Solution structure of Calmodulin-W-7 complex: the basis of diversity in molecular recognition¹

Abstract

The solution structure of calcium-bound calmodulin (CaM) complexed with an antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), has been determined by multidimensional NMR spectroscopy. The structure consists of one molecule of W-7 binding to each of the two domains of CaM. In each domain, the W-7 chloronaphthalene ring interacts with four methionine methyl groups and other aliphatic or aromatic side-chains in a deep hydrophobic pocket, the site responsible for CaM binding to CaM-dependent enzymes such as myosin light chain kinases (MLCKs) and CaM kinase II. This competitive binding at the same site between W-7 and CaM-dependent enzymes suggests the mechanism by which W-7 inhibits CaM to activate the enzymes. The orientation of the W-7 naphthalene ring in the N-terminal pocket is rotated approximately 40 degrees with respect to that in the C-terminal pocket. The W-7 ring orientation differs significantly from the Trp800 indole ring of smooth muscle MLCK bound to the C-terminal pocket and the phenothiazine ring of trifluoperazine bound to the N or C-terminal pocket. These comparative structural analyses demonstrate that the two hydrophobic pockets of CaM can accommodate a variety of bulky aromatic rings, which provides a plausible structural basis for the diversity in CaM-mediated molecular recognition.

Introduction

Calmodulin (CaM) is a ubiquitous Ca²⁺-binding protein of 148 residues that plays an important role in the Ca²⁺-dependent signaling pathways of eukaryotic cells Klee and Vanaman 1982, Means et al 1982. A wide range of physiological processes are mediated by CaM through Ca²⁺-dependent regulation of target enzymes such as myosin light chain kinase (MLCK), CaM-dependent kinases, protein phosphatase calcineurin, phosphodiesterase, nitric oxide synthase, Ca²⁺-ATPase pumps as well as cytoskeletal structural proteins Means et al 1991, Vogel 1994, James et al 1995. This broad specificity is manifested in the CaM binding regions of these target proteins, which differ significantly in their amino acid sequences. Related to this, a variety of small organic molecules, antagonists with distinct chemical structures have been found to inhibit the CaM-mediated processes by direct interaction with calcium-loaded CaM, Ca²⁺-CaM (Hait & Lazo, 1986). To understand this broad specificity, detailed structural analyses of Ca²⁺-CaM complexes with target proteins and small molecules are crucial. Furthermore, such analyses may improve our knowledge of how CaM mediates such calcium signaling processes especially in neural cells and how one can block a specific pathway, that would ultimately lead to therapeutic treatments for abnormal neural signal transduction.

CaM is composed of two domains (N-terminal and C-terminal domains) connected by a flexible linker, and has two EF-hand Ca²⁺-binding sites in each domain (Nakayama & Kretsinger, 1994). The three-dimensional structures of calcium-free (Ca²⁺-free) CaM and Ca²⁺-CaM have already been determined by NMR and X-ray crystallography Zhang et al 1995b, Kuboniwa et al 1995, Finn et al 1995, Babu 1985, Kretsinger et al 1986. A comparison of both forms reveals that Ca²⁺ binding to apo CaM changes the interhelical angle of the two helices of each EF-hand such that CaM undergoes a transition from the “closed” conformation to the “open” conformation Zhang et al 1995b, Kuboniwa et al 1995. This conformational change results in the creation of a hydrophobic pocket on the surface of each domain Babu 1985, Kretsinger et al 1986, which in turn is essential for CaM to bind target enzymes in a Ca²⁺-dependent manner Ikura et al 1992, Meador et al 1992, Meador et al 1993.

A number of three-dimensional structures of Ca²⁺-CaM complexed with peptides from target enzymes have been determined. The first was an NMR structure of the complex with a 26-residue peptide from skeletal muscle (sk) MLCK (Ikura et al., 1992), which was followed by the crystal structures of complexes with a 20-residue peptide from smooth muscle (sm) MLCK (Meador et al., 1992), and a 25-residue peptide from brain CaM kinase IIα (Meador et al., 1993). In each complex, the peptide adopts a helical conformation and the flexible domain linker of Ca²⁺-CaM allows its domains to clamp the target peptide.

CaM antagonists are frequently used to study Ca²⁺-CaM-dependent activation of various enzymes. In particular, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)-10 H-phenothiazine (trifluoperazine, TFP) have been extensively used for this purpose (Figure 1). Crystal structures of Ca²⁺-CaM complexed with TFP have been reported by two groups Cook et al 1994, Vandonselaar et al 1994, but their results differed significantly. In the crystal structure reported by Cook et al. (1994), only one TFP molecule was identified binding to the C-terminal hydrophobic pocket of CaM, while the crystal structure reported by Vandonselaar et al. (1994) had four TFP molecules: two of them binding to the hydrophobic pocket in each domain of CaM, respectively, and the other two located in the cleft between the CaM domains. Comparisons show that the orientation of the TFP phenothiazine ring binding to the C-terminal hydrophobic pocket of Ca²⁺-CaM differs by nearly 180° between the two structures. To further complicate matters, a previous NMR study indicated that two TFP molecules bind to one Ca²⁺-CaM molecule in solution Klevit et al 1981, Dalgarno et al 1984, Craven et al 1996. The stoichiometry of W-7 binding to Ca²⁺-CaM is also in a similarly complicated situation. Gel filtration experiments reported that three W-7 molecules bind to Ca²⁺-CaM with a dissociation constant of 11 μM (Hidaka et al., 1979), whereas a recent NMR study on the interaction of the CaM C-terminal half fragment with N-(8-aminooctyl)-5-iodo-1-naphthalene sulfonamide (J-8), a derivative of W-7, suggested that one J-8 molecule binds to the C-terminal domain with the terminal amino group of J-8 being highly mobile. However, the position and orientation of the J-8 naphthalene ring in the hydrophobic pocket remains unclear (Craven et al., 1996).

In this paper, we report the first three-dimensional structure of Ca²⁺-CaM complexed with W-7, using heteronuclear multidimensional NMR spectroscopy. The binding site and the orientation of W-7 in each domain of Ca²⁺-CaM have been clearly defined. Our present structure provides the first view of Ca²⁺-CaM recognizing a naphthalenesulfonamide derivative. The structural comparison of this structure with other published structures of CaM-peptide and CaM-ligand complexes helps to reveal the basis of diversity in molecular recognition by Ca²⁺-CaM.