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.

Membrane proteins, encoded by ~20% of genes in almost all organisms, including humans, are critical for cellular communication, electrical and ion balances, structural integrity of the cells and their adhesions, and other functions. Atomic-resolution structures of these proteins furnish important information for understanding their molecular organization and constitute major breakthroughs in our understanding of how they participate in physiological processes. However, obtaining structural information about these proteins has progressed slowly (1,2), mostly because of technical difficulties in the purification and handling of integral membrane proteins. Instability of the proteins in environments lacking phospholipids, the tendency for them to aggregate and precipitate, and/or difficulties with highly heterogeneous preparations of these proteins isolated from heterologous expression systems have hindered application of standard structure determination techniques to these molecules.Among membrane proteins, G-protein-coupled receptors (GPCRs) 1 are of special importance because they form one of the largest and most diverse groups of receptor proteins. More than 400 nonsensory receptors identified in the human genome are involved in the regulation of virtually all physiological processes. Drug addiction, mood control, and memory (via 5-HT6 or neuropeptide receptors) are just a short list of processes in which GPCRs are critically implicated. Another even larger group of GPCRs consist of sensory receptors involved in the fundamental process of translation of light energy (rhodopsin and cone pigments), the detection † This research was supported by NIH Grant EY-09339, awards from Research to Prevent Blindness, Inc. (RPB) SUPPORTING INFORMATION AVAILABLE Tabular listing of the following topics: tilt of the helices, bends within helices, possible hydrogen bonds between helices, closest atoms to the 11-cis-retinal, retinal atoms closest to these protein atoms, and torsion angles for the chromophore. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: GPCR, G-protein-coupled receptor; MAN, β-D-mannose; NAG, 2-N-acetyl-β-D-glucose; BNG, β-nonylglucoside; |F o |, observed structure factor; |F c |, calculated structure factor. R = ∑||F o | − |F c ||/∑|F o |. R cryst is the value for the working set of |F o |, while R free is that calculated for the 5% of reflections set aside for cross validation., NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2006 December 14. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of chemoattractant molecules, or the detection of compounds stimulating the taste buds (3,4). The activity of GPCRs comes about when binding of diffusable extracellular ligands causes them to switch from quiescent forms to an active conformation capable of interaction with hundreds of G-proteins. Their roles as extracellular ligand-binding proteins make them attractive targets for drug design. GPCRs account ...

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The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement. The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites. The enzyme is a trimer both in the crystal and in solution. The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands). Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart. Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer. The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.

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.

Rhodopsin is a retinal photoreceptor protein of bipartite structure consisting of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Studies on rhodopsin have unveiled many structural and functional features that are common to a large and pharmacologically important group of proteins from the G protein-coupled receptor (GPCR) superfamily, of which rhodopsin is the best-studied member. In this work, we focus on structural features of rhodopsin as revealed by many biochemical and structural investigations. In particular, the high-resolution structure of bovine rhodopsin provides a template for understanding how GPCRs work. We describe the sensitivity and complexity of rhodopsin that lead to its important role in vision.

We describe a class ofcationic structural proteins that associate specifically with intermediate filaments (IF) but not with other types ofcytoskeletal proteins. These proteins, for which the term filaggrin is introduced, are isolated from the stratum corneum of mammalian epidermis. They are species-distinct proteins; for example, rat and mouse filaggrin have different molecular weights and amino acid compositions, but are nevertheless chemically and functionally very similar. They interact in vitro with the IF of several different types of cells to form large fibers or macrofibrils in which many IF are highly aligned in parallel arrays. Stoichiometric analyses suggest that two molecules offilaggrin bind to each three-chain building block of the IF, possibly by ionic interactions with the coiled-coil et-helical regions of the IF.Intermediate filaments (IF) are ubiquitous constituents of the cytoskeleton of eukaryote cells. However, detailed studies of the IF isolated from a wide variety ofdifferent types ofcells have indicated broad differences in their solubility and immunological properties and in the size and complexity of their constituent subunits (1-3). Nevertheless, the IF studied to date appear to be structurally analogous. They are all a-type fibrous proteins and are composed of a common three-chain building block (4-7).Mammalian epidermis is unusual in that its principal differentiation products are filaments of the IF type (keratin filaments). These are synthesized and deposited intracellularly in the inner living cell layers as bundles or fibrils and eventually form the bulk ofthe terminally differentiated cells ofthe stratum corneum (8). The epidermis also produces significant amounts of another structural protein, which has distinctly different physicochemical properties from the IF. This protein is synthesized in the granular layer as a highly phosphorylated precursor and accumulates in amorphous keratohyalin granules, but it is dephosphorylated to become highly cationic when the granular cells differentiate to form the stratum corneum cells (9). The rat cationic protein, called stratum corneum basic protein (10), histidine-rich protein II (11), or histidine-rich basic protein (12), is thought to function in the stratum corneum as an interfilamentous matrix and thereby contribute to the formation ofthe characteristically insoluble protein complex ofthe epidermis, keratin. Support for this notion has been adduced from experiments in vitro. Mixing ofepidermal keratin IF with the rat cationic protein results in the formation of insoluble fibrous structures, or macrofibrils (12)(13)(14)(15). These macrofibrils consist of large numbers of keratin IF aligned in parallel arrays reminiscent of the keratin fibrils in the stratum corneum.In this paper, we report the isolation and characterization of a functionally similar although chemically distinct protein from mouse epidermis. Both the rat and mouse cationic proteins form macrofibrils when mixed with epidermal and inner root sheath keratin IF a...

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A 2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.The biological importance of factor XIII (fXIII) 1 (EC 2.3.2.13) lies in its ability to form new covalent bonds between protein chains. This activity was first recognized while studying blood coagulation; fXIII was required to form an insoluble clot (1). The activated form of fXIII covalently cross-links two fibrin molecules via an isopeptide bond between the side chains of a glutamine and a lysine located in the C-terminal region of the ␥-chain. Over a longer time period in coagulation, it also forms cross-links between the ␣-and ␥-chains of fibrin (2), and between ␣ 2 -anti-plasmin and fibrin (3). fXIII has been shown to react with more than fibrin (4), and it has recently been found in brain tumors (5) and arthritic joints (6), and there are cases of fXIII deficiencies (7,8). Factor XIII is a member in the family of transglutaminases (TGases), which have a wide range of biological functions (9).Like most coagulation factors, fXIII is synthesized as a zymogen and then cleaved by a protease to become an active enzyme. The structure of fXIII zymogen was determined several years ago (10, 11). In this crystal form, the active site cysteine, Cys-314, is inaccessible to solvent and is not available for catalysis.Physiologically, calcium ions are required for fXIII activation and for TGase activity. In the blood, activation of circulating fXIII requires thrombin cleavage, calcium ions (1.5 mM) (12-14), and fibrin(ogen) (15). High levels of calcium (Ͼ50 mM) can activate fXIII without the use of thrombin (15), and it has recently been shown that platelet fXIII can be activated nonproteolytically in vivo (16).Based on the amino acid sequence, the calcium binding site was predicted to be in a region (residues 468 -479) with high similarity to the EF-hand motif (17, 18). The main calcium binding site, as seen in the preliminary cryst...