Biology subjects

Amacher, J.

Publications and source records attributed to Amacher, J..

7 recordsLinked to original sources

bioRxiv2026

Mutation-induced heterogeneity of the β7-β8 loop of the Staphylococcus aureus class A sortase leading to enhanced catalytic efficiency characterized by NMR and enzyme kinetics

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

biochemistry↗

bioRxiv2026

A FRET Ligation Assay using Fluorescent Proteins for Bacterial Sortase Enzymes

Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

biochemistry↗

bioRxiv2025

An interaction between the transmembrane domains of Streptococcus pyogenes sortase A and its endogenous substrate M protein revealed by molecular dynamics simulations

Sortase enzymes are cysteine transpeptidases at the cell surface of gram-positive bacteria. Localized to distinct foci on the cell membrane, class A sortases (SrtAs) recognize a cell wall sorting signal (CWSS), and following cleavage at this specific binding motif, target proteins are ligated to precursors of the growing peptidoglycan layer. This activity of SrtA enzymes is utilized extensively in sortase-mediated ligation (SML) strategies, for a variety of protein engineering applications. Typically, engineered variants of SrtA are used for SML experiments considering the relatively low catalytic efficiency of this enzyme. Understandably, most biochemical studies are conducted with the isolated catalytic domain of SrtA enzymes from various bacteria, and the stereochemistry of the endogenous interaction between SrtA and substrate is not well understood. Here, we used AlphaFold2 to create a model of the full-length SrtA enzyme from Streptococcus pyogenes (spySrtA) with or without either a peptide substrate or a portion of M protein, a cellular target. We ran triplicate 500 ns molecular dynamics simulations for each model embedded in a lipid bilayer, which revealed several stereochemical features of this system. Contact map analyses revealed specific interactions between catalytic domain positions of spySrtA and the lipid bilayer, as well as between the enzyme and M protein residues outside the canonical LPXTG pentapeptide CWSS. We also characterized a potential transmembrane domain interaction between spySrtA and M protein that we predict orients and stabilizes substrate binding. Taken together, these interactions likely increase the catalytic efficiency of the enzyme for its substrates in vivo, and may provide important stereochemical insights for SML uses.

biochemistry↗

bioRxiv2024

The conformation of the nSrc specificity-determining loops in the Src and Abl SH3 domains are modulated by a WX conserved sequence motif

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 quite versatile, 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 the 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. We identified a position that contributes to the nSrc loop conformation, the amino acid immediately C-terminal to 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. The importance of this position for orienting the ligand is supported by analyses of previously deposited SH3 structures, multiple sequence alignment of SH3 domains in the human proteome, and our biochemical data of mutant Src and Abl SH3 domains. Overall, our work uses experimental approaches and structural modeling to better understand SH3 specificity determinants.

biochemistry↗

bioRxiv2024

Biochemical characterization of Bacillus anthracis sortase B: Use in sortase mediated ligation and substrate recognition dependent on residues beyond the canonical pentapeptide binding motif for sortase enzymes

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critical enzymes facilitate the attachment of proteins to the cell wall, and are potential targets for novel antibiotic development, as well as versatile tools in protein engineering applications. Although there are six classes of sortases recognized, class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes recognize the canonical Cell Wall Sorting Signal (CWSS), LPXTG, where X=any amino acid, although work in recent years identified additional promiscuity in multiple positions of this recognition motif. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes have previously been shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues at several positions. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. We also use chimeric proteins to identify a single site that improves baSrtB activity by [~]4-fold and use purified protein substrates to validate sortase-mediated ligation of two proteins using SrtB enzymes for the first time. Taken together, these studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.

biochemistry↗

bioRxiv2023

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

Protein-protein interactions that include recognition of short sequences of amino acids, or peptides, are critical in cellular processes. Protein-peptide interaction surface areas are relatively small and shallow, and there are often overlapping specificities in families of peptide-binding domains. Therefore, dissecting selectivity determinants can be challenging. PDZ domains are an example of a peptide-binding domain located in several intracellular signaling and trafficking pathways, which form interactions critical for the regulation of receptor endocytic trafficking, tight junction formation, organization of supramolecular complexes in neurons, and other biological systems. These domains are also directly targeted by pathogens, and a hallmark of many oncogenic viral proteins is a PDZ-binding motif. However, amidst sequences that target PDZ domains, there is a wide spectrum in relative promiscuity. For example, the viral HPV16 E6 oncoprotein recognizes over double the number of PDZ domain-containing proteins as the cystic fibrosis transmembrane conductance regulator (CFTR) in the cell, despite similar PDZ targeting-sequences and identical motif residues. Here, we determine binding affinities for PDZ domains known to bind either HPV16 E6 alone or both CFTR and HPV16 E6, using peptides matching WT and hybrid sequences. We also use energy minimization to model PDZ-peptide complexes and use sequence analyses to investigate this difference. We find that while the majority of single mutations had a marginal effect on overall affinity, the additive effect on the free energy of binding accurately describes the selectivity observed. Taken together, our results describe how complex and differing PDZ interactomes can be programmed in the cell.

biochemistry↗

bioRxiv2021

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative {beta}7-{beta}8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1 position of the substrate motif, underlined in LPXTG, in contrast to the strict P1 Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of {beta}7-{beta}8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified {beta}7-{beta}8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. We argue that a greater understanding of individual SrtA sequence and structural determinants of target selectivity can facilitate the design or discovery of improved sortagging tools.

biochemistry↗