Comparative Analysis and Ancestral Sequence Reconstruction of Bacterial Sortase Family Proteins Generates Functional Ancestral Mutants with Different Sequence Specificities

Abstract

Gram-positive bacteria are some of the earliest known life forms, diverging from gram-negative bacteria 2 billion years ago. These organisms utilize sortase enzymes to attach proteins to their peptidoglycan cell wall, a structural feature that distinguishes the two types of bacteria. The transpeptidase activity of sortases make them an important tool in protein engineering applications, e.g., in sortase-mediated ligations or sortagging. However, due to relatively low catalytic efficiency, there are ongoing efforts to create better sortase variants for these uses. Here, we use bioinformatics tools, principal component analysis and ancestral sequence reconstruction, in combination with protein biochemistry, to analyze natural sequence variation in these enzymes. Principal component analysis on the sortase superfamily distinguishes previously described classes and identifies regions of relatively high sequence variation in structurally-conserved loops within each sortase family, including those near the active site. Using ancestral sequence reconstruction, we determined sequences of ancestral Staphylococcus and Streptococcus Class A sortase proteins. Enzyme assays revealed that the ancestral Streptococcus enzyme is relatively active and shares similar sequence variation with other Class A Streptococcus sortases. Taken together, we highlight how natural sequence variation can be utilized to investigate this important protein family, arguing that these and similar techniques may be used to discover or design sortases with increased catalytic efficiency and/or selectivity for sortase-mediated ligation experiments.

Biochemical characterization ofBacillus anthracissortase B: Use in sortase-mediated ligation and substrate recognition dependent on residues beyond the canonical pentapeptide binding motif for sortase enzymes

“…60 In addition, biochemical and structural studies by ourselves in recent years investigated the structurally-conserved b7-b8 loop of sortases, specifically amongst Streptococcus SrtA proteins, finding that residues in this loop can have a major impact on the specificity and activity of a given enzyme. 13,17,41,42,53 Here, we chose to turn our attention to the understudied class B sortases, which had previously been shown to have relatively poor activity as compared to class A sortases from the same organisms. 24,26,30,37 Based on initial experiments with 4 enzymes, we chose Bacillus anthracis SrtB to use as a model enzyme to investigate general properties of this sortase class and to test if our previous approach of engineering b7-b8 loop chimeras would elucidate a better understanding of class B sortases.…”

“…However, >10,000 sortase sequences have been identified from over 1,000 bacterial species, which are generally grouped into six classes, A-F, based on primary sequence. [12][13][14] The sortase catalytic mechanism proceeds in a number of distinct steps via the action of a conserved triad of residues (His-Cys-Arg), with recent work also suggesting a key role for a conserved Thr residue immediately preceding the active site Cys. 1,[15][16][17][18] Class A sortases generally recognize and anchor proteins containing a LPXTG motif within their Cell Wall Sorting Signal (CWSS), where X=any amino acid and Leu=P4, Pro=P3, X=P2, T=P1, and G=P1'.…”

A unique binding mode of P1′ Leu-containing target sequences for Streptococcus pyogenes sortase A results in alternative cleavage

“…The purification protocols were also those used previously. 5,23,25,28 Briefly, following induction by IPTG and overexpression, the cells were harvested in lysis buffer [0.05 M Tris pH 7.5, 0.15 M NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA)], lysed via sonication, and clarified using centrifugation, followed by filtration of the supernatant. Initial purification was conducted via immobilized metal affinity chromatography (IMAC) using a 5 mL HisTrap HP column (Cytiva), with wash [0.05 M tris pH 7.5, 0.15 M NaCl, 0.02 M imidazole, 0.001 M TCEP] and elution [wash buffer with 0.3 M imidazole] buffers.…”

Additive energetic contributions of multiple peptide positions determine the relative promiscuity of viral and human sequences for PDZ domain targets

“…Briefly, all PDZ domains were recombinantly expressed from pET vectors (pET16b for CAL, N1P1, N2P2, and TIP-1, pET28a(+) for all others) in Escherichia coli (BL21(DE3) RIL for CAL and TIP-1, BL21(DE3) for all others), and purified using protein chromatography, using identical or similar protocols as those previously described. 34, 36, 41, 51, 71 Most PDZ domains included His 10 (CAL, N1P1, N2P2, and TIP-1) or His 6 (all others) sequences and a HRV 3C (CAL, N1P1, N2P2, and TIP-1) or TEV (all others) protease cleavage site. MAGI1-2 and PTPN3 were additionally expressed as N-terminal SUMO-fusion proteins.…”