RT Journal Article
SR Electronic
T1 A second specificity-determining loop in Class A sortases: Biochemical characterization of natural sequence variation in chimeric SrtA enzymes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.27.437355
DO 10.1101/2021.03.27.437355
A1 Isabel M. Piper
A1 Sarah A. Struyvenberg
A1 Jordan D. Valgardson
A1 D. Alex Johnson
A1 Melody Gao
A1 Katherine Johnston
A1 Justin E. Svendsen
A1 Hanna M. Kodama
A1 Kelli L. Hvorecny
A1 John M. Antos
A1 Jeanine F. Amacher
YR 2021
UL http://biorxiv.org/content/early/2021/03/28/2021.03.27.437355.abstract
AB Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice for SML reactions. However, the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. Here, we use principal component analysis to identify a structurally conserved loop with a high degree of variability in all classes of sortases. We investigate the contribution of this β7-β8 loop, located between the catalytic cysteine and arginine residues and immediately adjacent to the target binding cleft, by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of Class A sortases from 8 species engineered into the SrtA sequence from Streptococcus pneumoniae (spSrtA). While some of our chimeric enzymes mimic the activity and selectivity of the wild-type protein from which the loop sequence is derived (e.g., that of saSrtA), others result in chimeric spSrtA enzymes able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural, and sequence analyses, we identify three interactions facilitated by β7-β8 loop residues that appear to be broadly characteristic of Class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.Competing Interest StatementThe authors have declared no competing interest.