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

Mechanism of Activation and Regulation of BY-Kinases, a Unique Family of P-Loop Enzymes

The bacterial tyrosine kinases (BY-kinase) represent a class of membrane-bound enzymes that utilize a cycle of auto-phosphorylation and de-phosphorylation to drive the synthesis and export of exopolysaccharides in both Gram-positive and Gram-negative bacteria. The catalytic domain of BY-kinases utilizes a P-loop nucleoside triphosphatase (NTPase) fold that is commonly used by NTPases and small molecule kinases, being the only protein kinase to do so. In the work presented in this thesis, we aimed to obtain an understanding of the mechanisms of the BY-kinases’ unconventional deployment of P-loop scaffold to phosphorylate on tyrosine. We used the BY-kinase of Escherichia coli (K12) as our model and an array of theoretical and experimental approaches to investigate the regulatory dynamics of BY-kinases. Given that BY-kinases belong to the ancient P-loop family, we analyzed regulatory dynamics of a broad class of P-loop enzymes to test whether features of these dynamics are retained despite structural diversification beyond the catalytic site. Our findings reveal that all P-loop enzymes, including BY-kinases, populate global conformations that are correlated to the relative orientations of conserved catalytic elements. These conformational variations modulate the ability to engage Mg2+ and thereby the associated substrate NTP on the path to a chemistry compatible state. These conformational transitions couple to the overall structure of the specific P-loop enzyme through secondary features unique to its context. In the case of BY-kinases, these secondary features involve the interfaces that facilitate oligomerization, a requirement for tyrosine autophosphorylation in trans. Finally, we relate these regulatory properties of BY-kinases to functional phenotypes reported in the literature and provide specific hypotheses that are amenable to future experimental verification