BY-kinases: Protein tyrosine kinases like no other.

BY-kinases (for bacterial tyrosine kinases) constitute a family of protein tyrosine kinases that are highly conserved in the bacterial kingdom and occur most commonly as essential components of multicomponent assemblies responsible for the biosynthesis, polymerization, and export of complex polysaccharides involved in biofilm or capsule formation. BY-kinase function has been attributed to a cyclic process involving formation of an oligomeric species, its disassembly into constituent monomers, and subsequent reassembly, depending on the overall phosphorylation level of a C-terminal cluster of tyrosine residues. However, the relationship of this process to the active/inactive states of the enzyme and the mechanism of its integration into the polysaccharide production machinery remain unclear. Here, we synthesize the substantial body of biochemical, cell-biological, structural, and computational data, acquired over the nearly 3 decades since the discovery of BY-kinases, to suggest means by which they fulfill their physiological function. We propose a mechanism involving temporal coordination of the assembly/disassembly process with the autokinase activity of the enzyme and its ability to be dephosphorylated by its counteracting phosphatase. We speculate that this temporal control enables BY-kinases to function as molecular timers that coordinate the diverse processes involved in the synthesis, polymerization, and export of complex sugar derivatives. We suggest that BY-kinases, which deploy distinctive catalytic domains resembling P-loop nucleoside triphosphatases, have uniquely adapted this ancient fold to drive functional processes through exquisite spatiotemporal control over protein-protein interactions and conformational changes. It is our hope that the hypotheses proposed here will facilitate future experiments targeting these unique protein kinases.

Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb.

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.

The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse-chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

A Conserved Structural Role for the Walker-A Lysine in P-Loop Containing Kinases.

Bacterial tyrosine kinases (BY-kinases) and shikimate kinases (SKs) comprise two structurally divergent P-loop containing enzyme families that share similar catalytic site geometries, most notably with respect to their Walker-A, Walker-B, and DxD motifs. We had previously demonstrated that in BY-kinases, a specific interaction between the Walker-A and Walker-B motifs, driven by the conserved "catalytic" lysine housed on the former, leads to a conformation that is unable to efficiently coordinate Mg2+•ATP and is therefore incapable of chemistry. Here, using enhanced sampling molecular dynamics simulations, we demonstrate that structurally similar interactions between the Walker-A and Walker-B motifs, also mediated by the catalytic lysine, stabilize a state in SKs that deviates significantly from one that is necessary for the optimal coordination of Mg2+•ATP. This structural role of the Walker-A lysine is a general feature in SKs and is found to be present in members that encode a Walker-B sequence characteristic of the family (Coxiella burnetii SK), and in those that do not (Mycobacterium tuberculosis SK). Thus, the structural role of the Walker-A lysine in stabilizing an inactive state, distinct from its catalytic function, is conserved between two distantly related P-loop containing kinase families, the SKs and the BY-kinases. The universal conservation of this element, and of the key characteristics of its associated interaction partners within the Walker motifs of P-loop containing enzymes, suggests that this structural role of the Walker-A lysine is perhaps a widely deployed regulatory mechanism within this ancient family.

Expression of Active Staphylococcus aureus Tyrosine Kinases in a Human Cell Line.

Many bacteria encode tyrosine kinases that are structurally unrelated to their eukaryotic counterparts and are termed BY-kinases. Two BY-kinases, CapB1 and CapB2, have been identified in the Staphylococcus aureus genome. Although CapB1 and CapB2 share more than 70% homology, earlier studies with purified enzymes did not find any evident kinase activity in CapB1, whereas CapB2 was autophosphorylated on a C-terminal tyrosine cluster in the presence of the kinase modulator proteins CapA1 or CapA2. For the convenient analysis of BY-kinases, we attempted to express CapB2 in an active form in a mammalian cell line. To this end, the C-terminal activation domain of CapA1 was attached to the N-terminus of CapB2, and the resulting CapA1/CT-CapB2 chimera was further fused with various tags and transfected into HEK293T cells. Immunoblotting analyses showed that when fluorescent protein tags were attached to the N-terminus, CapA1/CT-CapB2 was both expressed and tyrosine phosphorylated in HEK293T cells. Mutation of the ATP-binding lysine abrogated tyrosine phosphorylation, indicating that tyrosine phosphorylation was catalyzed by the transfected bacterial kinase and not by endogenous cellular enzymes. Unexpectedly, mutation of the C-terminal tyrosine cluster did not abolish autophosphorylation. Further analyses revealed that CapA1/CT-CapB2 phosphorylated not only itself but also the attached fluorescent protein tag. Several domains and residues important for tyrosine kinase activity were identified from the production of various mutants. We also present data that CapB1, which was previously thought to be catalytically inert, may possess intrinsic kinase activity.

