Crystal structures of human 108V and 108M catechol O-methyltransferase.

Catechol O-methyltransferase (COMT) plays important roles in the metabolism of catecholamine neurotransmitters and catechol estrogens. The development of COMT inhibitors for use in the treatment of Parkinson's disease has been aided by crystallographic structures of the rat enzyme. However, the human and rat proteins have significantly different substrate specificities. Additionally, human COMT contains a common valine-methionine polymorphism at position 108. The methionine protein is less stable than the valine polymorph, resulting in decreased enzyme activity and protein levels in vivo. Here we describe the crystal structures of the 108V and 108M variants of the soluble form of human COMT bound with S-adenosylmethionine (SAM) and a substrate analog, 3,5-dinitrocatechol. The polymorphic residue 108 is located in the alpha5-beta3 loop, buried in a hydrophobic pocket approximately 16 A from the SAM-binding site. The 108V and 108M structures are very similar overall [RMSD of C(alpha) atoms between two structures (C(alpha) RMSD)=0.2 A], and the active-site residues are superposable, in accord with the observation that SAM stabilizes 108M COMT. However, the methionine side chain is packed more tightly within the polymorphic site and, consequently, interacts more closely with residues A22 (alpha2) and R78 (alpha4) than does valine. These interactions of the larger methionine result in a 0.7-A displacement in the backbone structure near residue 108, which propagates along alpha1 and alpha5 toward the SAM-binding site. Although the overall secondary structures of the human and rat proteins are very similar (C(alpha) RMSD=0.4 A), several nonconserved residues are present in the SAM-(I89M, I91M, C95Y) and catechol- (C173V, R201M, E202K) binding sites. The human protein also contains three additional solvent-exposed cysteine residues (C95, C173, C188) that may contribute to intermolecular disulfide bond formation and protein aggregation.

Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors.

The crystal structure of rhodopsin has provided the first three-dimensional molecular model for a G-protein-coupled receptor (GPCR). Alignment of the molecular model from the crystallographic structure with the helical axes seen in cryo-electron microscopic (cryo-EM) studies provides an opportunity to investigate the properties of the molecule as a function of orientation and location within the membrane. In addition, the structure provides a starting point for modeling and rational experimental approaches of the cone pigments, the GPCRs in cone cells responsible for color vision. Homology models of the cone pigments provide a means of understanding the roles of amino acid sequence differences that shift the absorption maximum of the retinal chromophore in the environments of different opsins.

Localization of the C-terminus of rat glutathione S-transferase A1-1: crystal structure of mutants W21F and W21F/F220Y.

Twelve C-terminal residues of human glutathione S-transferase A1-1 form a helix in the presence of glutathione-conjugate, or substrate alone, and partly cover the active site. According to X-ray structures, the helix is disordered in the absence of glutathione, but it is not known if it is helical and delocalized, or in a random-coil conformation. Mutation to a tyrosine of residue 220 within this helix was previously shown to affect the pK(a) of Tyr-9 at the active site, in the apo form of the enzyme, and it was proposed that an on-face hydrogen bond between Tyr-220 and Tyr-9 provided a means for affecting this pK(a). In the current study, X-ray structures of the W21F and of the C-terminal mutation, W21F/F220Y, with glutathione sulfonate bound, show that the C-terminal helix is disordered (or delocalized) in the W21F crystal but is visible and ordered in a novel location, a crystal packing crevice, in one of three monomers in the W21F/F220Y crystal, and the proposed hydrogen bond is not formed. Fluorescence spectroscopy studies using an engineered F222W mutant show that the C-terminus remains delocalized in the absence of glutathione or when only the glutathione binding site is occupied, but is ordered and localized in the presence of substrate or conjugate, consistent with these and previous crystallographic studies. Proteins 2001;42:192-200.

The 1.9 A crystal structure of the "as isolated" rubrerythrin from Desulfovibrio vulgaris: some surprising results.

