. (e) The phylogenetic tree constructed from the MSA of CheY chemotaxis proteins. 56 CheY sequences. The non-polar 18R substitutions localize at the internal tree nodes. Roman numerals denote major branches. Circle diameter = branch size. Note difference in scale with the EV-Trace tree (a).
Figure 5 The evolutionary importance of ALPF 18R in CheY evolution. (a) The phylogenetic tree for E. coli CheY. Based on 443 sequences used by EV-Trace. Scale bar = 2.0. (b) Top (10%) residue positions. The E. coli CheY residue positions were ranked by rv.ET score. Asterisks denote residues known previously to be important for chemotaxis. (c). Residue conservation of the top rv.ET ranked positions. From an MSA of 56 sequences from 12 distinct species. (d) The 3D CheY structure color-coded by ev.ET score. The residues oxidized by XFMS are labelled (stick sidechains). Spheres mark the residues 12D and 18R (spheres) important for CheY evolution under selection for chemotaxis. (e) The phylogenetic tree constructed from the MSA of CheY chemotaxis proteins. 56 CheY sequences. The non-polar 18R substitutions localize at the internal tree nodes. Roman numerals denote major branches. Circle diameter = branch size. Note difference in scale with the EV-Trace tree (a).CC BY · PubMed Central
Figure 6 Semi-automated algorithm for determination of PFs and the SASA–Log (PF) relation. There are three distinct stages to the MATLAB code. The code is generally applicable. 1. The proteolyzed (tryptic) peptides from the XF experiment were analyzed by LC-MS and MS/MS. The pipeline illustrated for one peptide was iterated for all peptides. The resulting Mass Hunter (LC-MS) and MASCOT (MS/MS) files were matched by RT (retention time) to parse out the oxidized and unoxidized peak areas (black sections) for each residue from unrelated peaks in the lists obtained by manual entry (red) of the MASCOT m/z values. 2. A list with the oxidized and unoxidized peak areas for all detected residues in the protein was compiled. 3. Computed oxidation rates were divided by intrinsic reactivities for determination of residue PFs. Data for all residues were combined for the SASA versus Log (PF) relations. The SASAs were obtained from the protein crystal structure or Alpha-fold model. Residues with Log (PF) values below the 95% confidence limit for the best-fit linear relation were designated as ALPRs (anomalous protection factor residues). See Appendix A for details.
Figure 6 Semi-automated algorithm for determination of PFs and the SASA–Log (PF) relation. There are three distinct stages to the MATLAB code. The code is generally applicable. 1. The proteolyzed (tryptic) peptides from the XF experiment were analyzed by LC-MS and MS/MS. The pipeline illustrated for one peptide was iterated for all peptides. The resulting Mass Hunter (LC-MS) and MASCOT (MS/MS) files were matched by RT (retention time) to parse out the oxidized and unoxidized peak areas (black sections) for each residue from unrelated peaks in the lists obtained by manual entry (red) of the MASCOT m/z values. 2. A list with the oxidized and unoxidized peak areas for all detected residues in the protein was compiled. 3. Computed oxidation rates were divided by intrinsic reactivities for determination of residue PFs. Data for all residues were combined for the SASA versus Log (PF) relations. The SASAs were obtained from the protein crystal structure or Alpha-fold model. Residues with Log (PF) values below the 95% confidence limit for the best-fit linear relation were designated as ALPRs (anomalous protection factor residues). See Appendix A for details.CC BY · PubMed Central

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Metadata

Authors
Hamid, M., Khalid, M. F., Chaudhary, S. U., & Khan, S.
Affiliations
  • Lahore University of Management Sciences (PK)
  • Lawrence Berkeley National Laboratory (US)
Corresponding author
  • Chaudhary, Safee Ullah
  • Khan, Shahid
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2022-10-23
Volume
23
Issue
21
Pages
12771
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms232112771
PMC
PMC9659070
OpenAlex
W4307267327
Cited by
7
References
4
Citations by year
2026: 2 · 2025: 5
FWCI
0.41
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Higher Education Commission (HEC) of Pakistan (20-3629/NRPU/R, D/HEC/14/585, RF-NCBC-015)
  • National Center for Bigdata and Cloud Computing (NCBC) (20-3629/NRPU/R, D/HEC/14/585, RF-NCBC-015)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
CheY, phosphorylation sites, solvation, hydrogen bond network, structured water, allosteric activation, bacterial chemotaxis, response regulator, evolutionary trace, signal transduction
MeSH terms
Methyl-Accepting Chemotaxis Proteins, Bacterial Proteins, Chemotaxis, Escherichia coli, Membrane Proteins, Mutation, Phosphorylation, Water, Escherichia coli Proteins

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Hamid, M., Khalid, M. F., Chaudhary, S. U., & Khan, S. (2022). The Solvation of the E. coli CheY Phosphorylation Site Mapped by XFMS. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms232112771