Functional analysis of conserved motifs in a bacterial tyrosine kinase, BtkB, from Myxococcus xanthus.

Myxococcus xanthus has two bacterial protein-tyrosine (BY) kinases, BtkA and BtkB. Autophosphorylation in C-terminal tyrosine-rich clusters and poly(Glu, Tyr) kinase activities of cytoplasmic catalytic domains of BtkA and BtkB were activated by the intracellular juxtamembrane regions of the second transmembrane helices. Protein kinase activity against poly(Glu, Tyr) of cytoplasmic fragment of BtkB (CF-BtkB) containing an activator region was not inhibited by serine/threonine protein kinase inhibitors. However, addition of tyrosine protein kinase inhibitors, genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), at a concentration of 0.2 mM, inhibited the CF-BtkB kinase activity by 20 and 64%, respectively. A CF-BtkB mutant constructed by replacing all C-terminal tyrosine residues with phenylalanines, did not undergo autophosphorylation. Further, this mutation did not significantly affect poly(Glu, Tyr) kinase activity, suggesting that M. xanthus BtkB kinase activity is not dependent on autophosphorylation in the C-terminal tyrosine cluster. A conserved motif (ExxRxxR) of BY kinases is involved in the self-association of catalytic domains of BY kinases, necessary to accomplish trans-phosphorylation. An ExxRxxR motif mutant of CF-BtkB led to loss of autophosphorylation and poly(Glu, Tyr) kinase activities. These observations provide insights into the regulation mechanism of M. xanthus BY kinase activity.

Alr5068, a Low-Molecular-Weight protein tyrosine phosphatase, is involved in formation of the heterocysts polysaccharide layer in the cyanobacterium Anabaena sp. PCC 7120.

The filamentous cyanobacterium Anabaena sp. PCC 7120 forms nitrogen-fixing heterocysts after deprivation of combined nitrogen. Under such conditions, vegetative cells provide heterocysts with photosynthate and receive fixed nitrogen from the latter. Heterocyst envelope contains a glycolipid layer and a polysaccharide layer to restrict the diffusion of oxygen into heterocysts. Low-Molecular-Weight protein tyrosine phosphatases (LMW-PTPs) are involved in the biosynthesis of exopolysaccharides in bacteria. Alr5068, a protein from Anabaena sp. PCC 7120, shows significant sequence similarity with LMW-PTPs. In this study we characterized the enzymatic properties of Alr5068 and showed that it can dephosphorylate several autophosphorylated tyrosine kinases (Alr2856, Alr3059 and All4432) of Anabaena sp. PCC 7120 in vitro. Several conserved residues among LMW-PTPs are shown to be essential for the phosphatase activity of Alr5068. Overexpression of alr5068 results in a strain unable to survive under diazotrophic conditions, with the formation of morphologically mature heterocysts detached from the filaments. Overexpression of an alr5068 allele that lost phosphatase activity led to the formation of heterocyst with an impaired polysaccharide layer. The alr5068 gene was upregulated after nitrogen step-down and its mutation affected the expression of hepA and hepC, two genes necessary for the formation of the heterocyst envelope polysaccharide (HEP) layer. Our results suggest that Alr5068 is associated with the production of HEP in Anabaena sp. PCC 7120.