Exploring Centrin targets: KRR1, and the Centrin-SFI1p21 complex salt-bridge interaction

Abstract

Centrins are small calcium binding proteins and ubiquitous centrosome components. Centrins play a fundamental role in the structure and function of centrioles, basal bodies, primary cilia and the microtubule-organizing center of lower eukaryotes. In humans, four centrin isoforms have been identified (Hscen1, Hscen2, Hscen3, and Hscen4). The structure of these small proteins is comprised of two rounded domains containing two EF-hands and tethered by a helix. The EF-hands act as calcium sensors within cells. The particularity of centrin’s structure provides the ability to refine interactions with numerous biological targets. In this study, we explore two centrin targets. First the expression and purification of the novel protein Krr1 was established. Krr1 is a human processome subunit that co-localizes with Hscen2 in the nucleus. Krr1 is known to be involved in ribosome biogenesis and RNA processing, nevertheless information regarding Krr1 is limited and human Krr1 research remains a novel area of study. Secondly the complex formation between Hscen1 and human Sfi1 was examined. Human Sfi1 is one of the centrin biological targets that co-localizes to centrioles and basal bodies. The aim was to study the existence of a potential salt bridge interaction between human centrin and Sfi1p. Using two dimensional infrared (2D IR) correlation spectroscopy and the labeled isotope 15N2-Arg16Hs_Sfi1p21 we were able to establish unequivocally the existence of this key salt-bridge interaction, crucial to elucidating the mechanism of complex formation and the role of the bridge interaction within the Hscen1-15N2-Arg16Hs_Sfi1p21 complex.