Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

3-POS Board 3 A TUG-OF-WAR MECHANISM DRIVES THE ALLOSTERIC ACTIVATION OF PROTEIN KINASE A Lihui Bai 1 ; Jeneffer England 1 ; Susan S Taylor 2 ; Rodrigo Maillard 1 ; 1 Georgetown University, Chemistry, Washington , DC, USA 2 University of California, San Diego, Pharmacology, San Diego, La Jolla, CA, USA Protein Kinase A (PKA) is an important regulatory enzyme in many signal transduction pathways. This study focuses on the mechanisms of allosteric communication between the PKA regulatory and catalytic subunits. The regulatory subunit establishes two sets of interactions with the catalytic subunit to form an inactive holoenzyme: 1) via the inhibitory sequence that docks into the active site of the catalytic subunit and is stabilized by ATP and two Mg 2+ ions, and 2) via surface contacts between two cyclic nucleotide binding (CNB-A and CNB-B) domains. We use optical tweezers to investigate the crosstalk between the inhibitory sequence, ATP and Mg 2+ with the surface contacts established between the CNB domains and the catalytic subunit. When ATP and Mg 2+ are in saturation, we find that the CNB domains bound to the catalytic subunit are in equilibrium between two conformational states, a predominant conformation in which the CNB domains unfold independently of each other and at high force, and a minor conformation wherein the CNB domains unfold near-simultaneously or cooperatively and at low force. In the absence of ATP or Mg 2+ , a redistribution of the PKA ensemble occurs where the CNB domains only unfold cooperatively and at low force. Moreover, we investigate how the CNB-B domain controls the interaction of the CNB-A domain with the catalytic subunit. We show that the truncated CNB-A domain has a stronger interaction with the catalytic subunit compared to the CNB-A domain as part of the regulatory subunit. This result indicates that the CNB-B domain weakens the interaction between the CNB-A domain and the catalytic subunit. Altogether, this study portrays a PKA allosteric activation mechanism in which the CNB-A domain experiences a tug-of-war due to stabilizing effects conferred by the IS and ATP-Mg 2+ , and destabilizing effects due to the presence of the CNB-B domain.

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