Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase

Chungyu Chang; HyLam Ton-That; Jerzy Osipiuk; Andrzej Joachimiak; Asis Das; Hung Ton-That

doi:10.1016/j.jbc.2024.107329

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase

J Biol Chem. 2024 Apr 26:107329. doi: 10.1016/j.jbc.2024.107329. Online ahead of print.

Authors

Chungyu Chang¹, HyLam Ton-That², Jerzy Osipiuk³, Andrzej Joachimiak⁴, Asis Das⁵, Hung Ton-That⁶

Affiliations

¹ Division of Oral & Systemic Health Sciences, School of Dentistry, University of California, Los Angeles, California, USA.
² Department of Chemistry, University of California, Irvine, Irvine, California, USA.
³ Center for Structural Biology of Infectious Diseases (CSBID), Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA; Structural Biology Center, Argonne National Laboratory, Lemont, IL, USA.
⁴ Center for Structural Biology of Infectious Diseases (CSBID), Consortium for Advanced Science and Engineering, University of Chicago, Chicago, IL, USA; Structural Biology Center, Argonne National Laboratory, Lemont, IL, USA; Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA.
⁵ Department of Medicine, Neag Comprehensive Cancer Center, University of Connecticut Health Center, Farmington, CT, USA.
⁶ Division of Oral & Systemic Health Sciences, School of Dentistry, University of California, Los Angeles, California, USA; Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, California, USA. Electronic address: htonthat@dentistry.ucla.edu.

PMID: 38679328
DOI: 10.1016/j.jbc.2024.107329

Abstract

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

Keywords: Actinomyces oris; cell wall anchoring; pilus assembly; secretion; signal peptide sequence; sortase.

Grants and funding

R01 DE017382/DE/NIDCR NIH HHS/United States