Abstract
GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.
Highlights
Guanylyl cyclase activating proteins (GCAPs)2 belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily [1,2,3] and regulate Ca2ϩ-sensitive activity of retinal guanylyl cyclase (RetGC) in rod and cone cells (4 – 6)
The NMR spectrum of Ca2ϩ-free/Mg2ϩ-bound GCAP1V77E contains one downfield NMR peak at ϳ10.5 ppm assigned to Gly-69, indicating that 1 Mg2ϩ is bound to GCAP1V77E at EF2. (Fig. 2A, inset)
The lower affinity Ca2ϩ binding to EF2 may result from a loss of protein dimerization for GCAP1V77E compared with the dimeric wild type protein [22]
Summary
Expression and Purification of GCAP1 and Mutants—Mutations were introduced in a bovine GCAP1 coding plasmid using a “splice by overlap extension” approach as previously described [24]. The expression and purification of isotopically labeled GCAP1 and mutants were described previously [22, 25]. Trp Fluorescence Spectroscopy—The intrinsic Trp fluorescence of GCAP1 and its mutants was recorded in the presence of variable-free Mg2ϩ and Ca2ϩ concentrations as previously described in detail [19, 23]. NMR Spectroscopy—Samples for NMR analyses were prepared by dissolving unlabeled, 15N-labeled, 13C,15N-labeled, or 13C,2H,15N-labeled GCAP1 proteins in 0.5 ml of 90% H2O, 10% [2H]H2O containing 10 mM [2H11]Tris (pH 7.4) and either 5 mM MgCl2 (Mg2ϩ-bound) or 5 mM CaCl2 (Ca2ϩ-bound). NMR experiments and backbone assignments for Ca2ϩ-saturated GCAP1 were described elsewhere [22]. Backbone NMR resonance assignments of the Ca2ϩ-free/ Mg2ϩ-bound GCAP1V77E activator state (Mg2ϩ bound at EF2). Two-dimensional 1H,15N HSQC with 2048 (1H) ϫ 256 (15N) data points, threedimensional HNCACB with 1024 (1H) ϫ 64 (15N) ϫ 120 (13C)
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