Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold |
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Authors: | Lee Grace; Chan Winnie; Hurle Mark R; DesJarlais Renee L; Watson Felicia; Sathe Ganesh M; Wetzel Ronald |
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Affiliation: | 1Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals 709 Swedeland Road, King of Prussia, PA 19406, USA
2Molecular Genetics Department, SmithKline Beecham Pharmaceuticals 709 Swedeland Road, King of Prussia, PA 19406, USA
3Physical and Structural Chemistry Department SmhhKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA
4 Present address: PerSeptive Biosystems, Cambridge, MA, USA |
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Abstract: | In order to probe the structural constraints on binding of RGDsequences to the platelet receptor IIbß3 we have usedrecombinant DNA techniques to install the RGD sequence intopresentation scaffolds, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinVL domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to IIbß3 up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 4257 or 4455from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC50s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto IIbß3, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents. |
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Keywords: | fibronectin/ immunoglobulin light chain variable domain/ interleukin-1ß / kistrin |
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