The structure of rice weevil pectin methylesterase.

Rice weevils (Sitophilus oryzae) use a pectin methylesterase (EC 3.1.1.11), along with other enzymes, to digest cell walls in cereal grains. The enzyme is a right-handed ß-helix protein, but is circularly permuted relative to plant and bacterial pectin methylesterases, as shown by the crystal structure determination reported here. This is the first structure of an animal pectin methylesterase. Diffraction data were collected to 1.8 Šresolution some time ago for this crystal form, but structure solution required the use of molecular-replacement techniques that have been developed and similar structures that have been deposited in the last 15 years. Comparison of the structure of the rice weevil pectin methylesterase with that from Dickeya dandantii (formerly Erwinia chrysanthemi) indicates that the reaction mechanisms are the same for the insect, plant and bacterial pectin methylesterases. The similarity of the structure of the rice weevil enzyme to the Escherichia coli lipoprotein YbhC suggests that the evolutionary origin of the rice weevil enzyme was a bacterial lipoprotein, the gene for which was transferred to a primitive ancestor of modern weevils and other Curculionidae. Structural comparison of the rice weevil pectin methylesterase with plant and bacterial enzymes demonstrates that the rice weevil protein is circularly permuted relative to the plant and bacterial molecules.

Atomic resolution studies of carbonic anhydrase II.

Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 A resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 A) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 A in some zinc-protein and zinc-ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available.

Release of 11-cis-retinal from cellular retinaldehyde-binding protein by acidic lipids.

PURPOSE: To determine molecular mechanisms for the release of 11-cis-retinal from the binding pocket of cellular retinaldehyde-binding protein (CRALBP). METHODS: Binding of CRALBP to lipid surfaces was assessed with a lipid-immunoblot assay. Lipids were presented to CRALBP as small unilamellar vesicles (SUVs) consisting of phosphatidylcholine (PC) plus other lipids. Release of 9-cis-retinal or 11-cis-retinal from CRALBP was measured with spectral and high performance liquid chromatography (HPLC) assays based on the protection of the protein-bound retinal carbonyl group from reaction with NH(2)OH. The electrostatic surface potential of CRALBP was calculated from a model of its structure using the program CCP4mg. RESULTS: Incubation of CRALBP.11-cis-retinal with lipids absorbed on nitrocellulose revealed binding to the acidic lipids, phosphatidic acid (PA)>phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]>phosphatidylserine (PS)> PI(4,5)P(2) and little or no binding to PC, phosphatidylethanolamine (PE), or PI(4)P. 11-cis-retinal was released during incubation of CRALBP with SUVs consisting of PC plus 50 mol% PA but not during incubation with those composed of 100 mol% PC. The efficacy of release of 9-cis-retinal or 11-cis-retinal from CRALBP by phospholipid-containing SUVs generally paralleled that of the binding of CRALBP to the lipids (PA>PS>PI>>PC). Examination of the electrostatic surface potential of the protein structure revealed a basic recess on one face of the protein, which may bind acidic lipids. CONCLUSIONS: Our results identify the first physiologic substances that release 11-cis-retinal from CRALBP. PA and PS are relatively minor membrane lipids that can be generated in the cytoplasmic leaflet of the plasma membrane in response to various signal transduction pathways, where they could interact with cytosolic CRALBP. The mechanism for release of retinal from CRALBP by acidic lipids remains to be determined but could involve binding of the acidic lipid in the 11-cis-retinal binding site or to the positive basic recess on the protein surface. These results open a new facet in our understanding of how CRALBP functions in the regeneration of visual pigments.

Reprint of "Crystal packing analysis of Rhodopsin crystals" [J. Struct. Biol. 158 (2007) 455-462].

Oligomerization has been proposed as one of several mechanisms to regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallographic analyses of two new crystal forms of rhodopsin reveal an interaction surface which may be involved in the formation of functional dimers or oligomers. New crystallization conditions lead to the formation of two crystal forms with similar rhodopsin-rhodopsin interactions, but changes in the crystal lattice are induced by the addition of different surfactant additives. However, the intermolecular interactions between rhodopsin molecules in these crystal structures may reflect the contacts necessary for the maintenance of dimers or oligomers in rod outer segment membranes. Similar contacts may assist in the formation of dimers or oligomers in other GPCRs as well. These new dimers are compared with other models proposed by crystallography or EM and AFM studies. The inter-monomer surface contacts are different for each model, but several of these models coincide in implicating helix I, II, and H-8 as contributors to the main contact surface stabilizing the dimers.

