Solution structure and backbone dynamics of the defunct domain of calcium vector protein

CaVP (calcium vector protein) is a Ca2+ sensor of the EF-hand protein famil y which is highly abundant in the muscle of Amphioxus. Its three-dimensiona l structure is not known, but according to the sequence analysis, the prote in is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81 -Ser161) and characterized the large conformational and dynamic changes ind uced by Ca2+ binding. In contrast, the N-terminal domain (Ala1-Asp86) has l ost the capacity to bind the metal ion due to critical mutations and insert ions in the two calcium loops. In this paper, we report the solution struct ure of the N-terminal domain and its backbone dynamics based on NMR spectro scopy, nuclear relaxation, and molecular modeling. The well-resolved three- dimensional structure is typical of a pair of EF-hand motifs, joined togeth er by a short antiparallel beta -sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel a-h elices, similar to other EF-hand pairs in the absence of calcium ions. To c haracterize the internal dynamics of the protein, we measured the N-15 nucl ear relaxation rates and the heteronuclear NOE effect in N-15-labeled N-CaV P at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisot ropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha -helices and beta -strands unde rgo highly restricted movements on a picosecond to nanosecond time scale. T he amide groups in Ca2+ binding loops and in the linker fragment also displ ay rapid fluctuations with slightly increased amplitudes.