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Abstract Bacterial sortases are a family of cysteine transpeptidases in Gram‐positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase‐mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition byStaphylococcus aureusSrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β‐carbon by W194, a tryptophan located in the β7‐β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET‐based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D‐Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D‐Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase‐mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies. 

Cellular signaling networks are modulated by multiple protein-protein interaction domains that coordinate extracellular inputs and processes to regulate cellular processes. Several of these domains recognize short linear motifs, or SLiMs, which are often highly conserved and are closely regulated. One such domain, the Src homology 3 (SH3) domain, typically recognizes proline-rich SLiMs and is one of the most abundant SLiM-binding domains in the human proteome. These domains are often described as quiteversatile, and indeed, SH3 domains can bind ligands in opposite orientations dependent on target sequence. Furthermore, recent work has identified diverse modes of binding for SH3 domains and a wide variety of sequence motifs that are recognized by various domains. Specificity is often attributed to the RT and nSrc loops near the peptide-binding cleft in this domain family, particularly for Class I binding, which is defined as RT and nSrc loop interactions with the N-terminus of the ligand. Here, we used the Src and Abl SH3 domains as a model to further investigate the role of the RT and nSrc loops in SH3 specificity. We created chimeric domains with both the RT and nSrc loop sequences swapped between these SH3 domains, and used fluorescence anisotropy assays to test how relative binding affinities were affected for Src SH3- and Abl SH3-specific ligands. We also used Alphafold–Multimer to model our SH3:peptide complexes in combination with molecular dynamics simulations. We identified a position that contributes to the nSrc loop conformation in Src SH3, the amino acid immediately following a highly conserved Trp that creates a hydrophobic pocket critical for SH3 ligand recognition. We defined this as the WX motif, where X = Trp for Src and Cys for Abl. A broad importance of this position for modulating nSrc loop conformation in SH3 domains is suggested by analyses of previously deposited SH3 structures, multiple sequence alignment of SH3 domains in the human proteome, and our biochemical and computational data of mutant Src and Abl SH3 domains. Overall, our work uses experimental approaches and structural modeling to better understand specificity determinants in SH3 domains. 

Vogelet al.reveals the stereochemical basis of alternative substrate cleavage byS. pyogenesSrtA for ligands with a P1′ Leu residue. The substrate adopts puckered alternative binding, whereby cleavage occurs between the P1′ and P2′ positions. 

Abstract High school science fairs provide an exceptional opportunity for students to gain experience with scientific research, and participation has positive outcomes with respect to chosen careers in the sciences. However, it can be challenging to engage high school students in university‐level research outside of formal internship programs. Here, we describe an experimental pipeline for a computational structural biology project that engages high school students. Students are involved at every step of the investigation and utilize freely available software to dock inhibitors onto protein homologues, and then analyze the resulting complexes. Bacterial sortases are transpeptidases on the cell surface of Gram‐positive bacteria and are a potential target for the development of antibiotics. Students modeled inhibitors bound to sortases from several organisms, asking questions about affinity and selectivity. Their project was ranked in the top 10% at both regional and state science fairs. This project design is easily adaptable to countless other protein systems and provides a pipeline for collaborative high school student/university professor inquiry. 

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. 

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 μM affinity, a value commonly considered the threshold for cellular PDZ–peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.