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Cited by 3Heat shock proteins researchBacterial Genetics and BiotechnologyProtein Structure and Dynamics

The DOI matches its Crossref record; 79 of the 79 references it cites were found in a registry or an index.

Bioinformatic identification of ClpI, a distinct class of Clp unfoldases in Actinomycetota

Open access
Jiang, Jialiu, Schmitz, Karl R.·2023·Frontiers in Microbiology
DOI 10.3389/fmicb.2023.1161764PMC PMC10149685

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Abstract

All clades of bacteria possess Hsp100/Clp family unfoldase enzymes that contribute to aspects of protein quality control. In Actinomycetota, these include ClpB, which functions as an independent chaperone and disaggregase, and ClpC, which cooperates with the ClpP1P2 peptidase to carry out regulated proteolysis of client proteins. We initially sought to algorithmically catalog Clp unfoldase orthologs from Actinomycetota into ClpB and ClpC categories. In the process, we uncovered a phylogenetically distinct third group of double-ringed Clp enzymes, which we term ClpI. ClpI enzymes are architecturally similar to ClpB and ClpC, with intact ATPase modules and motifs associated with substrate unfolding and translation. While ClpI possess an M-domain similar in length to that of ClpC, its N-terminal domain is more variable than the strongly conserved N-terminal domain of ClpC. Surprisingly, ClpI sequences are divisible into sub-classes that either possess or lack the LGF-motifs required for stable assembly with ClpP1P2, suggesting distinct cellular roles. The presence of ClpI enzymes likely provides bacteria with expanded complexity and regulatory control over protein quality control programs, supplementing the conserved roles of ClpB and ClpC.

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Crossref · 10.3389/fmicb.2023.1161764

