Search for: All records

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 structure presents 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. 

BY-kinases represent a highly conserved family of protein tyrosine kinases unique to bacteria without eukaryotic orthologs. BY-kinases are regulated by oligomerization-enabled transphosphorylation on a C-terminal tyrosine cluster through a process with sparse mechanistic detail. Using the catalytic domain (CD) of the archetypal BY-kinase, Escherichia coli Wzc, and enhanced-sampling molecular dynamics simulations, isothermal titration calorimetry and nuclear magnetic resonance measurements, we propose a mechanism for its activation and nucleotide exchange. We find that the monomeric Wzc CD preferentially populates states characterized by distortions at its oligomerization interfaces and by catalytic element conformations that allow high-affinity interactions with ADP but not with ATP·Mg 2+ . We propose that oligomer formation stabilizes the intermonomer interfaces and results in catalytic element conformations suitable for optimally engaging ATP·Mg 2+ , facilitating exchange with bound ADP. This sequence of events, oligomerization, i.e., substrate binding, before engaging ATP·Mg 2+ , facilitates optimal autophosphorylation by preventing a futile cycle of ATP hydrolysis.