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

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Gram-positive bacteria utilize class C sortase enzymes to assemble surface-displayed, covalently cross-linked pilus polymers.
• These enzymes catalyze a two-step protein ligation, involving LPXTG motif cleavage and subsequent isopeptide bond formation.
• The specific enzyme determinants enabling pilus construction by class C sortases remain largely uncharacterized.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying pilus assembly by class C sortases.
• To identify key determinants within sortase enzymes responsible for pilus-specific polymerization.
• To establish an in vitro system for studying pilus assembly and its biotechnological applications.

Main Methods:

• High-resolution crystal structures of corynebacterial pilus-specific sortase A (SrtA) and its activating mutant (SrtA2M).
• In vitro reconstitution of pilus polymerization using SrtA2M and the major pilin SpaA.
• Mass spectrometry, electron microscopy, and biochemical assays to characterize the synthesized pilus fibers and isopeptide bonds.
• Structural modeling of polymerization intermediates and site-directed mutagenesis of conserved motifs.

Main Results:

• SrtA2M successfully reconstituted in vitro polymerization of SpaA, forming pili with correct Lys-Thr isopeptide bonds via a thioacyl intermediate.
• Structural modeling revealed SrtA2M positioned between SpaA domains, highlighting interactions crucial for polymerization.
• A conserved TP(Y/L)XIN(S/T)H motif following the catalytic cysteine was identified; alanine substitutions abrogated cross-linking but not LPXTG cleavage.
• SrtA2M demonstrated the ability to terminate pilus polymerization and ligate isolated SpaA domains in vitro.

Conclusions:

• The study identifies critical determinants within class C sortases that enable pilus polymerization.
• The developed in vitro system provides a versatile platform for studying pilus assembly and engineering.
• These findings have significant implications for biotechnology, particularly in protein engineering and bio-conjugation applications.