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Cited by 18Tuberculosis Research and EpidemiologyBacterial Genetics and BiotechnologyEnzyme Structure and Function

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Identification of Arginine Phosphorylation in Mycolicibacterium smegmatis

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Ogbonna, Emmanuel C., Anderson, Henry R., Schmitz, Karl R.·2022·Microbiology Spectrum
DOI 10.1128/spectrum.02042-22PMC PMC9604228

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Abstract

Mycobacteria that cause tuberculosis infections employ proteolytic pathways that modulate cellular behavior by destroying specific proteins in a highly regulated manner. Some proteolytic enzymes have emerged as novel antibacterial targets against drug-resistant tuberculosis infections.

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FIG 1 Indirect evidence for phosphoarginine in actinobacteria. (A) The domain organization of ClpC1 is shown above a plot of amino acid conservation among actinobacterial ClpC1 orthologs, where conservation at each alignment position is plotted as a purple vertical strip. The apparent poor conservation at the beginning of the NTD (marked by an asterisk) likely reflects misannotation of the start site in some entries. (B) The sequence of the Bacillus subtilis ClpC NTD is shown above equivalent regions from several actinobacterial ClpC orthologs. The sequence logo below shows amino acid conservation in actinobacterial ClpC1 orthologs. Arrows mark positions reported to be important for phosphoarginine binding to site 1 (orange) or site 2 (yellow) (34). Phosphoarginines from the crystal structure of the B. subtilis ClpC NTD (PDB code 5HBN) were modeled on the M. tuberculosis ClpC1 NTD (PDB code 6PBQ) putative phosphoarginine-binding sites 1 (C) and 2 (D) (47). (E) Binding of phosphoarginine to M. smegmatis ClpC1NTD was measured by microscale thermophoresis. Data were fit to a noncooperative binding model (gray curve), yielding a KD (equilibrium dissociation constant) of 1.4 ± 0.6 μM. Values are averages of three technical replicates ± 1 SD. (F) Dot blots of Mycolicibacterium smegmatis, Corynebacterium jeikeium, Bacillus subtilis, and Escherichia coli cell lysates, probed with anti-phosphoarginine antibody (48) or stained with Ponceau S.
FIG 1 Indirect evidence for phosphoarginine in actinobacteria. (A) The domain organization of ClpC1 is shown above a plot of amino acid conservation among actinobacterial ClpC1 orthologs, where conservation at each alignment position is plotted as a purple vertical strip. The apparent poor conservation at the beginning of the NTD (marked by an asterisk) likely reflects misannotation of the start site in some entries. (B) The sequence of the Bacillus subtilis ClpC NTD is shown above equivalent regions from several actinobacterial ClpC orthologs. The sequence logo below shows amino acid conservation in actinobacterial ClpC1 orthologs. Arrows mark positions reported to be important for phosphoarginine binding to site 1 (orange) or site 2 (yellow) (34). Phosphoarginines from the crystal structure of the B. subtilis ClpC NTD (PDB code 5HBN) were modeled on the M. tuberculosis ClpC1 NTD (PDB code 6PBQ) putative phosphoarginine-binding sites 1 (C) and 2 (D) (47). (E) Binding of phosphoarginine to M. smegmatis ClpC1NTD was measured by microscale thermophoresis. Data were fit to a noncooperative binding model (gray curve), yielding a KD (equilibrium dissociation constant) of 1.4 ± 0.6 μM. Values are averages of three technical replicates ± 1 SD. (F) Dot blots of Mycolicibacterium smegmatis, Corynebacterium jeikeium, Bacillus subtilis, and Escherichia coli cell lysates, probed with anti-phosphoarginine antibody (48) or stained with Ponceau S.CC BY · PubMed Central
FIG 2 Comparative analysis of mass spectrometry output. (A) Workflow for arginine phosphoproteomics in Mycolicibacterium smegmatis. (B) Number of proteins obtained across biological replicates from cells grown under heat stress (50°C) and normal (37°C) conditions. (C) Volcano plot showing enrichment of proteins observed by LC-MS/MS in heat stress versus normal growth conditions. Plotted on the x axis is the log2 of the ratio of average Sequest HT score of stressed to unstressed samples. The y axis shows −log10 of P values obtained by Student’s t test. The horizontal green line indicates a cutoff P value = 0.05; the vertical blue line indicates score ratio of 1. Highlighted in red are notable chaperone proteins with stressed/unstressed ratio of >1.
FIG 2 Comparative analysis of mass spectrometry output. (A) Workflow for arginine phosphoproteomics in Mycolicibacterium smegmatis. (B) Number of proteins obtained across biological replicates from cells grown under heat stress (50°C) and normal (37°C) conditions. (C) Volcano plot showing enrichment of proteins observed by LC-MS/MS in heat stress versus normal growth conditions. Plotted on the x axis is the log2 of the ratio of average Sequest HT score of stressed to unstressed samples. The y axis shows −log10 of P values obtained by Student’s t test. The horizontal green line indicates a cutoff P value = 0.05; the vertical blue line indicates score ratio of 1. Highlighted in red are notable chaperone proteins with stressed/unstressed ratio of >1.CC BY · PubMed Central
FIG 3 Identification of arginine phosphosites in M. smegmatis proteome. (A) Localized arginine phosphosites in six M. smegmatis proteins. Localization probability is reported as PhosphoRS/ptmRS score. (B and C) Representative secondary fragmentation spectra show arginine-phosphorylated peptides from PhoU1 (B) and hemerythrin domain-containing protein (C). Additional spectra are shown in Fig. S2.
FIG 3 Identification of arginine phosphosites in M. smegmatis proteome. (A) Localized arginine phosphosites in six M. smegmatis proteins. Localization probability is reported as PhosphoRS/ptmRS score. (B and C) Representative secondary fragmentation spectra show arginine-phosphorylated peptides from PhoU1 (B) and hemerythrin domain-containing protein (C). Additional spectra are shown in Fig. S2.CC BY · PubMed Central
FIG 4 Structural characteristics of phosphoarginine sites. (A) Sequence alignment of 20 residues flanking the arginine phosphosites. (B to F) Structural models of Msmeg_1293 (B), Msmeg_1547 (C), PhoU1 (D), Kgd (E), and Msmeg_0907 (F) are shown with the phosphorylated arginine colored magenta and marked with an arrow. All models were generated by AlphaFold2 (59).
FIG 4 Structural characteristics of phosphoarginine sites. (A) Sequence alignment of 20 residues flanking the arginine phosphosites. (B to F) Structural models of Msmeg_1293 (B), Msmeg_1547 (C), PhoU1 (D), Kgd (E), and Msmeg_0907 (F) are shown with the phosphorylated arginine colored magenta and marked with an arrow. All models were generated by AlphaFold2 (59).CC BY · PubMed Central
FIG 5 Sequence conservation of phosphorylated arginine residues. Sequence logos show conservation near phosphorylated arginines in Kgd R433 (A), Msmeg_0907 R34 (B), Msmeg_0879 R31 (C), Msmeg_1547 R326 (D), PhoU1 R149 (E), and Msmeg_1293 R475 (F). Percentages indicate positional conservation of the phosphorylated arginine; “n” indicates the number of aligned orthologous sequences used to generate the sequence logo.
FIG 5 Sequence conservation of phosphorylated arginine residues. Sequence logos show conservation near phosphorylated arginines in Kgd R433 (A), Msmeg_0907 R34 (B), Msmeg_0879 R31 (C), Msmeg_1547 R326 (D), PhoU1 R149 (E), and Msmeg_1293 R475 (F). Percentages indicate positional conservation of the phosphorylated arginine; “n” indicates the number of aligned orthologous sequences used to generate the sequence logo.CC BY · PubMed Central

