and the ¶Institut fü r Physikalische Chemie, Schlossplatz 7, Universitä t Mü nster, 48149 Mü nster, GermanyWe have recombinantly expressed a soluble form of human ␣ 2 ␤ 1 integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble ␣ 2 ␤ 1 integrin binds to its collagen ligands the same way as the wildtype ␣ 2 ␤ 1 integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrinactivating antibody. However, it does not bind to rhodocytin, a postulated agonist of ␣ 2 ␤ 1 integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble ␣ 2 ␤ 1 integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble ␣ 2 ␤ 1 integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble ␣ 2 ␤ 1 integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to ␣ 2 ␤ 1 integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with ␣ 2 ␤ 1 integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to ␣ 2 ␤ 1 integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking ␣ 2 ␤ 1 integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, ␣ 2 ␤ 1 integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, ␣ 2 ␤ 1 integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit ␣ 2 ␤ 1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.Integrins are cell adhesion molecules that consist of two noncovalently associated subunits, ␣ and ␤ (for review see Refs. 1 and 2). The subfamily of integrins sharing the ␤ 1 subunit are well known receptors for extracellular matrix molecules, such as collagens, laminins, and fibronectin. The subfamily of ␤ 3 subunit containing cytoadhesins comprise the platelet integrin, ␣ IIb ␤ 3 which binds fibrinogen/fibrin (3) and the vitronectin receptor ␣ V ␤ 3 . The latter ones, along with several ␤ 1 integrin, such as the fibronectin receptor ␣ 5 ␤ 1 integrin, recognize a linear arginyl-glycyl-aspartyl sequence within their respective ...

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Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.

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