Interaction of CRP L124 with cAMP affects CRP cAMP binding constants, cAMP binding cooperativity, and CRP allostery.
A cyclic nucleotide-binding pocket of the CRP dimer is composed of amino acid residues contributed by both subunits. Leucine (L) 124 of one subunit packs against the adenine ring of cAMP bound to the opposing subunit. We have undertaken a study designed to evaluate the role of L124 in CRP allostery. Wild-type (WT) apo-CRP is a 47 kDa protease-resistant dimer composed of identical subunits that exhibits a biphasic isotherm in cAMP titration studies. The WT CRP-cAMP complex is a protease-sensitive dimer degraded by protease to a dimer core that ranges between 26.5 and 30.5 kDa. Substitution of L124 with isoleucine (I), valine (V), cysteine (C), or alanine (A) generated a series of CRP variants that exhibited unique differences in apo-CRP resistance to protease, the mass of the core fragments generated in protease digestion reactions, cAMP-mediated allostery, and CRP-cAMP complex functionality. Differences in the affinity of the position 124 CRP variants for cAMP were observed. The binding constants that drive the formation of the WT and L124I CRP-cAMP complexes deviated by not more than a factor of 1.5. In contrast, the L124V, L124A, and L124C forms of CRP exhibited both a decreased K(cAMP1)(app) and an increased K(cAMP2)(app) to produce 2.4-, 55-, and 204-fold reductions, respectively, in the difference between these two parameters compared to that observed for WT CRP. The data indicate that the van der Waals volume and/or the hyrophobicity of the L124 side chain are important determinants of CRP cAMP binding properties and affect, either directly or indirectly, cAMP-mediated conformation changes in CRP.
Tomlinson
Steven R
SR
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
Tutar
Yusuf
Y
Harman
James G
JG
D000495:Allosteric Site; D019943:Amino Acid Substitution; D001665:Binding Sites; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D021122:Protein Subunits; D013816:Thermodynamics; D001616:beta-Galactosidase / Q000378:metabolism
D002373:Cyclic AMP Receptor Protein; D017931:DNA Primers; D004269:DNA, Bacterial; D021122:Protein Subunits; D000242:Cyclic AMP; D001616:beta-Galactosidase