Rubrerythrin is a non-heme iron dimeric protein isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris. Each monomer has one mononuclear iron center similar to rubredoxin and one dinuclear metal center similar to hemerythrin or ribonucleotide reductase. The 1.88 A X-ray structure of the "as isolated" molecule and a uranyl heavy atom derivative have been solved by molecular replacement techniques. The resulting model of the native "as isolated" molecule, including 164 water molecules, has been refined giving a final R factor of 0.197 (R(free) = 0.255). The structure has the same general protein fold, domain structure, and dimeric interactions as previously found for rubrerythrin [1, 2], but it also has some interesting undetected differences at the metal centers. The refined model of the protein structure has a cis peptide between residues 78 and 79. The Fe-Cys4 center has a previously undetected strong seventh N-H...S hydrogen bond in addition to the six N-H...S bonds usually found in rubredoxin. The dinuclear metal center has a hexacoordinate Fe atom and a tetracoordinate Zn atom. Each metal is coordinated by a GluXXHis polypeptide chain segment. The Zn atom binds at a site distinctly different from that found in the structure of a diiron rubrerythrin. Difference electron density for the uranyl derivative shows an extremely large peak adjacent to and replacing the Zn atom, indicating that this particular site is capable of binding other atoms. This feature/ability may give rise to some of the confusing activities ascribed to this molecule.

Crystal structure of rhodopsin: A G protein-coupled receptor.

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.

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system.

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.

X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles.

Rhodopsin, a prototypic G protein-coupled receptor responsible for absorption of photons in retinal rod photoreceptor cells, was selectively extracted from bovine rod outer segment membranes, employing mixed micelles of nonyl beta-d-glucoside and heptanetriol. Highly purified rhodopsin was crystallized from solutions containing varying amounts of detergent and amphiphile. The crystals contained ground state rhodopsin molecules as judged by their red color and the linear dichroism originating from the 11-cis-retinal chromophore. However, when exposed to visible light, even at 4 degrees C, rhodopsin was bleached and the crystals decomposed. Reflections in the diffraction pattern were observed out to 3.5-A resolution at 100 K for the most ordered crystals. Diffraction data have been processed to 3.85-A resolution. The symmetry of the diffraction pattern and the systematic absences indicate that the crystals have tetragonal symmetry, space group P4(1)22 or P4(3)22, a = b = 96.51 A, c = 148.55 A. A value of 4.12 A(3)/Da for V(M) was obtained for one monomer in the asymmetric unit (eight molecules per unit cell). Our study is the first characterization of a three-dimensional crystal of a G protein-coupled receptor and may be valuable for future structural studies on related receptors of this important superfamily.

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

Atomic resolution structure of biotin-free Tyr43Phe streptavidin: what is in the binding site?

The streptavidin-biotin system is an example of a high-affinity protein-ligand pair (Ka approximately 10(13) mol-1). The thermodynamic and structural properties have been extensively studied as a model system for protein-ligand interactions. Here, the X-ray crystal structure of a streptavidin mutant of a residue hydrogen bonding to biotin [Tyr43Phe (Y43F)] is reported at atomic resolution (1.14 A). The biotin-free structure was refined with anisotropic displacement parameters (SHELXL97 program package). The high-resolution data also allowed interpretation of side-chain and residue disorder in 41 residues where alternate conformations were refined. The Y43F mutation is unambiguously observed in difference maps, although only a single O atom per monomer is altered. The atomic resolution enabled the identification of 2-methyl-2, 4-pentanediol (MPD) molecules in the biotin-binding pocket for the first time. Electron density for MPD was observed in all four subunit binding sites of the tetrameric protein. This was not possible with data at lower resolution (1.8-2.3 A) for wild-type streptavidin or mutants in the same crystal form using MPD in the crystallization. The impact of MPD binding on these studies is discussed.

Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography.

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 A2 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.

X-ray crystallographic studies of streptavidin mutants binding to biotin.

On the basis of high resolution crystallographic studies of streptavidin and its biotin complex, three principal binding motifs have been identified that contribute to the tight binding. A flexible binding loop can undergo a conformational change from an open to a closed form when biotin is bound. Additional studies described here of unbound wild-type streptavidin have provided structural views of the open conformation. Several tryptophan residues packing around the bound biotin constitute the second binding motif, one dominated by hydrophobic interactions. Mutation of these residues to alanine or phenylalanine have variable effects on the thermodynamics and kinetics of binding, but they generate only small changes in the molecular structure. Hydrogen bonding interactions also contribute significantly to the binding energetics of biotin, and the D128A mutation which breaks a hydrogen bond between the protein and a ureido NH group results in a significant structural alteration that could mimic an intermediate on the dissociation pathway. In this review, we summarize the structural aspects of biotin recognition that have been gained from crystallographic analyses of wild-type and site-directed streptavidin mutants.

