Solution Structure of Human Mts1 (S100A4) As Determined by NMR Spectroscopy † Kristen M. Vallely, ‡ Richard R. Rustandi, ‡ Karen C. Ellis, ‡ Olga Varlamova, § Anne R. Bresnick,* ,§ and David J. Weber* ,‡ Department of Biochemistry and Molecular Biology, UniVersity of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201, and Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park AVenue, Bronx, New York 10461 ReceiVed May 16, 2002; ReVised Manuscript ReceiVed August 7, 2002 ABSTRACT: Mts1 is a member of the S100 family of Ca 2+ -binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca 2+ -dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1′, and 4′) to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca 2+ -binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel -sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF- hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family. Mts1, also known as S100A4, p9Ka, calvasculin, metasta- sin, 18A2, pEL98, and CAPL, belongs to the S100 family of Ca 2+ -binding proteins (1-4). S100 proteins are small (10- 12 kDa per subunit), acidic proteins that are characterized by their solubility in 100% ammonium sulfate (5). There are currently 21 known S100 family members (Figure 1), most of which are expressed in a highly tissue specific manner (1, 3, 6). While mts1 is expressed in a wide range of normal tissues (1), high expression levels correlate with the metastatic potential of tumor cells. For example, meta- static rat and mouse mammary tumor cells contain higher levels of mts1 than nonmetastatic cells (7), and the level of mts1 expression is higher in malignant human breast tumors than in benign tumors (8), which correlates strongly with poor patient survival (9, 10). Furthermore, overexpression of mts1 in nonmetastatic rat and mouse mammary tumor cells confers a metastatic phenotype, whereas in metastatic cells, a reduction in the level of mts1 expression suppresses metastasis (11, 12). In transgenic mouse models of breast cancer, overexpression of mts1 in murine mammary tumor virus-induced (13) or in neu oncogene-induced (14) benign mammary tumors induces lung metastasis. These findings directly implicate mts1 in the establishment of the metastatic phenotype. As with other S100 family members, mts1 activity is regulated by intracellular calcium ion concentrations. Mts1 displays Ca 2+ -dependent interactions with target proteins that include the p53 tumor suppressor protein, non-muscle myosin II, tropomyosin, and F-actin, which may be related to its role in cancer and metastasis (15-18). This Ca 2+ -dependent regulation occurs in mts1 via two helix-loop-helix Ca 2+ - binding domains known as EF-hands (19). As with other S100 proteins, the C-terminal binding domain of mts1 (typical EF-hand) contains 12 residues and binds calcium with a higher affinity (K d < 50 µM) than the N-terminal domain (pseudo-EF-hand or S100-hand), which contains 14 residues (K d > 50 µM) (20, 21). Structural studies of several S100 proteins (i.e., S100B, S100A1, and calcyclin) indicate that the orientation of helix 3 in the typical EF-hand changes upon calcium binding (22-25). However, the magnitude of the conformational change differs among the S100 proteins due to variations in the position of helix 3 in the apo state (23, 25). For example, helix 3 of S100B undergoes a very large conformational change when going from the apo to the calcium-bound state, whereas the change in the orienta- † This work was supported by the National Institutes of Health (Grant GM58888 to D.J.W.), the American Cancer Society (Grant RPG0004001- CCG to D.J.W.), and the Department of Defense Breast Cancer Research Program (Grant DAMD17-01-1-0122 to A.R.B.). This work also made use of the UMB NMR facility that was supported by the Shared Instrumentation Grant Program (Grants RR10441, RR15741, and RR16812 to D.J.W.). * To whom correspondence should be addressed. D.J.W.: telephone, (410) 706-4354; fax, (410) 706-0458; e-mail, dweber@umaryland.edu. A.R.B.: telephone, (718) 430-2741; fax, (718) 430-8565; e-mail, bresnick@aecom.yu.edu. ‡ University of Maryland School of Medicine. § Albert Einstein College of Medicine. 12670 Biochemistry 2002, 41, 12670-12680 10.1021/bi020365r CCC: $22.00 © 2002 American Chemical Society Published on Web 09/27/2002