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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The procollagen C-proteinase (PCP) is a zinc peptidase of the astacin family and the metzincin superfamily. The enzyme removes the C-terminal propeptides of fibrillar procollagens and activates other matrix proteins. Besides its catalytic protease domain, the procollagen C-proteinase contains several C-terminal CUB modules (named after complement factors C1r and C1s, the sea urchin UEGF protein, and BMP-1) and EGF-like domains. The two major splice forms of the C-proteinase differ in their overall domain composition. The longer variant, termed mammalian tolloid (mTld, i.e., PCP-2), has the protease-CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5 composition, whereas the shorter form termed bone morphogenetic protein 1 (BMP-1, i.e., PCP-1) ends after the CUB3 domain. Two related genes encode proteases similar to mTld in humans and have been termed mammalian tolloid like-1 and -2 (mTll-1 and mTll-2, respectively). For mTll-1, it has been shown that it has C-proteinase activity. We demonstrate that recombinant EGF1-CUB3, CUB3, CUB3-EGF2, EGF2-CUB4, and CUB4-CUB5 modules of the procollagen C-proteinase can be expressed in bacteria and adopt a functional antiparallel beta-sheet conformation. As shown by surface plasmon resonance analysis, the modules bind to procollagen I in a 1:1 stoichiometry with dissociation constants (K(D)) ranging from 622.0 to 1.0 nM. Their binding to mature collagen I is weaker by at least 1 order of magnitude. Constructs containing EGF domains bind more strongly than those consisting of CUB domains only. This suggests that a combination of CUB and EGF domains serves as the minimal functional unit. The binding affinities of the EGF-containing modules for procollagen increase in the order EGF1-CUB3< CUB3-EGF2 < EGF2-CUB4. In the context of the full length PCP, this implies that a given module has an affinity that continues to increase the more C-terminally the module is located within the PCP. The tightest binding module, EGF2-CUB4 (K(D) = 1.0 nM), is only present in mTld, which might provide a quantitative explanation for the different efficiencies of BMP-1 and mTld in procollagen C-proteinase activity.

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

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