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Cited by

Metadata

Authors
Ogbonna, E. C., Anderson, H. R., & Schmitz, K. R.
Affiliations
University of Delaware (US)
Corresponding author
Schmitz, Karl R.
ORCID
Venue
Microbiology Spectrum
Publisher
American Society for Microbiology
Published
2022-10-26
Volume
10
Issue
5
Pages
e0204222-e0204222
ISSN
2165-0497 (ISSN-L)
DOI
10.1128/spectrum.02042-22
PMC
PMC9604228
OpenAlex
W4304005546
Cited by
18
References
3
Citations by year
2026: 3 · 2025: 3 · 2024: 8 · 2023: 4
FWCI
1.83
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbiology Spectrum (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
HHS | NIH | National Institute of General Medical Sciences (P20GM104316)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Tuberculosis Research and Epidemiology
  • 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
Mycobacterium smegmatis, mycobacteria, proteolytic enzymes, protein degradation, drug-resistant tuberculosis, antibiotic targets, protein chemical modification
MeSH terms
Anti-Bacterial Agents, Arginine, Bacterial Proteins, Humans, Mycobacterium tuberculosis, Organophosphorus Compounds, Phosphopeptides, Phosphorylation, Tuberculosis, Mycobacterium smegmatis, Proteome, ATP-Dependent Proteases

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