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Figures

Figure 1 Clp enzyme architecture and domain organization. (A) A cartoon of ClpC illustrates notable features of Clp enzymes. In the context of proteolysis by ClpCP1P2, client substrates are recognized by ClpC, unfolded, and translocated through the axial pore into the peptidase degradation chamber. (B) ClpB and ClpC proteins contain a family-specific N-terminal domain (NTD), followed by two AAA+ ATPase modules (D1 and D2) with conserved Walker A and B motifs. The D1 ATPase module is interrupted by a helical M-domain that projects upward toward the NTD. ClpC enzymes additionally possess an LGF loop that makes stabilizing contacts with the ClpP2 ring of the peptidase. (C) Sequence alignment of select ClpC orthologs from Actinomycetota shows strong conservation of Walker A and B motifs. Notably, a subset of sequences possesses a short M-domain and distinct LGF loop regions, with or without identifiable LGF-like motifs.
Figure 1 Clp enzyme architecture and domain organization. (A) A cartoon of ClpC illustrates notable features of Clp enzymes. In the context of proteolysis by ClpCP1P2, client substrates are recognized by ClpC, unfolded, and translocated through the axial pore into the peptidase degradation chamber. (B) ClpB and ClpC proteins contain a family-specific N-terminal domain (NTD), followed by two AAA+ ATPase modules (D1 and D2) with conserved Walker A and B motifs. The D1 ATPase module is interrupted by a helical M-domain that projects upward toward the NTD. ClpC enzymes additionally possess an LGF loop that makes stabilizing contacts with the ClpP2 ring of the peptidase. (C) Sequence alignment of select ClpC orthologs from Actinomycetota shows strong conservation of Walker A and B motifs. Notably, a subset of sequences possesses a short M-domain and distinct LGF loop regions, with or without identifiable LGF-like motifs.CC BY · PubMed Central
Figure 2 BLAST analysis of ClpC/B/A orthologs. (A) Actinomycetota ClpC orthologs were compared to Mycolicibacterium smegmatis ClpB (UniProt ID: A0QQF0_MYCS2) and ClpC1 (CLPC1_MYCS2) reference sequences. The x axis represents the BLAST bit-score resulting from alignment of dataset sequences with the ClpC1 reference, while the y axis represents the BLAST bit-score associated with alignment to the ClpB reference. The density of hits in a region of the plot is shown as a heatmap. Typical ClpC orthologs cluster within the purple box, typical ClpB orthologs within the pink box, and atypical sequences with intermediate features cluster within the blue box. (B) Proteobacterial ClpA orthologs were compared to E. coli ClpA (CLPA_ECOLI) and ClpB (CLPB_ECOLI) references as in A. Conventional ClpA sequences lie within the orange box, conventional ClpB sequences lie within the green box, and orthologs of ClpV, a translocase associated with Type IV secretion systems, occupy the cyan box.
Figure 2 BLAST analysis of ClpC/B/A orthologs. (A) Actinomycetota ClpC orthologs were compared to Mycolicibacterium smegmatis ClpB (UniProt ID: A0QQF0_MYCS2) and ClpC1 (CLPC1_MYCS2) reference sequences. The x axis represents the BLAST bit-score resulting from alignment of dataset sequences with the ClpC1 reference, while the y axis represents the BLAST bit-score associated with alignment to the ClpB reference. The density of hits in a region of the plot is shown as a heatmap. Typical ClpC orthologs cluster within the purple box, typical ClpB orthologs within the pink box, and atypical sequences with intermediate features cluster within the blue box. (B) Proteobacterial ClpA orthologs were compared to E. coli ClpA (CLPA_ECOLI) and ClpB (CLPB_ECOLI) references as in A. Conventional ClpA sequences lie within the orange box, conventional ClpB sequences lie within the green box, and orthologs of ClpV, a translocase associated with Type IV secretion systems, occupy the cyan box.CC BY · PubMed Central
Figure 3 Sequence abundance and variability within orders. For each category of Clp unfoldases, sequences are grouped by taxonomic order and the BLAST bit-score calculated against M. smegmatis ClpB is plotted on the y axis.
Figure 3 Sequence abundance and variability within orders. For each category of Clp unfoldases, sequences are grouped by taxonomic order and the BLAST bit-score calculated against M. smegmatis ClpB is plotted on the y axis.CC BY · PubMed Central
Figure 4 Phylogenetic analysis of ClpC paralogs. Distance methods were used to generate a phylogenetic tree of ~ 250 representative sequences of Actinomycetota ClpB (orange), ClpC (violet), ClpIa (blue), and ClpIb (green). Leaves are labeled with UniProt accession IDs.
Figure 4 Phylogenetic analysis of ClpC paralogs. Distance methods were used to generate a phylogenetic tree of ~ 250 representative sequences of Actinomycetota ClpB (orange), ClpC (violet), ClpIa (blue), and ClpIb (green). Leaves are labeled with UniProt accession IDs.CC BY · PubMed Central
Figure 5 Comparison of sequence conservation patterns. Multiple sequence alignments of Clp unfoldase orthologs are shown as bitmaps with each residue represented as a single color-coded pixel. Residues are colored according to their similarity to the overall amino acid frequency at that alignment position. Pairs of sequence alignments are cross-aligned. A cross-comparison heatmap illustrates the similarity in residue frequency at each position in the top and bottom alignments: (A) ClpB is aligned to ClpC, (B) ClpB to ClpI, (C) ClpIa to ClpIb, and (D) ClpC to ClpI. Strong conservation within or across alignments appears blue; low conservation appears red.
Figure 5 Comparison of sequence conservation patterns. Multiple sequence alignments of Clp unfoldase orthologs are shown as bitmaps with each residue represented as a single color-coded pixel. Residues are colored according to their similarity to the overall amino acid frequency at that alignment position. Pairs of sequence alignments are cross-aligned. A cross-comparison heatmap illustrates the similarity in residue frequency at each position in the top and bottom alignments: (A) ClpB is aligned to ClpC, (B) ClpB to ClpI, (C) ClpIa to ClpIb, and (D) ClpC to ClpI. Strong conservation within or across alignments appears blue; low conservation appears red.CC BY · PubMed Central
Figure 6 Surface conservation of Clp unfoldase NTDs. (A) ClpB sequence conservation was projected onto the NTD surface from the AlphaFold model AF-P9WPD1-F1 of M. tuberculosis ClpB. (B) ClpC sequence conservation was projected onto the surface of the crystal structure M. tuberculosis ClpC1 NTD (PDB ID: 3WDB). Phosphoarginine, shown as ball-and-sticks, was modeled into conserved binding sties (Ogbonna et al., 2022) as observed in the B. subtilis NTD, PDB ID: 5HBN. (C) ClpI sequence conservation was projected onto the NTD from the AlphaFold model AF-A0A4P6UA74-F1 of Rhodococcus sp. ABRD24 ClpIa.
Figure 6 Surface conservation of Clp unfoldase NTDs. (A) ClpB sequence conservation was projected onto the NTD surface from the AlphaFold model AF-P9WPD1-F1 of M. tuberculosis ClpB. (B) ClpC sequence conservation was projected onto the surface of the crystal structure M. tuberculosis ClpC1 NTD (PDB ID: 3WDB). Phosphoarginine, shown as ball-and-sticks, was modeled into conserved binding sties (Ogbonna et al., 2022) as observed in the B. subtilis NTD, PDB ID: 5HBN. (C) ClpI sequence conservation was projected onto the NTD from the AlphaFold model AF-A0A4P6UA74-F1 of Rhodococcus sp. ABRD24 ClpIa.CC BY · PubMed Central

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  1. Van Rossum, 2009, Python 3 Reference Manual

Metadata

Authors
Jiang, J., & Schmitz, K. R.
Affiliations
University of Delaware (US)
Corresponding author
Schmitz, Karl R.
ORCID
Schmitz, Karl R.
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2023-04-17
Volume
14
Pages
1161764-1161764
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2023.1161764
PMC
PMC10149685
OpenAlex
W4366208117
Cited by
3
References
79
Citations by year
2026: 1 · 2024: 2
FWCI
0.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Microbiology (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
National Institute of General Medical Sciences (P20GM104316)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
Keywords
actinobacteria, ClpB, ClpC, protein quality control, M-domain, protein disaggregation

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