Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.

The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors.G protein-coupled receptor ͉ G protein-coupled receptor activation ͉ phototransduction ͉ membrane protein structure G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in animals, accounting for Ϸ3% of the genome (1). GPCRs are involved in detecting a large variety of chemical and physical signals, and they are the targets of Ϸ50% of current therapeutics. Structural information on GPCRs has been limited because of difficulties with their expression, purification, intrinsic chemical heterogeneity, and instability. These biochemical problems were overcome by using rhodopsin as a model GPCR, as it is highly expressed in a homogeneous form in rod photoreceptors and stabilized in the ground state by its covalently bound chromophore, 11-cis-retinal (2).The first crystal structure of rhodopsin revealed the arrangement of helices, the interhelical connections, the chromophore binding site, the extracellular ''plug,'' interactions involved in ligand binding in other GPCRs, and cytoplasmic helix 8 (3). Further improvements in the rhodopsin crystals yielded higher-resolution diffraction data that provided details about the effects of water molecules located close to the chromophore and more precise descriptions of the cytoplasmic loops. However, the improved crystals did not elucidate the mechanism of activation (4, 5). The arrangement of the seven transmembrane helices of rhodopsin differs from that in the more completely structurally studied bacterial retinoid-binding protein, bacteriorhodopsin (6).Upon absorption of a single photon of light, rhodopsin's chromophore, 11-cis-retinal, isomerizes to form all-trans-retinal, a covalently bound, full agonist. Once all-trans-retinal is formed, the protein portion of rhodopsin progresses through a series of photostates, including bathorhodopsin, lumirhodopsin, and metarhodopsin I (Met...

Summary The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multi-protein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the β-sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around ligand like a finger trap toy. Thus, β-sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

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Background: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (␣␤␥) 2 , held together by 29 disulfide bonds. The globular C terminus of the ␥ chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. Results:The X-ray crystallographic structure of the 30 kDa globular C terminus of the ␥ chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 Å or 2.1 Å resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. Conclusions:The polymerization domain in the ␥ chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.