Ligand-bound Conformation Research Articles

Monoclonal Antibody 9EG7 Defines a Novel β1 Integrin Epitope Induced by Soluble Ligand and Manganese, but Inhibited by Calcium

The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin beta 1 chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9051-9055). Here we show that treatment of beta 1 integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This epitope was also induced upon EGTA treatment to remove calcium, and the addition of calcium inhibited 9EG7 epitope induction by manganese or by ligand. Further emphasizing the importance of the 9EG7 epitope, the 9EG7 antibody itself stimulated adhesion mediated by multiple beta 1 integrins, and conversely, ligands for alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, and alpha 5 beta 1 all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits beta 1 integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances beta 1 integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other beta 1-stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliable detect the ligand-bound conformation of beta 1 integrins, its expression does not always correlate with integrin "activation". Finally, mouse/chicken beta 1 chimeric molecules were used to map the 9EG7 epitope to beta 1 residues 495-602 within the cysteine-rich region, and antibody cross-blocking studies showed that the 9EG7 epitope is distinct from all previously defined human beta 1 epitopes.

Development of a Receptor Peptide Antagonist to Human γ-Interferon and Characterization of Its Ligand-bound Conformation Using Transferred Nuclear Overhauser Effect Spectroscopy

Polyclonal anti-idiotypic antibody raised to a synthetic discontinuous peptide derived from the human gamma-interferon (huIFN-gamma) sequence recognizes soluble human gamma-interferon receptor (Seelig, G. F., Prosise, W. W., and Taremi, S. S. (1994) J. Biol. Chem. 269, 358-363). We sought to use this reagent to identify a ligand-binding domain within IFN-gamma-receptor. To do this, the neutralizing anti-idiotypic antibody was used to probe overlapping linear peptide octamers of the extracellular domain of the huIFN-gamma receptor. A 22-amino-acid residue receptor segment 120-141 identified by the antibody was synthesized. CD and NMR analysis indicates that peptide 120-141 has no apparent secondary structure in water or in water containing 50% trifluoroethanol. The synthetic receptor peptide inhibited huIFN-gamma induced expression of HLA/DR antigen on Colo 205 cells with an approximate IC50 of 35 microM. Immobilized peptide specifically bound recombinant huIFN-gamma but did not bind human granulocyte-macrophage colony-stimulating factor on a microtiter plate in a direct binding enzyme-linked immunosorbent assay. The binding results are supported by two-dimensional transferred nuclear Overhauser effect (TRNOE) NMR data obtained on the peptide in the presence of recombinant huIFN-gamma. Characterization of the conformation of the bound peptide by TRNOE suggests that this peptide assumes a distinct conformation. Intramolecular interactions within the bound peptide were detected at two non-contiguous regions and at a third region comprising a beta-turn formed by the sequence DIRK. We believe that this represents the structure of the receptor within the ligand-binding domain.

The nature of ferrous haem protein complexes prepared by photolysis

Ultra-low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the ground-state magnetic properties of deoxymyoglobin, -haemoglobin and reduced cytochrome c oxidase, all of which contain the high-spin ferrous haem ( S = 2). In addition the MCD properties have been studied of the fully photolysed forms, frozen at 1.5 K, of the carbonmonoxy derivatives of these three haemoproteins. In these cases the use of very low temperatures prevents both the thermal recombination of CO and haem and the relaxation of the haem and its associated protein-bound ligand, imidazole. MCD spectra and magnetisation curves show clearly that the state of the haem produced by photolytic removal of the CO ligand and prior to relaxation of the haem centre is the high-spin ( S = 2) ferrous form. However, the zero-field splitting parameters of the ground states of the deoxy and photolysed-CO forms of Hb are quantitatively different. This attests to the presence of significant conformational changes at the haem which occur after the sixth ligand has been removed. The same is found by comparison of the MCD of deoxy-Hb and photolysed-CO-Hb. This is not unexpected, since various physical techniques have revealed that the ligand-bound and deoxy conformations of these two proteins are different at the haem site. We have shown that MCD spectroscopy can detect this difference sensitively. However, in marked contrast the MCD properties of the high-spin ferrous ( S = 2) cytochrome a 3in the reduced and the photolysed CO form of cytochrome c oxidase are quite indistinguishable. This is attributed to the absence of any significant structural or conformational change at the cytochrome a 3 site when the ligand CO becomes bound (or unbound). This suggests that there is no iron movement in- or out-of-plane or any haem doming in cytochrome a 3 when CO is photolysed off. These observations account for the lower binding affinity of CO for cytochrome c oxidase and the absence of a ‘quickly reacting’ form of this enzyme.