Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference

Abstract

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from cleaves LPTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPTG-containing substrates and Gly-terminated nucleophiles occurs as well as in the presence of Ca and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPTG specificity restriction, and Ca requirement (particularly for applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LATG recognition motif over LPTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Anti-SARS-CoV-1 and −2 nanobody engineering towards avidity-inspired therapeutics

“…The “X” is preferentially aspartic acid (D), glutamic acid (E), alanine (A), asparagine (N), glutamine (Q) or lysine (K). The transpeptidation mechanism is mostly Ca 2+ dependent, but there is a Ca 2+ independent variant available [167] . In view of this, several applications associated with protein multi-functionalization [161] , protein cyclization [168] , protein lipidation [169] , and cell-surface labelling [170] , [171] have been attempted.…”

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“…Sortase E would act as the housekeeping sorting enzyme in T. pyogenes , as sortases A and E are never found in the same organism. Sortases E recognize substrates containing the LAXTG motif [ 35 , 36 ]. However, there is no published evidence that they can act on LSXTG, although it cannot be discarded.…”

Challenges in the use of sortase and other peptide ligases for site-specific protein modification

“…10 Sortases can be divided into six distinct families (A-F) on the basis of structure and substrate dependence. [11][12][13] The sortase A family is best characterised, and members of this family are present in almost all Gram-positive bacteria. 14 This class of sortase enzymes performs a housekeeping role in the bacterial cell, anchoring a large number of functionally-distinct proteins to the cell wall.…”