Abstract
Ca2+-binding proteins play pivotal roles in both eukaryotic and prokaryotic cells. CcbP from cyanobacterium Anabaena sp. strain PCC 7120 is a major Ca2+-binding protein involved in heterocyst differentiation, a process that forms specialized nitrogen-fixing cells. The three-dimensional structures of both Ca2+-free and Ca2+-bound forms of CcbP are essential for elucidating the Ca2+-signaling mechanism. However, CcbP shares low sequence identity with proteins of known structures, and its Ca2+-binding sites remain unknown. Here, we report the solution structures of CcbP in both Ca2+-free and Ca2+-bound forms determined by nuclear magnetic resonance spectroscopy. CcbP adopts an overall new fold and contains two Ca2+-binding sites with distinct Ca2+-binding abilities. Mutation of Asp38 at the stronger Ca2+-binding site of CcbP abolished its ability to regulate heterocyst formation in vivo. Surprisingly, the β-barrel subdomain of CcbP, which does not participate in Ca2+-binding, topologically resembles the Src homology 3 (SH3) domain and might act as a protein-protein interaction module. Our results provide the structural basis of the unique Ca2+ signaling mechanism during heterocyst differentiation.