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

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

doi:10.1073/pnas.1800954115

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

Journal Article · Tue May 29 00:00:00 EDT 2018 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1800954115· OSTI ID:1464631

Chang, Chungyu ^[1]; Amer, Brendan R. ^[2]; Osipiuk, Jerzy ^[3]; McConnell, Scott A. ^[2]; Huang, I-Hsiu ^[4]; Hsieh, Van ^[2]; Fu, Janine ^[2]; Nguyen, Hong H. ^[2]; Muroski, John ^[2]; Flores, Erika ^[1]; ^[2]; Loo, Joseph A. ^[2]; Putkey, John A. ^[1]; Joachimiak, Andrzej ^[3]; Das, Asis ^[5]; Clubb, Robert T. ^[2]; ^[1]

Univ. of Texas Health Science Center, Houston, TX (United States)
Univ. of California, Los Angeles, CA (United States). Dept. of Chemistry and Biochemistry and UCLA-DOE Inst. for Genomics and Proteomics
Argonne National Lab. (ANL), Argonne, IL (United States); Univ. of Chicago, IL (United States)
National Cheng Kung Univ., Tainan (Taiwan)
Univ. of Connecticut Health Center, Farmington, CT (United States)

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 (SrtA^2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Further, 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: Center for Structural Genomics of Infectious Diseases (CSGID); National Institute of Allergy and Infectious Diseases (NIAID); National Institute of Dental and Craniofacial Research (NIDCR); National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); USDHHS

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1464631

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 24 Vol. 115; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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

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