6BWE

Sortase A from Corynebacterium diphtheriae, lid mutant

Summary for 6BWE

• Entry DOI: 10.2210/pdb6bwe/pdb
• Related: 5k9a
• Descriptor: Putative fimbrial associated sortase-like protein (2 entities in total)
• Functional Keywords: sortase, structural genomics, idp58949, center for structural genomics of infectious diseases, csgid, hydrolase
• Biological source: Corynebacterium diphtheriae (strain ATCC 700971 / NCTC 13129 / Biotype gravis)
• Total number of polymer chains: 2
• Total formula weight: 46745.86

Authors

Osipiuk, J.,Chang, C.,Huang, I.H.,Ton-That, H.,Anderson, W.F.,Joachimiak, A.,Center for Structural Genomics of Infectious Diseases (CSGID) (deposition date: 2017-12-14, release date: 2017-12-27, Last modification date: 2024-10-23)

Primary citation

Chang, C.,Amer, B.R.,Osipiuk, J.,McConnell, S.A.,Huang, I.H.,Hsieh, V.,Fu, J.,Nguyen, H.H.,Muroski, J.,Flores, E.,Ogorzalek Loo, R.R.,Loo, J.A.,Putkey, J.A.,Joachimiak, A.,Das, A.,Clubb, R.T.,Ton-That, H.
In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking.
Proc. Natl. Acad. Sci. U.S.A., 115:E5477-E5486, 2018

PubMed 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 (SrtA), 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 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 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 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.

PubMed: 29844180
DOI: 10.1073/pnas.1800954115

Experimental method

X-RAY DIFFRACTION (1.85 Å)