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Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes
Melody Gao1, D Alex Johnson1, Isabel M Piper1
1Department of Chemistry, Western Washington University, Bellingham, Washington, USA.
Protein Science : a Publication of the Protein Society
|December 23, 2021
Summary
Bacterial sortases (SrtA) show sequence variation affecting protein attachment. New structures reveal how the β7-β8 loop influences substrate selectivity, aiding in developing better protein engineering tools.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Bacterial sortases (SrtA) are surface enzymes crucial for Gram-positive bacteria, involved in cell wall anchoring and protein engineering via sortase-mediated ligations (SML).
- Sequence variation within sortase families, particularly Class A, significantly impacts enzyme activity and substrate recognition.
- Previous work identified β7-β8 loop interactions in Streptococcus pneumoniae SrtA influencing P1' substrate recognition, but a structural model was lacking.
Purpose of the Study:
- To elucidate the structural basis of substrate selectivity in Class A sortases from Streptococcus species.
- To provide structural insights into the role of the β7-β8 loop in modulating SrtA function and specificity.
- To facilitate the design of improved sortagging tools for protein engineering.
Main Methods:
- X-ray crystallography was used to determine the monomeric structure of *S. agalactiae* SrtA and three β7-β8 loop chimeras from *S. pyogenes* and *S. agalactiae* SrtA.
- Biochemical assays were performed to assess the functional impact of identified structural features on enzyme activity and substrate recognition.
Main Results:
- The first monomeric structure of *S. agalactiae* SrtA, including its C-terminus, was determined.
- Structures of three β7-β8 loop chimeras provided insights into loop-mediated interactions.
- Biochemical data corroborated the structural findings, confirming the role of the β7-β8 loop in SrtA substrate selectivity.
Conclusions:
- Structural and biochemical data confirm the importance of β7-β8 loop interactions in determining Class A sortase substrate selectivity.
- Understanding these sequence and structural determinants can guide the development of novel and enhanced sortagging technologies.
- This research advances the field of protein engineering by providing a deeper understanding of sortase enzyme mechanisms.
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