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

Abstract

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 β7-β8 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 0 position of the substrate motif, underlined in LPXTG, in contrast to the strict P1 0 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 β7-β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7-β8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition

“…Briefly, spySrtA protein containing the inactivating C208A mutation was expressed and purified as previously described for the wildtype protein and analyzed via SDS-PAGE and LC–electrospray ionization (ESI)–mass spectrometry (MS) ( Fig. S1 ) ( 23 ). Purified protein (at ∼1.1 mM) was incubated in a 1:1 ratio with 1 mM peptide for 1 h prior to crystallization by hanging drop vapor diffusion method.…”

“…2 A ). The former is an example of a FRET quencher probe that is commonly used for monitoring sortase enzymatic activity ( 9 , 15 , 23 , 29 , 30 , 31 ), whereas the latter is a simplified target containing an acetyl ( Ac -) cap and C-terminal primary amide (- NH 2 ). For both substrates, LC–ESI–MS was used to confirm that they were cleaved by wildtype spySrtA in a model transacylation reaction ( Fig.…”

“…Expression and purification of spySrtA protein. Wild-type spySrtA, C208A spySrtA, T207A spySrtA, and R216A spySrtA genes were recombinantly expressed using Escherichia coli BL21 (DE3) cells in the pET28a(+) vector (Genscript), as previously described (23). The wild-type sequence used matches that of the published spySrtA structure, PDB ID 3FN5 (17).…”

“…Like other Class A sortases, the majority of predicted and verified in vivo targets of spySrtA possess LPXTG substrate sequences (24,25). In addition, prior work from ourselves and others has demonstrated that spySrtA readily accepts LPXTA and LPXTS substrates in vitro, despite the fact that these particular sequence variants do not appear to be present in naturally occurring spySrtA substrates in vivo (20,23,26). The spySrtA enzyme also accepts alanine-or serine-based nucleophiles, which is a characteristic that has been exploited for dual-labeling SML strategies and is consistent with the presence of N-terminal alanines in the interpeptide bridge of lipid II in S. pyogenes (17)(18)(19)(20)(21)(22)27).…”

“…The apo structure of spySrtA was solved using X-ray crystallography in 2009, and its catalytic triad consists of His142, Cys208,and Arg216 (17). Using similar crystallization conditions, we were able to crystallize and solve the structures of a catalytically inactive C208A spySrtA mutant bound to the peptides LPATA and LPATS, which are sequences that are known to serve as spySrtA substrates in vitro (17)(18)(19)(20)(21)(22)(23). In addition, we synthesized a model peptide (LPAT-LII) of the ligation product between the LPAT fragment and the J o u r n a l P r e -p r o o f in vivo nucleophile lipid II, and solved the structures of two complexes between C208A spySrtA and LPAT-LII where the peptide is in the "Thr-in" and "Thr-out" conformations, terminology previously used to describe the side chain of the P1 Thr as protein-interacting ("Thr-in") or solventinteracting ("Thr-out") (8).…”