Core mutations that promote the calcium-induced allosteric transition of bovine recoverin

Recoverin is a small calcium binding protein involved in regulation of the phototransduction cascade in retinal rod cells. It functions as a calcium s ensor by undergoing a cooperative, ligand-dependent conformational change, resulting in the extrusion of the N-terminal myristoyl group from a hydroph obic pocket. To test the role of certain core residues in tuning this allos teric switch, we have made and characterized two mutants: W31K, which repla ces Trp31 with Lys; and a double mutant, I52A/Y53A, in which Ile52 and Tyr5 3 are both replaced by Ala. These mutations decrease the hydrophobicity of the myristoyl binding pocket. They are thus expected to make sequestering o f the myristoyl group less favorable and destabilize the Ca2+-free state. A s predicted, the myristoylated forms of the mutants exhibit increased affin ity for Ca2+, whether monitored by equilibrium binding of Ca-45(2+) (K-d = 17.2, 7.9, and 8.1 mu M for wild type, W31K, and I52A/Y53A, respectively) o r by the change in tryptophan fluorescence associated with the conformation al change (K-d = 17.9, 3.6, and 4.4 mu M for wild type, W31K, and I52A/Y53A , respectively). The mutants also exhibit decreased cooperativity of bindin g (Hill coefficient = 1.2 and 1.0 for W31K and I52A/Y53A vs 1.4 for wild ty pe). Binding of the mutant proteins to rod outer segment membranes occurs a t lower Ca2+ concentrations compared to wild-type protein (K-1/2 = 5.6, 2.2 , and 1.0 mu M for wild type, W31K, and I52A/Y53A, respectively). The unmyr istoylated forms of the mutants exhibit biphasic Ca2+ binding curves, nearl y identical to that observed for wild type. The binding data for the two mu tants can be explained by a concerted allosteric model in which the mutatio ns affect only the equilibrium constant L between the two allosteric forms, T (the Ca2+-free form) and R (the Ca2+-bound form), without affecting the intrinsic binding constants for the two Ca2+ sites. Two-dimensional NMR spe ctra of the Ca2+-free forms of the mutants have been compared to the wild-t ype spectrum, whose peaks have been assigned to specific residues (1). Many resonances assigned to residues in the C-terminal domain (residues 100-202 ) in the wild-type spectrum are identical in the mutant spectra, suggesting that the backbone structure of the C-terminal domain is probably unchanged in both mutants. The N-terminal domain, in which both mutations are locate d, reveals in each case numerous changes of undetermined spatial extent.