Sortases play a crucial role in anchoring surface proteins in Gram-positive bacteria, serving as potential targets for novel antibiotics and tools for protein engineering. While Class A sortases are well-studied for recognizing LPXTG motifs, Class B sortases, like Bacillus anthracis SrtB, target distinct sequences such as NPXTG or NPQTN. In this study, through biochemistry, structural analyses, and computational simulations, we delve into the substrate recognition mechanisms of baSrtB, revealing critical residues beyond the pentapeptide motif and showcasing the potential for enhancing baSrtB activity through mutagenesis. Our findings shed light on the substrate-binding specificity of SrtB enzymes and their potential in protein engineering applications.
Surface proteins are pivotal for bacterial functions, especially in pathogens, where they often serve as virulence factors. Sortases, like SrtA from Staphylococcus aureus, mediate the attachment of these proteins to the cell wall. With over 10,000 sortase sequences identified across various bacterial species, Class A sortases have been extensively studied for their LPXTG recognition motif within the Cell Wall Sorting Signal. In contrast, Class B sortases, exemplified by baSrtB targeting NPKTG, remain less understood. Our study focuses on unraveling the substrate recognition mechanisms of baSrtB to expand the knowledge of SrtB enzymes and their potential applications in protein engineering.
Structural modeling predicts a plausible enzyme-substrate complex for baSrtB, validated through mutagenesis studies targeting binding cleft residues. We discovered that residues flanking the pentapeptide recognition motif are crucial for baSrtB activity, with chimeric proteins revealing mutations that enhance baSrtB activity by approximately 4-fold. Moreover, our demonstration of sortase-mediated ligation using a baSrtB enzyme variant underscores the feasibility of leveraging SrtB enzymes in protein engineering applications. These findings not only enhance our understanding of SrtB-target binding but also highlight the potential for modifying SrtB enzymes to broaden the scope of suitable substrates and applications in sortase-mediated ligation.
Through activity assays of multiple SrtB enzymes, we identified baSrtB as the most active, paving the way for in-depth investigations into its enzymatic properties. Further analysis revealed the pH dependence, preferred nucleophiles, and position-specific recognition of the substrate by baSrtB. Our exploration of extended sequence motifs elucidated the importance of specific amino acids outside the pentapeptide motif for baSrtB activity, showcasing the intricate substrate specificity of SrtB enzymes.
To delve deeper into the stereochemistry of substrate binding, we employed structural modeling and molecular dynamics simulations to investigate the baIsdC-baSrtB complex. Our models provided insights into potential interactions that impact baSrtB activity, highlighting specific contacts at different peptide positions crucial for substrate recognition. Additionally, our molecular dynamics simulations confirmed the stability of the baSrtB protein-peptide complex, shedding light on the dynamics of substrate binding and the stability of the recognition motif within the binding pocket.
In conclusion, our comprehensive study unravels the substrate recognition mechanisms of Bacillus anthracis Sortase B beyond the canonical pentapeptide binding motif, offering valuable insights into the enzymology of SrtB enzymes. By elucidating critical residues and enhancing baSrtB activity through mutagenesis, we pave the way for leveraging SrtB enzymes in protein engineering applications. This research not only expands our understanding of SrtB enzymes but also underscores their potential as versatile tools in sortase-mediated ligation and protein engineering endeavors.
Tags: chromatography, protein purification, mass spectrometry, upstream
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