In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

Proceedings of the National Academy of Sciences of the United States of America
Chungyu ChangHung Ton-That

Abstract

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA har...Continue Reading

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Citations

May 24, 2020·Scientific Reports·Jason E GosschalkRobert T Clubb
Dec 9, 2020·Chembiochem : a European Journal of Chemical Biology·Christian Freund, Dirk Schwarzer
Mar 17, 2021·Proceedings of the National Academy of Sciences of the United States of America·Scott A McConnellRobert T Clubb
Jun 6, 2020·Trends in Microbiology·Nicholas A RamirezHung Ton-That
Feb 10, 2019·Microbiology Spectrum·Olaf Schneewind, Dominique Missiakas
Jun 22, 2018·Journal of the American Chemical Society·Scott A McConnellRobert T Clubb

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