Unraveling the Dynamics of the EF1 Hand Upon Ca2+ Binding in Neurocalcin Delta

Abstract

Neuronal calcium sensor (NCS) proteins comprise a family of related proteins that mediate signal transduction in response to calcium, primarily in neurons. Neurocalcin delta (NCALD) is a member of this family. Though NCALD, like other NCS proteins, has 4 EF-hand motifs that could potentially bind calcium, it has been demonstrated that the first EF hand, EF1, does not bind calcium. NCALD has been purified from the brain and the retina and is thought to play critical roles in signaling. However, the details of the calcium-dependent dynamics remain to be elucidated, despite the availability of its crystal structure. To probe the calcium-dependent changes, we carried out both experimental and computational analyses. The experimental investigations comprised of analyses of local conformational changes through tryptophan fluorescence, global conformational changes through mobility on native gels, and dimerization state through gel filtration chromatography. The wild-type as well as different mutant versions of the protein were used in these analyses. Meanwhile, atomistic molecular dynamics (MD) simulations with the explicit solvent model were performed to explore the structure and dynamics of the NCALD monomer with and without Ca2+ ions. In this report, the joint results are presented to provide evidence to support a major role for EF1 hand (which does not bind calcium) in determining the response of the protein to calcium.