Structural Insight into Human Zn 2+ -Bound S100A2 from NMR and Homology Modeling Antonio Randazzo,* Christian Acklin,† Beat W. Scha ¨ fer,† Claus W. Heizmann,† and Walter J. Chazin* ,1 *Department of Biochemistry and Department of Physics and Center for Structural Biology, Vanderbilt University, 896 MRB II, Nashville, Tennessee 37232-0146; and †Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, CH 8032 Zurich, Switzerland Received September 24, 2001 The S100 subfamily of EF-hand proteins is distin- guished by the binding of Zn 2 in addition to Ca 2 . In an effort to understand the role of Zn 2 in modulating the activity of S100 proteins, we have carried out het- eronuclear NMR studies of Zn 2 -bound S100A2 and ob- tained near complete resonance assignments. This analysis revealed an equilibrium between multiple isoforms due to cis-trans isomerism of proline residues in flexible regions of the protein. The secondary struc- ture of S100A2 has been determined based on the NMR chemical shift index (CSI) technique. The protein is found to possess essentially the same secondary struc- ture found in other S100 proteins such as S100A6 and S100B. Homology models have been built based on the high resolution three-dimensional structures of other S100 proteins. The models predict two Zn 2 binding clusters, one involving residues His17-Cys21-Cys93 and the other Cys2-His39, and with Cys86 participat- ing in either the N-terminal or the C-terminal binding site. © 2001 Academic Press Key Words: S100A2; proline isomerism; calcium- binding protein; zinc-binding protein; NMR; assign- ment; secondary structure; homology model. The S100 protein family constitutes a large subgroup of the EF-hand family of Ca 2+ -binding proteins (CaBPs). These proteins contain two distinct helix- loop-helix (EF-hand) motifs joined by a central linker region. Unlike ubiquitous calmodulin, the expression of S100 proteins is cell- and tissue-specific; over 20 different S100 proteins have been discovered to date. S100 proteins have been implicated in pleiotropic cel- lular events, with specific functions for each of the family members, such as cell cycle regulation, cell growth, cell differentiation, and mobility. Deregulated expression of S100 genes is a hallmark of a wide range of human diseases. A number of S100 proteins have physiologically rel- evant Zn 2+ affinities, and Zn 2+ is known to modulate both the spectroscopic properties and the biological activities of some of these proteins. In fact, certain S100 proteins (e.g., S100A2, S100A3) have very low Ca 2+ affinities but much higher Zn 2+ affinities, suggest- ing that Zn 2+ controls their activities. The structure of one S100 protein (S100A7) with Zn 2+ -bound has been published (1). The overall fold of Ca 2+ -bound S100A7 in the absence and presence of Zn 2+ is the same symmetric homodimer as that ob- served in all other S100 protein structures determined to date. In this structure, there is a canonical Ca 2+ - filled site in each of the two C-terminal EF-hands of the S100A7 dimer, and two Zn 2+ sites at symmetrically disposed locations at the interface between the two subunits. For each of the Zn 2+ sites, two His ligands are provided by one subunit and a third His and a biden- tate Asp by the other. The binding of Zn 2+ is seen to have only very limited effect on the well-packed helical core of the protein, with substantial structural changes only for three consecutive residues in the Ca 2+ -free N-terminal binding loop. There were virtually no ef- fects on the C-terminal EF-hand, which remains Ca 2+ - filled in both structures. The identification of the Zn 2+ site in S100A7 combined with consideration of se- quence homologies suggests that Zn 2+ should be bound in homologous sites in S100A9 and S100A12 and pos- sibly in S100A8 and S100B. S100A2, S100A6, and other S100 proteins do not bind Zn 2+ in this man- ner due to substitutions at the critical Zn 2+ -ligating residues. S100A2 has attracted particular interest based on its potential function as tumor suppressor-related gene (2). Human S100A2 (also known as S100L) was first detected at high levels in a subset of cells in lung and kidney and at moderate levels in liver, cardiac muscle, 1 To whom correspondence should be addressed. Fax: 615-936- 2211. E-mail: walter.chazin@vanderbilt.edu. Biochemical and Biophysical Research Communications 288, 462– 467 (2001) doi:10.1006/bbrc.2001.5793, available online at http://www.idealibrary.com on 462 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.