Structure of guanylyl cyclase activator protein 1 (GCAP1) mutant V77E in a Ca2+-free/Mg2+-bound activator state

Abstract

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca2+-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 (GCAP1V77E) in the Ca2+-free/Mg2+-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca2+-dependent differences for residues 170-174. An NMR-derived model of GCAP1V77E contains Mg2+ bound at EF2 and looks similar to Ca2+ saturated GCAP1 (root mean square deviations = 2.0 Å). Ca2+-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca2+ switch helix). Ca2+-induced shortening of the Ca2+ switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca2+ 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 Ca2+ binding affinity and abolished RetGC1 activation. We conclude that Ca2+-dependent conformational changes in the Ca2+ switch helix are important for activating RetGC1 and provide further support for a Ca2+-myristoyl tug mechanism.