Hydropathic Complementarity Research Articles

Determination of the Putative Binding site for Fibronectin on Platelet Glycoprotein IIb-IIIa Complex through a Hydropathic Complementarity Approach

We have applied the principle of complementary hydropathy to the prediction of the binding site for fibronectin (FN) and for the alpha-chain of fibrinogen in the platelet receptor complex glycoprotein (GP) IIb-IIIa. Since both ligands bind to it through their respective RGDS (Arg-Gly-Asp-Ser) domains and since both have been cloned, we were able to deduce the amino acid sequence of the binding site from the nucleotide sequence coding for RGDS in both proteins. The deduced peptides were very similar. Antibodies raised against a synthetic peptide WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) deduced from the cloned rat FN RGDS domain block ADP-mediated platelet aggregation; this block can be overcome by additional fibrinogen. In Western blots of whole cell platelet extracts run under reducing conditions, this antibody binds to a 108-kDa band. It also binds to affinity-purified GP IIIa. Furthermore, it reacts strongly with GP IIIa immunoprecipitated by a commercially available anti-GP IIb-IIIa monoclonal antibody. Binding of affinity-purified GP IIb-IIIa complex to fibronectin is inhibited by the 110-kDa FN fragment. Similar inhibitions can be effected by WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) and GAVSTA (Gly-Ala-Val-Ser-Thr-Ala) predicted from the rat and human fibronectin nucleotide sequences, respectively. GAGSTA (Gly-Ala-Gly-Ser-Thr-Ala) and GARSTA (Gly-Ala-Arg-Ser-Thr-Ala) related to the human peptide but with discrepant hydropathies are noninhibitory.

Recognition Properties of Peptides Hydropathically Complementary to Residues 356–375 of the c-raf Protein

Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.

Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach.

It has been shown that a significant correlation is seen when the hydropathy scores of amino acids encoded by the coding strand of double-helical DNA are plotted against those of the noncoding strand. Thus, peptides encoded by complementary DNA strands might form amphiphilic structures and bind one another. We have used this approach to study the interaction between fibronectin (FN) and its cell receptor. Taking into consideration the nucleotide sequence from published rat cDNA clones that corresponds to the cell binding site (Arg-Gly-Asp-Ser) in the FN molecule, the deduced amino acid sequence found for the putative receptor binding site was Trp-Thr-Val-Pro-Thr-Ala. This peptide was chemically synthesized and coupled to an AH-Sepharose column. FN bound appreciably to this column and was eluted much more efficiently by a solution of Arg-Gly-Asp-Ser-containing peptide than by a solution of related but inactive Arg-Gly-Glu-Ser-containing peptide. Binding of labeled FN to receptor-rich MG63 human osteosarcoma cells was inhibited by the hexapeptide. The hexapeptide Gly-Ala-Val-Ser-Thr-Ala predicted similarly from the nucleotide sequence of human FN was equally efficient in such inhibition. Antibodies produced against Trp-Thr-Val-Pro-Thr-Ala recognized with equal efficiency Gly-Ala-Val-Ser-Thr-Ala in an ELISA assay. Furthermore, they were able to recognize a single 140-kDa band in whole-cell extracts from Chinese hamster ovary cells, attesting to their specificity. Identification of the recognized protein was provided by showing that this antibody was also able to bind to affinity-purified FN receptor from human osteosarcoma MG63 cells.