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Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation
Open accessPreprint — published as 10.1073/pnas.2201141119 · Paper page
Abstract
Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus , acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site, and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein. SIGNIFICANCE STATEMENT: The Gram-positive pathogen Staphylococcus aureus is a leading cause of antibiotic resistance-associated death in the United States. Many antibiotics used to treat S. aureus , including the beta-lactams, target biogenesis of the essential peptidoglycan (PG) cell wall. Some hydrolases play important roles in cell wall construction and are potential antibiotic targets. The amidase LytH, which requires a protein partner, ActH, for activity, is one such hydrolase. Here, we uncover how the extracellular domain of ActH binds to LytH to stabilize metals in the active site for catalysis. This work advances our understanding of how hydrolase activity is controlled to contribute productively to cell wall synthesis.
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Highly accurate protein structure prediction with AlphaFold
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Metadata
- Authors
- Page, J. E., Skiba, M. A., Do, T., Kruse, A. C., & Walker, S.
- Affiliations
- Harvard University (US)
- Corresponding author
- Walker, Suzanne
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- openRxiv
- Published
- 2022-01-22
- Volume
- 119
- Issue
- 26
- Pages
- e2201141119-e2201141119
- ISSN
- 0027-8424 (ISSN-L)
- PMC
- PMC9245657
- OpenAlex
- W4283326347
- Cited by
- 12
- References
- 1
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 6 · 2023: 3
- FWCI
- 1.66
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- cc-by-nc-nd
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- National Science Foundation (DE-AC02–06CH11357, 1144152)
- U.S. Department of Energy (DE‐ AC02‐06CH11357, Contract No. DE-AC02-06CH11357., AC02‐06CH11357, 06CH11357, No. DE-AC02-06CH11357, DE-AC02-)
- Helen Hay Whitney Foundation
- Dartmouth College
- National Institutes of Health (DGE1144152, ACB-12002, 1s10od012289-01a1, DE-AC02–06CH11357, AGM-12006)
- Office of Science (Contract No. DE-AC02-06CH11357, AC02‐06CH11357, DE-AC02, No. DE-AC02-06CH11357., 06CH11357, DE-AC02-06CH11357)
- National Cancer Institute (DE-AC02-06CH11357, ACB12002, AGM-12006)
- National Institute of General Medical Sciences (R01GM076710, S10 OD012289, T32GM007753, DE-AC02-06CH11357, AGM-12006, ACB-12002, GM007753, P30GM138396, AGM-12006, P30GM138396)
- National Institute of Allergy and Infectious Diseases (F30AI156972, R01AI139011, R01AI148752)
- Argonne National Laboratory (DE- AC02-06-CH11357, DE-AC02, AC02–06CH11357, No. DE-AC02-06CH11357, 06CH11357, Contract No. DE-AC02-06CH11357., S10 OD012289)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
- Keywords
- ACTH, peptidoglycan hydrolase, Staphylococcus aureus, tetratricopeptide repeats, protein-protein complex structure, cell wall remodeling, cell size control, antibiotic targets
- MeSH terms
- Protein Domains, Bacterial Proteins, Cell Wall, Enzyme Activation, Enzyme Stability, Membrane Proteins, Metals, N-Acetylmuramoyl-L-alanine Amidase, Peptidoglycan, Protein Binding
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