The N-Terminal Domain of Human Centrin 2 Has a Closed Structure, Binds Calcium with a Very Low Affinity, and Plays a Role in the Protein Self-Assembly^,

Abstract

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.

Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a Role in mRNA and Protein Export

“…on May 11, 2018 by guest http://mcb.asm.org/ structs, a human centrin 2 N-terminal half (aa 1 to 98) and a human centrin 2 C-terminal half (aa 94 to 172), based on the published structures of these domains (83,139). We found that the expression of either half of the human centrin 2 led to nuclear poly(A) ϩ RNA accumulation in ϳ20 to 25% of the transfected cells (Fig.…”

Centrin4 coordinates cell and nuclear division inT. brucei

“…Although this variety of functions can be achieved by a single centrin gene in organisms such as Saccharomyces cerevisiae, multiple centrin genes have been found in many other organisms, including mammals. Structural analyses suggest different biochemical properties for different centrin isoforms (Hu and Chazin, 2003;Sheehan et al, 2006;Yang et al, 2006) and different intracellular localizations and functions have also been reported (Giessl et al, 2004;Ruiz et al, 2005;Hu et al, 2006;Gogendeau et al, 2008).…”

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

“…Another solution structure of a HsCen-2 fragment (26), including residues Met 84 -Trp 172 shows that a helical portion of the N-terminal domain lies within a hydrophobic binding cavity of the C-terminal domain. A calcium-free solution structure of the HsCen-2 N-terminal domain was just published with a closed conformation (27).…”

“…The affinity of apocalmodulin for Ca 2ϩ increases over 1000-fold when bound to target proteins (52). Ca 2ϩ affinities for an N-terminal domain fragment of HsCen-2 are reported as ϳ10 2 to 10 3 M Ϫ1 in the absence of bound polypeptide (27).…”