A model of the seven transmembrane helical domain (7-TM) of the human lutropin receptor was constructed from the 2D electron density map of bovine rhodopsin and a set of geometric parameters derived from a published analysis of 204 G-protein coupled receptor sequences. The Self-Consistent Ensemble Optimization method was applied to predict overall side chain packing. The model is consistent with the general helical packing properties expected of transmembrane proteins and suggests possible structural and functional effects of constitutively activating mutations. A tightly packed hydrophobic cluster formed between the intracellular halves of TM5 and TM6, as well as a specific H-bonding network formed between the central regions of TM6 and TM7, is proposed to be critical for stabilizing the inactive form of the receptor. The model suggests that single activating mutations perturb the specific interactions of TM6 with TM5 and TM7, either by disrupting the hydrophobic packing between TM5 and TM6, or by weakening the H-bonds between TM6 and TM7.

Factor Xa (FXa) is an important serine protease in the blood coagulation cascade. Small synthetic competitive inhibitors of FXa are under development as potential anticoagulants. To better understand FXa structural features and molecular recognition mechanisms, we have constructed three dimensional models of FXa-inhibitor complex structures via a new search approach that samples conformational space and binding space simultaneously for DABE and DX-9065a, two bis amidinoaryl derivatives that are among the most potent and selective FXa inhibitors reported to date. We find the most probable binding modes for the two inhibitors to be a folded conformation, with one distal amidino group extending into the S1 pocket, forming a salt-bridge ~ith FXa Asp-189, and the other positively charged group fitting into the $4 subsite, and stabilized by a cation-~r interaction. We propose as a hypothesis that the cavity-like $4 subsite formed by the three ~r-faces of the aromatic residues Tyr-99, Phe-174 and Trp-215 is sufficiently rich in ~r electrons that it is not only a hydrophobic pocket, but also forms a cation recognition site. This proposed cation-~r binding mechanism is one of the first proposed for enzymatic molecular recognition, and for which experimental verification can be obtained without any complicating charge compensation mechanism. Our models provide plausible explanations of the structure-activity relationships observed for these inhibitors, and suggest that cation-lr interactions may provide a novel mechanism for molecular recognition.

HIV gp120 is specifically cleaved at a single site in the V, loop between Arg315 and Ala316 by thrombin. Previous observations by others have indicated that binding to CD4 enhances the rate of Vg loop cleavage, and that this cleavage is a prerequisite for HIV infection. Other observations also suggest that the cleavage site is in a type II B-turn centered at Pro313 -Gly314. However, our docking experiments indicate that this conformation cannot dock to thrombin and other trypsin-like serine proteases. Thus, based on the thrombin-bound conformation of peptide substrates, we propose that CD4 binding, at a site remote from the V, loop, induces and stabilizes a trans to cis isomerization of the highly conserved residue Pro"', and that this conformational shift is a prerequisite for cleavage by a 'thrombin-like' cellular protease and subsequent infection.

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Tryptophan and 5-bromotryptophan (5-BrTrp) are relatively potent inhibitors of sickle-haemoglobin polymerization. The binding sites of these compounds to normal and sickle haemoglobin (HBA and HBS) have been suggested, but not firmly established, through the use of spin-labelled derivatives and/or computer modeling. In the present study we approached the problem by utilizing the technique of photoaffinity labelling. The cyanomet forms of HBA and HBS were subjected to photoaffinity labelling with N alpha-(4-azidotetrafluorobenzoyl)tryptophan and N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan respectively. Both irradiated samples of HBA and HBS were denatured, digested with trypsin, and then separated by reversed-phase HPLC. A labelled tryptic peptide was isolated from the photolabelling of HBS with N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan. The peptide was identified to be Val1(alpha)-Lys7(alpha), with the label attached to Val1(alpha), by virtue of amino acid analysis and sequencing, in conjunction with fast-atom-bombardment MS. The binding mode of N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan is proposed and its relevance to the potency of the 5-BrTrp-based anti-sickling agents is discussed.

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