Structural and Motional Changes Induced in apo-S100A1 Protein by the Disulfide Formation between Its Cys 85 Residue and -Mercaptoethanol †,‡ Igor Zhukov, §,| Andrzej Ejchart, § and Andrzej Bierzyn ´ski* ,§ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin ´ skiego 5A, 02-106 Warsaw, Poland, and SloVenian NMR Centre, National Institute of Chemistry, HajdrihoVa 19, P.O.B. 660, SI-1001 Ljubljana, SloVenia ReceiVed September 4, 2007; ReVised Manuscript ReceiVed October 26, 2007 ABSTRACT: Recently, we have shown (Goch, G., Vdovenko, S., Kozlowska, H., and Bierzyn ´ ski, A. (2005) FEBS J. 272, 2557-2565) that the chemical modification of Cys 85 residue of S100A1 protein by disulfide bond formation with small thiols such as glutathione, cysteine, or -mercaptoethanol (ME) leads to a dramatic increase of the protein affinity for calcium. Therefore, the biological function of S100A1 as a calcium signal transmitter is probably regulated by the redox potential within the cell. Systematic, structural studies of various mixed disulfides of S100A1 in the apo and holo states are necessary to elucidate the mechanism of this phenomenon. Using NMR methods we have determined the structure of apo-S100A1-ME and, on the basis of 15 N nuclear magnetic relaxation data, we have characterized the structural dynamics of both: modified and unmodified molecules of apo-S100A1. The following effects of ME modification have been observed: (1) Helices IV and IV′ of two protein subunits are elongated by five residues (85-89). (2) Conformation of the calcium binding N-terminal loops is dramatically changed, and structural flexibility of the N-loops as well as C-loops markedly increases. (3) The angle between helices I and IV increases by ∼20° and between helices IV and IV′ decreases by ∼35°. All these observations lead to the conclusion that ME modification of apo-S100A1 makes its structure more similar to that of holo-S100A1, so that it becomes much better adjusted for calcium coordination. Calcium ion is one of the most important messengers regulating numerous vital biological processes. A crucial role in the calcium signal transduction is played by EF-hand proteins that coordinate calcium and change their conforma- tion exposing hydrophobic patches to which a large variety of target proteins bind. S100 is a subfamily of EF-hand proteins found only in mammals and avians, implicated in cell growth, motility, and cell differentiation, by a variety of processes such as regulation of some enzyme as well as transcription factor activities, Ca 2+ homeostasis, and regulation of cytoskeleton dynamics. Some of them have extracellular cytokine-like activities (1, 2). Recently, S100 proteins have received increasing attention due to their close association with several human diseases including cardiomyopathy, neurodegenera- tive disorders, and cancer (2). S100A1 protein, intensively studied since its discovery in 1965 (3), is, in every respect, a typical representative of the S100 family. The structure of S100A1 in the apo state has been determined by Rustandi et al. (4) using NMR methods. Recently, its structure in the holo state has also been solved (5). The protein molecule is a symmetrical homodimer, strongly stabilized by hydrophobic interactions between its two R subunits. Each subunit contains two EF-hand motives that coordinate calcium ions in a noncooperative way with the binding constants, at physiological pH (7.2) and NaCl concentration (100 mM), of the order of 10 3 M -1 for C-terminal motives and 10 2 M -1 for N-terminal ones (6). These values are too low for a calcium-signaling protein, because the intracellular calcium concentration of Ca 2+ ions never exceeds ≈1 µM, even during the calcium stress (7). We have found, though, that the formation of a mixed disulfide between glutathione, cysteine, or -mercaptoethanol (ME) 1 and the single cysteine residue present in position 85 in both protein subunits leads to a dramatic increase of S100A1 affinity for calcium (6). This observation suggests that the calcium-dependent biological activity of the protein can be regulated by thionylation, most probably glutathio- nylation, because of high concentration of glutathione in the cell (∼10 mM). Therefore, S100A1 can play the role of a linker between the two most important signaling pathways, i.e., the redox- and calcium-dependent ones. What is the structural mechanism responsible for this phenomenon? To answer this question it is necessary to compare the structures of S100A1 and its mixed disulfides † This work was supported by Polish Committee for Scientific Research Grant 6 P04 009 16. ‡ The coordinates have been deposited in the Protein Data Bank (code 2JPT). * To whom correspondence should be addressed. Telephone: (48 22) 592-23-71. Fax: (48 22) 823-71-94. E-mail: ajb@ibb.waw.pl. § Polish Academy of Sciences. | National Institute of Chemistry. 1 Abbreviations: ME, -mercaptoethanol; DSS-d4, 3-trimethylsilyl- 2,2,3,3-tetradeuteropropionic acid sodium salt; EDTA, ethylenedi- aminetetraacetic acid; HSQC, heteronuclear single quantum coherence; HPLC, high performance liquid chromatography; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; S100A1-ME, mixed disulfide of apo-S100A1 protein with -mercaptoethanol; TRIS- d 11, perdeuterated 2-amino-2-(hydroxymethyl)1,3-propanediol. 640 Biochemistry 2008, 47, 640-650 10.1021/bi701762v CCC: $40.75 © 2008 American Chemical Society Published on Web 12/19/2007