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

Chungyu Chang, Brendan R. Amer, Jerzy Osipiuk, Scott A. McConnell, I-Hsiu Huang, Van Hsieh, Janine Fu, Hong H. Nguyen, John Muroski, Erika Flores, Rachel R.Ogorzalek Loo, Joseph A. Loo, John A. Putkey, Andrzej Joachimiak, Asis Das, Robert T. Clubb, Hung Ton-That

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2 Citations (Scopus)

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 (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 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 crosslinking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M 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.

Original languageEnglish
Pages (from-to)E5477-E5486
JournalProceedings of the National Academy of Sciences of the United States of America
Volume115
Issue number24
DOIs
Publication statusPublished - 2018 Jun 12

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Fimbriae Proteins
Polymerization
Protein Subunits
Ligation
Enzymes
Peptidyl Transferases
Protein Engineering
Gram-Positive Bacteria
Biotechnology
In Vitro Techniques
Alanine
Mass Spectrometry
Electron Microscopy
Polymers
Mutation
Membranes

All Science Journal Classification (ASJC) codes

  • General

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Chang, Chungyu ; Amer, Brendan R. ; Osipiuk, Jerzy ; McConnell, Scott A. ; Huang, I-Hsiu ; Hsieh, Van ; Fu, Janine ; Nguyen, Hong H. ; Muroski, John ; Flores, Erika ; Loo, Rachel R.Ogorzalek ; 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. In: Proceedings of the National Academy of Sciences of the United States of America. 2018 ; Vol. 115, No. 24. pp. E5477-E5486.
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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 (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 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 crosslinking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M 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.",
author = "Chungyu Chang and Amer, {Brendan R.} and Jerzy Osipiuk and McConnell, {Scott A.} and I-Hsiu Huang and Van Hsieh and Janine Fu and Nguyen, {Hong H.} and John Muroski and Erika Flores and Loo, {Rachel R.Ogorzalek} and Loo, {Joseph A.} and Putkey, {John A.} and Andrzej Joachimiak and Asis Das and Clubb, {Robert T.} and Hung Ton-That",
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Chang, C, Amer, BR, Osipiuk, J, McConnell, SA, Huang, I-H, Hsieh, V, Fu, J, Nguyen, HH, Muroski, J, Flores, E, Loo, RRO, Loo, JA, Putkey, JA, Joachimiak, A, Das, A, Clubb, RT & Ton-That, H 2018, '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, vol. 115, no. 24, pp. E5477-E5486. https://doi.org/10.1073/pnas.1800954115

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; Loo, Rachel R.Ogorzalek; Loo, Joseph A.; Putkey, John A.; Joachimiak, Andrzej; Das, Asis; Clubb, Robert T.; Ton-That, Hung.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, No. 24, 12.06.2018, p. E5477-E5486.

Research output: Contribution to journalArticle

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T1 - In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

AU - Chang, Chungyu

AU - Amer, Brendan R.

AU - Osipiuk, Jerzy

AU - McConnell, Scott A.

AU - Huang, I-Hsiu

AU - Hsieh, Van

AU - Fu, Janine

AU - Nguyen, Hong H.

AU - Muroski, John

AU - Flores, Erika

AU - Loo, Rachel R.Ogorzalek

AU - Loo, Joseph A.

AU - Putkey, John A.

AU - Joachimiak, Andrzej

AU - Das, Asis

AU - Clubb, Robert T.

AU - Ton-That, Hung

PY - 2018/6/12

Y1 - 2018/6/12

N2 - 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 (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 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 crosslinking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M 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.

AB - 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 (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 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 crosslinking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M 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.

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U2 - 10.1073/pnas.1800954115

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