(Rcccived 25 Julyi2 October 1995) ~ E3B 95 122XilThe human CSa receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human CSa. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asnll9, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the CSaR (Asp37, Asp82, Arg175, Arg206, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282--+Ala) or partial loss of ligandbinding capacity (Argl75+Ala, Arg206+Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln206]CSaR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population ( < l o % ) of high-affinity sites ( K p 2 nM), which was functionally active at 20 nM in the heterologous Xencyus oocyte expression system after coexpression of Ga-16. The number of high-affinity sites of wild-type and [Gln206]C5aR in 293 cells could be up-regulated by coexpression of Gia-2 and downregulated by GTP[ yS ]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg206 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human CSaR by affecting the intracellular G-protein coupling.Keywords: C5a receptor; receptor structure; guanine-nucleotide-binding-regulatory-protein coupling; site-directed mutagenesis.Human complement factor C5a (C5a) is a 74-amino-acid glycopeptide which is generated by proteolytic cleavage from the fifth component of complement. Binding of the CSa peptide to its receptor triggers major cell activation and degranulation events, such as chemotaxis of polymorphonuclear cells, induction of the oxidative burst, histamine and interleukin release rrom monocytes. C5a is therefore thought to be an important inflammation-promoting agent. It interacts with the C5a receptor (CSaR) found in the membrane of polymorphonuclear cells, monocytes, macrophages and the histiocytic U937 and HL-60 cell lines (for a recent review, see KBhl and Bitter-Suermann, 1993, and the literature cited therein).
The T <--> R transition in the insulin hexamer is an outstanding model for protein structural changes in terms of its extent and complexity: the limiting structures T(6), T(3)R(3) and R(6) have been defined by X-ray crystallography. The transition occurs cooperatively within trimers. It involves displacements of >30 A and a secondary structural rearrangement of 15% of the peptide chain between extended and helical conformations. Experimental data for the transition are plentiful. Theoretical methods to simulate pathways without constraints would never succeed with such substantial transitions. We have developed two approaches, targeted energy minimization (TEM) and targeted molecular dynamics (TMD). Previously successful in simulating the T <--> R transition of the insulin monomer, these procedures are also shown here to be effective in the hexamer. With TMD, more conformational space is explored and pathways are found at 500 kJ/mol lower energy than with TEM. Because the atoms have to meet distance constraints in sum rather than individually, a high degree of conformational freedom and independence is implied. T(6) --> T(3)R(3) and T(3)R(3) --> T(6) pathways do not coincide because the transformation is directed. One subunit enters a dead end pathway in one direction of the TMD simulation, which shows that constraint and freedom are critically balanced. The ensemble of productive pathways represents a plausible corridor for the transition. A video display of the transformations is available.
The structure of bacteriorhodopsin was used as a template to generate a model for G-protein coupled receptors. However, these receptors and the template are not related by sequence homology. Therefore a pragmatic and reproducible approach was developed to achieve an energetically favourable accommodation of receptor sequences to the backbone structure of bacteriorhodopsin. Improved interaction energy differences are used in a two step procedure analogous to a hypothetical folding mechanism for integral membrane proteins. The resulting model is in good agreement with existing data from structure-function studies.
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