Sequence variation in the β7–β8 loop of bacterial class A sortase enzymes alters substrate selectivity

Isabel M Piper,Sarah A Struyvenberg,Jordan D Valgardson,D Alex Johnson,Melody Gao,Katherine Johnston,Justin E Svendsen,Hanna M Kodama,Kelli L Hvorecny,John M Antos,Jeanine F Amacher

doi:10.1016/j.jbc.2021.100981

Isabel M Piper, Sarah A Struyvenberg + Show 9 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100981

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Abstract

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 to catalyze SML reactions. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this β7–β8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by β7–β8 loop residues that appear to be broadly conserved or converged upon in 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.

Highlights

Construction of the bacterial pilus [1, 4]
While the increase in enzyme activity afforded by these mutations included a 3.6-fold increase in kcat, the effect was dominated by a 33-fold decrease in KM, suggesting these loop residues may be important in cell wall sorting signal (CWSS) recognition [8]
We look at natural sequence variation in the β7–β8 loop of class A sortases, using Streptococcus pneumoniae SrtA as a model system

Summary

Introduction

Construction of the bacterial pilus (class C) [1, 4]. These enzymes recognize a cell wall sorting signal (CWSS) on the outer membrane of gram-positive bacteria [1, 5]. Substitution of the β6–β7 loop residues from saSrtB into the saSrtA enzyme alters substrate recognition to that of a sortase B protein (NPQTN) [18]. We look at natural sequence variation in the β7–β8 loop of class A sortases, using Streptococcus pneumoniae SrtA (spSrtA) as a model system.

Results

Conclusion