Crystal packing analysis of Rhodopsin crystals.

Oligomerization has been proposed as one of several mechanisms to regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallographic analyses of two new crystal forms of rhodopsin reveal an interaction surface which may be involved in the formation of functional dimers or oligomers. New crystallization conditions lead to the formation of two crystal forms with similar rhodopsin-rhodopsin interactions, but changes in the crystal lattice are induced by the addition of different surfactant additives. However, the intermolecular interactions between rhodopsin molecules in these crystal structures may reflect the contacts necessary for the maintenance of dimers or oligomers in rod outer segment membranes. Similar contacts may assist in the formation of dimers or oligomers in other GPCRs as well. These new dimers are compared with other models proposed by crystallography or EM and AFM studies. The inter-monomer surface contacts are different for each model, but several of these models coincide in implicating helix I, II, and H-8 as contributors to the main contact surface stabilizing the dimers.

Structural insights into the cellular retinaldehyde-binding protein (CRALBP).

Cellular retinaldehyde-binding protein (CRALBP) is an essential protein in the human visual cycle without a known three-dimensional structure. Previous studies associate retinal pathologies to specific mutations in the CRALBP protein. Here we use homology modeling and molecular dynamics methods to investigate the structural mechanisms by which CRALBP functions in the visual cycle. We have constructed two conformations of CRALBP representing two states in the process of ligand association and dissociation. Notably, our homology models map the pathology-associated mutations either directly in or adjacent to the putative ligand-binding cavity. Furthermore, six novel residues have been identified to be crucial for the hinge movement of the lipid-exchange loop in CRALBP. We conclude that the binding and release of retinoid involve large conformational changes in the lipid-exchange loop at the entrance of the ligand-binding cavity.

Rhodopsin: a structural primer for G-protein coupled receptors.

An overview of the rhodopsin crystal structure provides a structural basis for understanding the structures and functions of other G-protein coupled receptors (GPCRs). All of the structural details observed to date for rhodopsin will not necessarily carry over to other GPCRs, but major features such as the arrangement of the seven transmembrane helices, the retinal/ligand binding site, the D(E)RY and NPXXY sequence and structural motifs, and the bent helices are likely characteristics of the GPCRs most closely related to rhodopsin. A general view of these structural features is presented here.

Identification of CRALBP ligand interactions by photoaffinity labeling, hydrogen/deuterium exchange, and structural modeling.

Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cis-retinol dehydrogenase. Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses. Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr(179), Phe(197), Cys(198), Met(208), Lys(221), Met(222), Val(223), and Met(225). Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apo- and holo-rCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198-255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photo-labeled residues. A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins. In the model, all of the photolabeled residues line the ligand binding cavity except Met(208), which appears to reside in a flexible loop at the entrance/exit of the ligand cavity. Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions.

Evolutionary analysis of rhodopsin and cone pigments: connecting the three-dimensional structure with spectral tuning and signal transfer.

Extensive sequence data and structural sampling of expressed proteins from different species lead to the idea that entire molecules or specific domain folds belong to large superfamilies of proteins. A subset of G protein-coupled receptors, one of the largest families involved in cellular signaling, rod and cone opsins are involved in phototransduction in photoreceptor cells. Here, the evolutionary analysis of opsin sequences and structures predicts key residues involved in the transmission of the signal from the binding site of the chromophore to the cytoplasmic surface and residues that are involved in the spectral tuning of opsins to short wavelengths of light.

Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin.

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.

The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways activating cellular processes. Rhodopsin is a GPCR found in rod cells in retina where it functions as a photopigment. Its molecular structure is known from cryo-electron microscopic and X-ray crystallographic studies, and this has reshaped many structure/function questions important in vision science. In addition, this first GPCR structure has provided a structural template for studies of other GPCRs, including many known drug targets. After presenting an overview of the major structural elements of rhodopsin, recent literature covering the use of the rhodopsin structure in analyzing other GPCRs will be summarized. Use of the rhodopsin structural model to understand the structure and function of other GPCRs provides strong evidence validating the structural model.

G protein-coupled receptor rhodopsin: a prospectus.

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.