Physical Chemistry Seminar- Prof. Rodrigo Maillard, Georgetown University

Event Presenter:

Prof. Rodrigo Maillard

Institution:

Georgetown University

Categories:

Physical Chemistry, Seminar

“Mechanism of Allosteric Regulation in Protein Kinases: A Single Molecule Approach”

Protein kinases are dynamic switches composed of a structurally conserved catalytic subunit, and a regulatory subunit that controls allosterically the kinase phosphorylating activity. Despite their broad biological importance and high potential for therapeutic measures, the energetic and dynamic features underlying the allosteric regulatory mechanisms in kinases remain unclear and challenging to study. Here, we apply single molecule optical tweezers and FRET to study the cAMP-mediated activation mechanism of protein kinase A (PKA). The regulatory subunit of PKA harbors two cyclic-nucleotide binding (CNB) domains that oscillate between an inactive conformation when bound to the catalytic subunit, an active conformation when bound to cAMP, and a transient apo conformation. The mechanical manipulation of the CNB domains as truncated structures or as part of the regulatory subunit showed indistinguishable folding properties in the apo state, indicating negligible inter-domain interactions in the absence of cAMP. In contrast, the presence of cAMP activates an allosteric interaction network that results in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Interestingly, binding of the catalytic subunit reshapes entirely the folding energy landscape of the CNB domains, revealing two additional conformational states. The equilibrium between these two conformations is allosterically controlled by ATP and Mg²⁺, which are distantly located in the catalytic subunit active site and play key roles in the activation cycle of PKA. Single molecule FRET experiments show that ATP is critical for the assembly of catalytic and regulatory subunits to form the inactive PKA holoenzyme, and that the cAMP-dependent release of the catalytic subunit is fast and highly cooperative. Taken together, our single molecule approaches provide a new avenue to study the activation cycle and conformational plasticity that enables protein kinases to adapt and respond to multiple signaling molecules.

Rodrigo Maillard Seminar Poster