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

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 acylenzyme 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 (SrtA(2M)), 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 SrtA(2M) 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 SrtA(2M) sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavagemore » of LPXTG. These insights and our evidence that SrtA(2M) can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.« less

Authors:
; ; ; ; ; ; ; ; ; ; ORCiD logo; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
U. S. Department of Health and Human Services; National Institutes of Health (NIH) - National Institute of Allergy and Infectious Diseases (NIAID); National Institutes of Health (NIH) - National Institute of General Medical Sciences; National Institutes of Health (NIH) - National Institute of Dental and Craniofacial Research; Computation Institute - Center for Structural Genomics of Infectious Disease
OSTI Identifier:
1464631
DOE Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 115; Journal Issue: 24; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
English
Subject:
Corynebacterium diphtheriae; pilus polymerization; protein ligation; sortase; transpeptidation

Citation Formats

Chang, Chungyu, Amer, Brendan R., Osipiuk, Jerzy, McConnell, Scott A., Huang, I-Hsiu, Hsieh, Van, Fu, Janine, Nguyen, Hong H., Muroski, John, Flores, Erika, Ogorzalek Loo, Rachel R., Loo, Joseph A., Putkey, John A., Joachimiak, Andrzej, Das, Asis, Clubb, Robert T., and Ton-That, Hung. In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking. United States: N. p., 2018. Web. doi:10.1073/pnas.1800954115.
Chang, Chungyu, Amer, Brendan R., Osipiuk, Jerzy, McConnell, Scott A., Huang, I-Hsiu, Hsieh, Van, Fu, Janine, Nguyen, Hong H., Muroski, John, Flores, Erika, Ogorzalek Loo, Rachel R., Loo, Joseph A., Putkey, John A., Joachimiak, Andrzej, Das, Asis, Clubb, Robert T., & Ton-That, Hung. In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking. United States. doi:10.1073/pnas.1800954115.
Chang, Chungyu, Amer, Brendan R., Osipiuk, Jerzy, McConnell, Scott A., Huang, I-Hsiu, Hsieh, Van, Fu, Janine, Nguyen, Hong H., Muroski, John, Flores, Erika, Ogorzalek Loo, Rachel R., Loo, Joseph A., Putkey, John A., Joachimiak, Andrzej, Das, Asis, Clubb, Robert T., and Ton-That, Hung. Tue . "In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking". United States. doi:10.1073/pnas.1800954115.
@article{osti_1464631,
title = {In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking},
author = {Chang, Chungyu and Amer, Brendan R. and Osipiuk, Jerzy and McConnell, Scott A. and Huang, I-Hsiu and Hsieh, Van and Fu, Janine and Nguyen, Hong H. and Muroski, John and Flores, Erika and Ogorzalek Loo, Rachel R. and Loo, Joseph A. and Putkey, John A. and Joachimiak, Andrzej and Das, Asis and Clubb, Robert T. and Ton-That, Hung},
abstractNote = {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 acylenzyme 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 (SrtA(2M)), 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 SrtA(2M) 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 SrtA(2M) sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA(2M) can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.},
doi = {10.1073/pnas.1800954115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 24,
volume = 115,
place = {United States},
year = {2018},
month = {5}
}

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