Comparative Analysis of the Tyr-Kinases CapB1 and CapB2 Fused to Their Cognate Modulators CapA1 and CapA2 from Staphylococcus aureus

Abstract

A particular class of tyrosine-kinases sharing no structural similarity with eukaryotic tyrosine-kinases has been evidenced in a large array of bacterial species. These bacterial tyrosine-kinases are able to autophosphorylate on a C-terminal tyrosine-rich motif. Their autophosphorylation has been shown to play a crucial role in the biosynthesis or export of capsular polysaccharide. The analysis of the first crystal structure of the staphylococcal tyrosine kinase CapB2 associated with the activating domain of the transmembrane modulator CapA1 had brought conclusive explanation for both the autophosphorylation and activation processes. In order to explain why CapA1 activates CapB2 more efficiently than its cognate transmembrane modulator CapA2, we solved the crystal structure of CapA2B2 and compared it with the previously published structure of CapA1B2. This structural analysis did not provide the expected clues about the activation discrepancy observed between the two modulators. Staphylococcus aureus also encodes for a CapB2 homologue named CapB1 displaying more than 70% sequence similarity and being surprisingly nearly unable to autophosphorylate. We solved the crystal structure of CapA1B1 and carefully compare it with the structure of CapA1B2. The active sites of both proteins are highly conserved and the biochemical characterization of mutant proteins engineered to test the importance of small structural discrepancies identified between the two structures did not explain the inactivity of CapB1. We thus tested if CapB1 could phosphorylate other protein substrates or hydrolyze ATP. However, no activity could be detected in our in vitro assays. Taken together, these data question about the biological role of the homologous protein pairs CapA1/CapB1 and CapA2/CapB2 and we discuss about several possible interpretations.

Expression of Active <i>Staphylococcus aureus</i> Tyrosine Kinases in a Human Cell Line

“…Somewhat unexpectedly, and in contradiction to a previous report, 19) mutation of the four C-terminal tyrosines to phenylalanines resulted in no reduction of the phosphotyrosine signal (Fig. 3, lane 3).…”

“…The kinase activity of CapB2 was also significantly enhanced when it was fused to the C-terminal domain of CapA protein. [18][19][20] Here, we show that the S. aureus BY-kinase CapB2, and possibly CapB1, can be translated into active tyrosine kinases in a human cell line when they are fused to CapA C-terminal cytoplasmic activation domains. This approach provides an efficient and economical method to measure and analyze BYkinases, and could be useful in future enzyme characterization and inhibitor screening.…”

“…Mutation of this region in E. coli Wzc decreased autophosphorylation and polysaccharide production 23) but did not inhibit S. aureus CapB2 kinase activity. 20) We changed the three conserved residues to one aspartic acid and two lysines ( Fig. 5 lane 2, DKK) or to three alanines (lane 3, AAA) but did not notice any notable change in phosphotyrosine levels, suggesting that this motif is not essential for CapB2 kinase activity per se.…”

“…Previous reports have shown that the activity of the S. aureus BYkinase CapB2 is enhanced when it is fused to the C-terminal cytoplasmic tail of the activator CapA1. [18][19][20] Based on these reports, we constructed CapA1/CT-CapB2 by attaching the Cterminal 29 amino acids of CapA1 to the N-terminus of fulllength CapB2 (Fig. 1A).…”

The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

“…However, CapB2 showed significant activity, particularly in the presence of CapA1 (92), which is transcribed at a distant site on the chromosome. There has been a recent suggestion that CapB1 may in fact be a pseudokinase; a protein that while containing a kinase-like domain is actually catalytically inactive (34), and may perform other auxillary roles as has been seen for the pseudokinase, MviN of Mycobacterium tuberculosis (27). The role of these genes within the bacterial cell is yet to be investigated.…”