Allosteric Determinants of Guanine Nucleotide Binding Proteins and Methods to Crystallize the Cytosolic Domains of Adenylyl Cyclase




Hatley, Mark Edward

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The cytosolic domains of mammalian adenylyl cyclases, termed C1 and C2, are responsible for catalytic activity and most regulatory properties. Crystal structures of the soluble catalytic core of adenylyl cyclase bound to activators Gsa and forskolin were previously determined. However, structural information regarding low activity (non-Gsa or forskolin bound) states of the enzyme is lacking. Genetic and biochemical methods were utilized to overcome the low affinity of the cytosolic domains in the absence of activators. A genetic screen in Saccharomyces cerevisiae identified mutations that activate mammalian adenylyl cyclase in the absence of Gsa. The increased affinity of the K1014N-C2 mutant protein for the C1 domain in the absence of Gsa was exploited to isolate a complex containing C1 and C2 in the absence of Gsa. Unfortunately, this complex crystallized but failed to diffract due to heterogeneity. Intein-mediated protein ligation and expression of a C1-C2 fusion protein in adenylyl cyclase deficient Escherichia coli were explored to circumvent the low affinity of the domains. However, the yields of products were insufficient for crystallization. Members of the G protein superfamily contain nucleotide-dependent switches that dictate the specificity of their interactions with binding partners. Using a new sequence-based method termed statistical coupling analysis (SCA), I identified the allosteric core of these proteins - the network of amino acid residues that couples the domains responsible for nucleotide binding and protein-protein interactions. One-third of the 38 residues identified by SCA were mutated in the G protein Gsa, and the interactions of GTPgamma S- and GDP-bound mutant proteins were tested with both adenylyl cyclase (preferential binding to GTP-Gsa) and the G protein beta gamma subunit complex (preferential binding to GDP-Gsa). A two-state allosteric model predicts that mutation of residues that control the equilibrium between GDP- and GTP-bound conformations of the protein will cause the ratio of affinities of these species for adenylyl cyclase and beta gamma to vary in a reciprocal fashion. Observed results were consistent with this prediction. The network of residues identified by the SCA appears to comprise a core allosteric mechanism conferring nucleotide-dependent switching; the specific features of different G protein family members are built upon this core.

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