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.
Highlights
A new family of protein tyrosine-kinases harboring a P-loop type nucleotide binding-domain [1] with a Walker A motif [2] has been described. They have been called BY-kinases (Bacterialtyrosine-kinases) as they are only found in bacterial genomes [3,4]. They have been proposed to participate in the regulation of several cellular processes but to date, their best-documented cellular function concerns capsular polysaccharide synthesis in which they act as co-polymerase in the multiprotein capsule assembly machinery [5]
In order to find out the structural basis of this discrepancy, we solved the crystal structure of the chimera CapA2B2 [17]
Previous mass spectrometry analysis demonstrated [18] that each band corresponds to a different phosphorylation level, from 0 to 4 phosphorylated residues among the 4 tyrosines 221, 222, 224 and 225 of the Y-cluster (Figure 2). This result demonstrated that the Y-cluster of the CapA2B2 chimera from S. aureus autophosphorylated spontaneously during its production process in E. coli cells
Summary
A new family of protein tyrosine-kinases harboring a P-loop type nucleotide binding-domain [1] with a Walker A motif [2] has been described. They have been called BY-kinases (Bacterialtyrosine-kinases) as they are only found in bacterial genomes [3,4]. They have been proposed to participate in the regulation of several cellular processes but to date, their best-documented cellular function concerns capsular polysaccharide synthesis in which they act as co-polymerase in the multiprotein capsule assembly machinery [5]. BY-kinase autophosphorylation on a Cterminal tyrosine cluster, called Y-cluster [6] is crucial in regulating polysaccharide export. BY-kinase autophosphorylation and ability to phosphorylate polysaccharide-synthesizing enzymes represent a key regulatory tool of capsular polysaccharide synthesis and export
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