UC San Diego

Protein phosphorylation is a major mechanism of enzyme control in cell signaling systems. Phosphoryl transfer activity by cyclic adenosine mono-phosphate (cAMP) dependant protein kinase A (PKA) is inhibited by the regulatory (R) subunits. The binding of cAMP to the R- subunit disassociates the PKA holoenzyme complex and releases the C-subunit inhibition. The R-subunits consist of four non-redundant isoforms that vary in molecular weight, cAMP affinity, localization, and in their potential as PKA substrates. Although all known cAMP binding domains share the same basic structure, there is significant diversity among protein families and isoforms. RII[beta] is implicated in both Lupus and cancer, and knockout of RII[beta] in mice has unique phenotypes of obesity resistance and alcohol consumption. The crystal structure of the RII[beta] deletion mutant 108-268 bound to the C-subunit was determined at 1.6 Å. Crystallization of the holoenzyme complex with the non-hydrolyzable ATP analogue AMPPNP trapped the RII[beta] substrate in a transition state. This structure revealed the conserved and non-conserved regions between RII[beta] holoenzyme and previous structures of PKA holoenzymes. Biochemical affinity analysis determined the contribution of each domain of RII[beta] in holoenzyme formation. The RII[beta] linker region 102-107 was determined to be important for high affinity complex formation. The crystal structure of the RII[beta] deletion mutant 102-265 bound to the C- subunit was determined at 2.9 Å. This structure highlighted the structurally unique docking of RII[beta] 102-107 to the C-subunit. Solution based methods of hydrogen-deuterium exchange and small angle X-ray scattering demonstrated the isoform diversity of RII[beta] and allosteric communication in the holoenzyme complex. The cyclic nucleotide binding (CNB) domain is a highly conserved module that serves as the primary intracellular receptor for cAMP. Although similar in sequence and overall structure, cAMP binding domains vary in affinity and binding rates for cAMP. Additionally, analogues of cAMP show distinct preferences for binding different CNBs. A high throughput, isoform specific assay to screen cAMP analogues demonstrated the RII[beta] preference for N6 substituted analogues and RI[alpha] preference for C8 substituted analogues. The combination of X-ray structures and high throughput screening is a powerful technique to develop highly isoform specific cAMP analogues