Crystal Structure of the Disintegrin Heterodimer from Saw-Scaled Viper (Echis carinatus) at 1.9 Å Resolution Sameeta Bilgrami, ‡ Savita Yadav, ‡ Punit Kaur, ‡ Sujata Sharma, ‡ Markus Perbandt, § Christian Betzel, § and Tej P. Singh* ,‡ Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110 029, India, and Institute of Medical Biochemistry and Molecular Biology, Hamburg 22603, Germany ReceiVed May 9, 2005; ReVised Manuscript ReceiVed June 27, 2005 ABSTRACT: Disintegrins constitute a family of potent polypeptide inhibitors of integrins. Integrins are transmembrane heterodimeric molecules involved in cell-cell and cell-extracellular matrix interactions. They are involved in many diseases such as cancer and thrombosis. Thus, disintegrins have a great potential as anticancer and antithrombotic agents. A novel heterodimeric disintegrin was isolated from the venom of saw-scaled viper (Echis carinatus) and was crystallized. The crystals diffracted to 1.9 Å resolution and belonged to space group P4 3 2 1 2. The data indicated the presence of a pseudosymmetry. The structure was solved by applying origin shifts to the disintegrin homodimer schistatin solved in space group I4 1 22 with similar cell dimensions. The structure refined to the final R cryst /R free factors of 0.213/0.253. The notable differences are observed between the loops, (Gln39-Asp48) containing the important Arg42- Gly43-Asp44, of the present heterodimer and schistatin. These differences are presumably due to the presence of two glycines at positions 43 and 46 that allow the molecule to adopt variable conformations. A comparative analysis of the surface-charge distributions of various disintegrins showed that the charge distribution on monomeric disintegrins occurred uniformly over the whole surface of the molecule, while in the dimeric disintegrins, the charge is distributed only on one face. Such a feature may be important in the binding of two integrins to a single dimeric disintegrin. The phylogenetic analysis developed on the basis of amino acid sequence and three-dimensional structures indicates that the protein diversification and evolution presumably took place from the medium disintegrins and both the dimeric and short disintegrins evolved from them. Disintegrins are a family of small cysteine-rich proteins, found in Viperidae and Crotalidae snake venoms that bind to integrins of the 1 and 3 classes (1, 2). They can be classified into small, medium, long, and dimeric disintegrins depending on the size and the number of cysteines. Short disintegrins are 49-50 amino acids long and have 8 cysteines (3). The medium disintegrins contain approximately 70 amino acids with 12 cysteines. Most of the polypeptides that have been characterized so far belong to the class of medium disintegrins (4-10). Long disintegrins have ∼80 amino acid residues including 14 cysteines (11, 12). The dimeric disintegrins have been identified recently, and their number is increasing gradually. Each of the monomers of the dimeric disintegrins has approximately 67 amino acid residues including 10 cysteines. Each monomer contains two unpaired cysteine residues at the N terminus that form two disulfide bridges with the other monomer to generate the dimer (13- 21). An exception is bilitoxin-I (22), a homodimeric disin- tegrin, each monomer of which has 15 cysteine residues. The binding of disintegrins to integrins is typically characterized by the presence of the Arg-Gly-Asp motif, although muta- tions of Arg and Gly have been reported (15-19). The Asp residue is always conserved (23). The Arg-Gly-Asp is located at a long surface-exposed loop that extends out of the molecule and is highly flexible. Integrins are present on the cell surfaces and are known to be involved in cell-cell interactions and cell-extracellular matrix interactions (24, 25). The integrins have been reported to participate in a number of important biological processes such as angiogen- esis, tumor invasion, inflammatory responses, platelet ag- gregation, and tissue repair (26-31). It has been reported that the disintegrins interfere with the functions of integrins as antagonists (1) and hence act as anticancer and antithrom- botic agents. In view of such important functions of disintegrins, the elucidation of their detailed three-dimen- sional structures is necessary so that the mechanism of their actions could be understood at the structural level. It will help in the design of synthetic polypeptides as potent antagonists of integrins. Unfortunately, the structural infor- mation about disintegrins is still scanty because only one crystal (32) and a few NMR structures (33-37) of mono- meric medium disintegrins and only one structure of a dimeric disintegrin, schistatin, are known (23). The most important feature of dimeric disintegrins is having two Arg- Gly-Asp-binding sites that diverge away from each other in the dimer. It has been shown that, in comparison to monomers, the dimeric disintegrins show increased affinity for integrins (38). We have isolated a novel heterodimer of * To whom correspondence should be addressed. Telephone: 91- 11-2658-8931. Fax: 91-11-2658-8663. E-mail: tps@aiims.aiims.ac.in. ‡ All India Institute of Medical Sciences. § Institute of Medical Biochemistry and Molecular Biology. 11058 Biochemistry 2005, 44, 11058-11066 10.1021/bi050849y CCC: $30.25 © 2005 American Chemical Society Published on Web 07/28/2005