Reconstructing the Binding Site of Factor Xa in Trypsin Reveals Ligand-induced Structural Plasticity

Reyda, Sabine; Sohn, Christian; Klebe, G.; Rall, Kathrin; Ullmann, Dirk; Jakubke, Hans‐Dieter; Stubbs, Milton T.

doi:10.1016/s0022-2836(02)01337-2

Cited by 26 publications

(30 citation statements)

References 74 publications

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“…We have previously observed all these phenomena for variants of trypsin designed to behave like the closely related serine proteinase Factor Xa. 22,23 Clearly, more experimental data are required to understand the complex mechanisms driving protein-ligand affinity and the evolution of substrate specificity. Results from directed evolution may yield important clues for future tailoring of enzymatic reactions.…”

Section: Resultsmentioning

confidence: 99%

Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2+-Dependent O-Methyltransferases

Kopycki

Rauh

Chumanevich

et al. 2008

Journal of Molecular Biology

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Section: Resultsmentioning

confidence: 99%

Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2+-Dependent O-Methyltransferases

Kopycki

Rauh

Chumanevich

et al. 2008

Journal of Molecular Biology

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“…The construction of factor Xa-trypsin hybrids has been reported elsewhere: [25,26] both the rat and bovine trypsin have been used as a starting point for site-directed mutagenesis to produce the stepwise transfer of the factor Xa binding site into trypsin. The mutants obtained were characterized by crystal structure analysis and enzyme kinetics with respect to the binding of different inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Understanding Binding Selectivity toward Trypsin and Factor Xa: the Role of Aromatic Interactions

et al. 2007

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A congeneric series of four bis-benzamidine inhibitors sharing a dianhydrosugar isosorbide scaffold in common has been studied by crystal structure analysis and enzyme kinetics with respect to their binding to trypsin and factor Xa. Within the series, aromatic interactions are an important determinant for selectivity discrimination among both serine proteases. To study the selectivity-determining features in detail, we used trypsin mutants in which the original binding site is gradually substituted to finally resemble the factor Xa binding pocket. The influence of these mutations has been analyzed on the binding of the closely related inhibitors. We present the crystal structures of the inhibitor complexes obtained by co-crystallizing an "intermediate" trypsin mutant. They could be determined to a resolution of up to 1.2 A, and we measured the inhibitory activity (K(i)) of each ligand against factor Xa, trypsin, and the various mutants. From these data we were able to derive a detailed structure-activity relationship which demonstrates the importance of aromatic interactions in protein-ligand recognition and their role in modulating enzyme selectivity. Pronounced preference is experienced to accommodate the benzamidine anchor with meta topology in the S(1) specificity pocket. One ligand possessing only para topology deviates strongly from the other members of the series and adopts a distinct binding mode addressing the S(1)' site instead of the distal S(3)/S(4) binding pocket.

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“…Furthermore, estimation of secondary structure of Ole e 6 performed by theoretical methods and based on the amino acid sequence (46) gave an ␣-helix conformation for the segment at positions 34 -47. It is likely that the C-terminal tail adopts a defined structure upon binding to a ligand or receptor or in different experimental conditions from those used here, as has been described for other proteins (47,48).…”

Section: Discussionmentioning

confidence: 89%

NMR Solution Structure of Ole e 6, a Major Allergen from Olive Tree Pollen

Treviño

García-Mayoral²,

Barral³

et al. 2004

Journal of Biological Chemistry

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Ole e 6 is a pollen protein from the olive tree (Olea europaea) that exhibits allergenic activity with a high prevalence among olive-allergic individuals. The threedimensional structure of Ole e 6 has been determined in solution by NMR methods. This is the first experimentally determined structure of an olive tree pollen allergen. The structure of this 50-residue protein is based on 486 upper limit distance constraints derived from nuclear Overhauser effects and 24 torsion angle restraints. The global fold of Ole e 6 consists of two nearly antiparallel ␣-helices, spanning residues 3-19 and 23-33, that are connected by a short loop and followed by a long, unstructured C-terminal tail. Viewed edge-on, the structured N terminus has a dumbbell-like shape with the two helices on the outside and with the hydrophobic core, mainly composed of 3 aromatic and 6 cysteine residues, on the inside. All the aromatic rings lie on top of and pack against the three disulfide bonds. The lack of thermal unfolding, even at 85°C, indicates a high conformational stability. Based on the analysis of the molecular surface, we propose five plausible epitopes for IgE recognition. The results presented here provide the structural foundation for future experiments to verify the antigenicity of the proposed epitopes, as well as to design novel hypoallergenic forms of the protein suitable for diagnosis and treatment of type-I allergies. In addition, three-dimensional structure features of Ole e 6 are discussed to provide a basis for future functional studies.

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Reconstructing the Binding Site of Factor Xa in Trypsin Reveals Ligand-induced Structural Plasticity

Cited by 26 publications

References 74 publications

Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2+-Dependent O-Methyltransferases

Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2+-Dependent O-Methyltransferases

Understanding Binding Selectivity toward Trypsin and Factor Xa: the Role of Aromatic Interactions

NMR Solution Structure of Ole e 6, a Major Allergen from Olive Tree Pollen

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