Backbone and side-chain chemical shift assignments for the C-terminal domain of Tcb2, a cytoskeletal calcium-binding protein from Tetrahymena thermophila

Abstract

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.

Solution NMR structures of the C‐domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium‐induced structural rearrangements

“…This rearrangement is consistent with large changes in the backbone amide chemical shifts (>1 standard deviation) of residues in helix α3 and the linker region upon calcium binding [Ref. , Fig. (B)].…”

“…(A,D,E)], as expected from 1 H, 15 N weighted chemical shift changes between apo‐ and Ca 2+ ‐Tcb2‐C [Ref. , Fig. (B)].…”

“…Although the α1‐α2 interhelical angle does not change significantly upon addition of calcium, helix α2 is subtly repositioned due to the opening of the linker, altering many contacts with the rest of the structure, which is consistent with the large chemical shift differences in the presence versus absence of calcium of most residues in this helix [Ref. , Fig. (B)].…”

“…Unlabeled or uniformly‐labeled ( 15 N or 15 N/ 13 C) Tcb2‐C was cloned, expressed, and purified as described previously; details are given in Supplementary Methods. NMR samples contained 0.75 m M protein in 25 m M Tris, 50 m M KCl, 5 m M MgCl 2 , 1 m M EGTA, pH 7.5, in either 90% H 2 O/10% D 2 O or 100% D 2 O. Calcium‐bound Tcb2‐C samples also contained 5 m M CaCl 2 .…”

“…1 H‐ 15 N HSQC spectra of Ca 2+ ‐Tcb2‐C samples match an HSQC at the endpoint of a calcium titration (described below), indicative of fully saturated protein. Nearly complete resonance assignments were made using standard triple‐resonance methods and have been reported previously and deposited in the BMRB with accession codes 26723 (calcium‐free Tcb2‐C) and 26724 (Ca 2+ ‐Tcb2‐C). All NMR spectra were acquired at 298 K using a 600 MHz Varian INOVA NMR spectrometer equipped with a triple resonance gradient probe.…”