Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

Abstract

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Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition

“…In general, the binding site for the P1' position in spySrtA does not appear to be particularly selective, which is consistent with our previous work on S. pneumoniae SrtA (9,15). Due to the observed similarities in these Streptococcus SrtA proteins, as well as our previous work investigating the 7-8 loop in these proteins, we hypothesize that spySrtA is also non-selective at this position and can accommodate a wide variety of P1' amino acids (9,23). Overall, the observed location for the P1' Ser, as well as the adjacent P1 Thr, renders the LPATS peptide ideally positioned for nucleophilic attack by the catalytic cysteine residue.…”

“…For Class A sortases, the general consensus sequence, which is found within the cell wall sorting signal (CWSS), includes a pentapeptide motif, Leu-Pro-X-Thr-Gly (or LPXTG), where X = any amino acid (4). Positions are defined with respect to the location of the cleavage site between the threonine and glycine residues, with P1' = Gly, P1 = Thr, P2 = X, P3 = Pro, and P4 = Leu (9). For proteinanchoring to the bacterial cell surface, the nucleophile in the second step of the reaction J o u r n a l P r e -p r o o f mechanism is the cell-wall precursor lipid II, thus allowing for incorporation of the protein into the growing peptidoglycan layer (10).…”

“…These structures have revealed that sortases share a conserved core antiparallel 8-stranded -barrel structure, termed the sortase fold (8,11). This was first identified in the Staphylococcus aureus Class A sortase (saSrtA) structure and is consistently found in wild-type and chimeric sortase enzymes (9,11,12). As of early 2022, there were over 65 structures of sortases in the Protein Data Bank, including from all 6 classes and SrtA structures from 10 different organisms.…”

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

“…Piper et al 137 have reported the effect of mutation in the β7–β8 loop region on the activity of SpSrtA. As discussed above, this enzyme is able to act on an LPX 1 TX 2 sequence where X 2 is Ala, Ser or Gly but the activity is otherwise relatively low.…”

“…Wojcik et al had previously shown that grafting the β7–β8 loop from SaSrtA into SpSrtA generated an LPXTG specific enzyme 138 however Piper et al investigated the effect of creating SpSrtA chimeras where the β7–β8 loop from SrtA from a variety of other Gram-positive bacteria was grafted into the SpSrtA backbone. 137 Many of these chimeras such as SpSrtA faecilis (in which three amino acid) substitutions were able to catalyse reaction of LPX 1 TX 2 peptide substrates faster than SpSrtA. Most interestingly, some of these enzymes were also able to act on a wider range of amino-acid nucleophiles including SpSrtA faecilis which was shown to act on a LPXTV sortase recognition sequence.…”