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Structural determinants of the bifunctional corn Hageman factor inhibitor: x-ray crystal structure at 1.95 A resolution.

Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13.6 kDa protein isolated from corn seeds. It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulation as well as alpha-amylases from several insect species. Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases. We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution. Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution. The two proteins are essentially identical. The proteinase binding loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the family of 25 cereal trypsin/alpha-amylase inhibitors, we have made three-dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase inhibitor 0.19 [Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T. (1997) Biochemistry 36, 13503-13511]. From an analysis of the models and a structure-based sequence analysis, we propose a testable hypothesis for the regions of these proteins which bind alpha-amylase. In the course of the investigations, we have found that the cereal trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants. This is a new addition to the group which now consists of the trypsin/alpha-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family. Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.

Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex.

Previous thermodynamic and computational studies have pointed to the important energetic role of aromatic contacts in generating the exceptional binding free energy of streptavidin-biotin association. We report here the crystallographic characterization of single site tryptophan mutants in investigating structural consequences of alterations in these aromatic contacts. Four tryptophan residues, Trp79, Trp92, Trp108 and Trp120, play an important role in the hydrophobic binding contributions, which along with a hydrogen bonding network and a flexible binding loop give rise to tight ligand binding (Ka approximately 10(13) M-1). The crystal structures of ligand-free and biotin-bound mutants, W79F, W108F, W120F and W120A, in the resolution range from 1.9 to 2.3 A were determined. Nine data sets for these four different mutants were collected, and structural models were refined to R-values ranging from 0.15 to 0.20. The major question addressed here is how these mutations influence the streptavidin binding site and in particular how they affect the binding mode of biotin in the complex. The overall folding of streptavidin was not significantly altered in any of the tryptophan mutants. With one exception, only minor deviations in the unbound structures were observed. In one crystal form of unbound W79F, there is a coupled shift in the side-chains of Phe29 and Tyr43 toward the mutation site, although in a different crystal form these shifts are not observed. In the bound structures, the orientation of biotin in the binding pocket was not significantly altered in the mutant complex. Compared with the wild-type streptavidin-biotin complex, there were no additional crystallographic water molecules observed for any of the mutants in the binding pocket. These structural studies thus suggest that the thermodynamic alterations can be attributed to the local alterations in binding residue composition, rather than a rearrangement of binding site architectures.

Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system.

A circularly permuted streptavidin (CP51/46) has been designed to remove the flexible polypeptide loop that undergoes an open to closed conformational change when biotin is bound. The original termini have been joined by a tetrapeptide linker, and four loop residues have been removed, resulting in the creation of new N- and C-termini. Isothermal titration calorimetric studies show that the association constant has been reduced approximately six orders of magnitude below that of wild-type streptavidin to 10(7) M(-1). The deltaH degrees of biotin association for CP51/46 is reduced by 11.1 kcal/mol. Crystal structures of CP51/46 and its biotin complex show no significant alterations in the binding site upon removal of the loop. A hydrogen bond between Ser45 and Ser52 found in the absence of biotin is broken in the closed conformation as the side-chain hydroxyl of Ser45 moves to hydrogen bond to a ureido nitrogen of biotin. This is true in both the wild-type and CP51/46 forms of the protein, and the hydrogen bonding interaction might thus help nucleate closure of the loop. The reduced entropic cost of binding biotin to CP51/46 is consistent with the removal of this loop and a reduction in entropic costs associated with loop closure and immobilization. The reduced enthalpic contribution to the free energy of binding is not readily explainable in terms of the molecular structure, as the binding contacts are nearly entirely conserved, and only small differences in solvent accessible surfaces are observed relative to wild-type streptavidin.

The primary fibrin polymerization pocket: three-dimensional structure of a 30-kDa C-terminal gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro.

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the alpha chain of one fibrin molecule and the C-terminal region of a gamma chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) gamma chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the alpha chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.