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

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Title: Allosteric Determinants of Guanine Nucleotide Binding Proteins and Methods to Crystallize the Cytosolic Domains of Adenylyl Cyclase
Author: Hatley, Mark Edward
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /624
Date: 2004-05-04

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Allosteric Determinants of Guanine Nucleotide Binding Proteins and Methods to Crystallize the Cytosolic Domains of Adenylyl Cyclase. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /624 .

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