proteins STRUCTURE O FUNCTION O BIOINFORMATICS STRUCTURE NOTE Solution NMR structures of the C-domain of Tetrahymena cytoskeletal protein Tcb2 reveal distinct calcium-induced structural rearrangements Adina M. Kilpatrick, 1 * Jerry E. Honts, 2 Heidi M. Sleister, 2 and C. Andrew Fowler 3 1 Department of Physics and Astronomy, Drake University, Des Moines, Iowa 50311 2 Department of Biology, Drake University, Des Moines, Iowa 50311 3 Roy J. and Lucille A. Carver College of Medicine NMR Facility, University of Iowa, Iowa City, Iowa 52242 ABSTRACT 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 iso- lated from the Tetrahymena cytoskeleton. To gain insight into Tcb2’s structure-function relationship and contractile proper- ties, 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; 00:000–000. V C 2016 Wiley Periodicals, Inc. Key words: calcium-binding protein; calcium signaling; conformational change; protein structure; EF-hand; cytoskeleton; helical packing; hydrophobic surface; TCBP-25; calmodulin. INTRODUCTION Changes in intracellular calcium levels trigger an array of essential cellular processes, including muscle contraction, cell cycle regulation, gene transcription, and cytoskeleton assem- bly. Calcium-binding proteins (CaBPs) mediate many of these processes through calcium-dependent interactions with target proteins. The basic structural element of most CaBPs is a pair of helix-loop-helix EF-hand calcium-binding motifs connected by a linker of variable length. Structural changes in the EF-hands upon calcium binding are an important deter- minant of CaBP function. 1,2 In calcium sensors such as calmodulin (CaM), calcium binding triggers a dramatic conformational change and exposes a hydrophobic surface, which enables interactions with target proteins. In contrast, Additional Supporting Information may be found in the online version of this article. Grant sponsor: Drake NASA-ISGC EPSCoR Base Program; Grant number: S5937O; Grant sponsor: Drake University Faculty Research Grants; Grant sponsor: University of Iowa Carver College of Medicine FUTURE in Biomedicine program; Grant spon- sor: NSF IUSE; Grant number: 1431379. *Correspondence to: Adina M. Kilpatrick, Harvey Ingham Hall of Science, Room 24, Des Moines, IA 50311-4516. E-mail: adina.kilpatrick@drake.edu Received 28 April 2016; Revised 8 July 2016; Accepted 25 July 2016 Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.25111 V V C 2016 WILEY PERIODICALS, INC. PROTEINS 1