Sorting of LPXTG peptides by archetypal sortase A: role of invariant substrate residues in modulating the enzyme dynamics and conformational signature of a productive substrate

Tora Biswas, Vijaykumar S Pawale, Devapriya Choudhury, Rajendra P Roy
Biochemistry 2014 April 22, 53 (15): 2515-24
Transpeptidase sortase catalyzes the covalent anchoring of surface proteins to the cell wall in Gram-positive bacteria. Sortase A (SrtA) of Staphylococcus aureus is a prototype enzyme and considered a bona fide drug target because several substrate proteins are virulence-related and implicated in pathogenesis. Besides, SrtA also works as a versatile tool in protein engineering. Surface proteins destined for cell wall anchoring contain a LPXTG sequence located in their C-terminus which serves as a substrate recognition motif for SrtA. Recent studies have implicated substrate-induced conformational dynamics in SrtA. In the present work, we have explored the roles of invariant Leu and Pro residues of the substrate in modulating the enzyme dynamics with a view to understand the selection process of a catalytically competent substrate. Overall results of molecular dynamics simulations and experiments carried out with noncanonical substrates and site-directed mutagenesis reveal that the kinked conformation due to Pro in LPXTG is obligatory for productive binding but does not per se control the enzyme dynamics. The Leu residue of the substrate appears to play the crucial role of an anchor to the beta6-beta7 loop directing the conformational transition of the enzyme from an "open" to a "closed" state subsequent to which the Pro residue facilitates the consummation of binding through predominant engagement of the loop and catalytic motif residues in hydrophobic interactions. Collectively, our study provides insights about specificity, tolerance, and conformational sorting of substrate by SrtA. These results have important implications in designing newer substrates and inhibitors for this multifaceted enzyme.

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