RESUMO
Tcb2 is a calcium-binding protein that localizes to the membrane-associated skeleton of the ciliated protozoan Tetrahymena thermophila with hypothesized roles in ciliary movement, cell cortex signaling, and pronuclear exchange. Tcb2 has also been implicated in a unique calcium-triggered, ATP-independent type of contractility exhibited by filamentous networks isolated from the Tetrahymena cytoskeleton. To gain insight into Tcb2's structure-function relationship and contractile properties, we determined solution NMR structures of its C-terminal domain in the calcium-free and calcium-bound states. The overall architecture is similar to other calcium-binding proteins, with paired EF-hand calcium-binding motifs. Comparison of the two structures reveals that Tcb2-C's calcium-induced conformational transition differs from the prototypical calcium sensor calmodulin, suggesting that the two proteins play distinct functional roles in Tetrahymena and likely have different mechanisms of target recognition. Future studies of the full-length protein and the identification of Tcb2 cellular targets will help establish the molecular basis of Tcb2 function and its unique contractile properties. Proteins 2016; 84:1748-1756. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Calmodulina/química , Proteínas do Citoesqueleto/química , Proteínas de Protozoários/química , Tetrahymena thermophila/genética , Sequência de Aminoácidos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismo , Motivos EF Hand , Expressão Gênica , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Tetrahymena thermophila/metabolismoRESUMO
Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.
