The characteristic wavelength at which a visual pigment absorbs light is regulated by interactions between protein (opsin) and retinylidene Schiff base chromophore. By using site-directed mutagenesis, charged amino acids in bovine rhodopsin transmembrane helix C were systematically replaced. Substitution of glutamic acid-134 or arginine-135 did not affect spectral properties. However, substitution of glutamic acid-122 by glutamine or by aspartic acid formed pigments that were blue-shifted in light absorption (Amax = 480 nm and 475 nm, respectively). While the substitution of glutamic acid-113 by aspartic acid gave a slightly red-shifted pigment (Amax = 505 nm), replacement by glutamine formed a pigment that was strikingly blue-shifted in light absorption (Amax = 380 nm). The 380-nm species existed in a pH-dependent equilibrium with a 490-nm species such that at acidic pH all of the pigment was converted to Amax = 490 nm. We conclude that glutamic acid-113 serves as the retinylidene Schiff base counterion in rhodopsin. We believe that this opsin-chromophore interaction is an example of a general mechanism of color regulation in the visual pigments.
Stem cell homing and breast cancer metastasis are orchestrated by the chemokine SDF-1 and its receptor CXCR4. Here, we report the nuclear magnetic resonance (NMR) structure of a constitutively dimeric SDF-1 in complex with a CXCR4 fragment that contains three sulfotyrosine residues important for a high-affinity ligand-receptor interaction. CXCR4 bridged the SDF-1 dimer interface so that sulfotyrosines sTyr 7 and sTyr 12 of CXCR4 occupied positively charged clefts on opposing chemokine subunits. Dimeric SDF-1 induced intracellular Ca 2+ mobilization but had no chemotactic activity; instead, it prevented native SDF-1-induced chemotaxis, suggesting that it acted as a potent partial agonist. Our work elucidates the structural basis for sulfotyrosine recognition in the chemokine-receptor interaction and suggests a novel strategy for CXCR4-targeted drug development.
The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.
Many integral membrane proteins assemble to form oligomeric structures in biological membranes. In particular, seven-transmembrane helical G protein-coupled receptors (GPCRs) appear to self-assemble constitutively in membranes, but the mechanism and physiological role of this assembly are unknown. We developed and employed coarse-grain molecular dynamics (CGMD) models to investigate the molecular basis of how the physicochemical properties of the phospholipid bilayer membrane affect self-assembly of visual rhodopsin, a prototypical GPCR. The CGMD method is a mesoscopic simulation technique in which groups of atoms are mapped to particles on the basis of a four-to-one rule. This systematic reduction of the degrees of freedom allows for computationally efficient calculation of the structure and dynamics of molecular assemblies for larger time and length scales than accessible to atomistic models, providing here an unprecedented view of spontaneous protein assembly in biomembranes. Systems with up to 16 rhodopsin molecules at a protein-to-lipid ratio of 1:100 were simulated for time scales of up to 8 micros. The results obtained for four different phospholipid environments showed that localized adaptation of the membrane bilayer to the presence of receptors is reproducibly most pronounced near transmembrane helices 2, 4, and 7. This local membrane deformation appears to be a key factor defining the rate, extent, and orientational preference of protein-protein association. The implications of our findings are discussed within a framework of a generalized mechanism of membrane protein self-assembly.
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