Proline 96 of the copper ligand loop of amicyanin regulates electron transfer from methylamine dehydrogenase by positioning other residues at the protein-protein interface.

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a "hydrophobic patch" of amino acid residues including those that comprise a "ligand loop" that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K(d) for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H(AB)) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin, but notably, in addition to the change in Pro96, the side chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K(d), λ, and H(AB) for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner but also to optimize the orientation of proteins for interprotein ET.

Probing oxygen activation sites in two flavoprotein oxidases using chloride as an oxygen surrogate.

A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX·chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX·chloride complex and a ternary MSOX·chloride·MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase.

Methanol dehydrogenase is a heterotetrameric enzyme containing the prosthetic group pyrroloquinoline quinone (PQQ), which catalyzes the oxidation of methanol to formaldehyde. The crystal structure of methanol dehydrogenase from Methylophilus W3A1, previously determined at high resolution, exhibits a non-planar configuration of the PQQ ring system and lends support for a hydride transfer mechanism of the enzymatic reaction catalyzed by the enzyme. To investigate why PQQ is in the C5-reduced form and to better understand the catalytic mechanism of the enzyme, three structures of this enzyme in a new crystal form have been determined at higher resolution. Two of the three crystals were grown in the presence of 1 and 50 mM methanol, respectively, both structures of which show non-planar configurations of the PQQ ring system, confirming the previous conclusion; the other was crystallized in the presence of 50 mM ethanol, the structure of which displays a planar ring system for PQQ. Comparison of these structures reveals that the configuration change of PQQ is induced by the enzymatic reaction. The reaction takes place and the C5-reduced PQQ intermediate is produced when the enzyme co-crystallizes with methanol, but the enzymatic reaction does not take place and the PQQ ring retains a planar configuration of the oxidized orthoquinone form when ethanol instead of methanol is present in the crystallization solution.

Structural identification of the pathway of long-range communication in an allosteric enzyme.

Allostery is a common mechanism of regulation of enzyme activity and specificity, and its signatures are readily identified from functional studies. For many allosteric systems, structural evidence exists of long-range communication among protein domains, but rarely has this communication been traced to a detailed pathway. The thrombin mutant D102N is stabilized in a self-inhibited conformation where access to the active site is occluded by a collapse of the entire 215-219 beta-strand. Binding of a fragment of the protease activated receptor PAR1 to exosite I, 30-A away from the active site region, causes a large conformational change that corrects the position of the 215-219 beta-strand and restores access to the active site. The crystal structure of the thrombin-PAR1 complex, solved at 2.2-A resolution, reveals the details of this long-range allosteric communication in terms of a network of polar interactions.

Structural characterization of mutations at the oxygen activation site in monomeric sarcosine oxidase .

Oxygen reduction and sarcosine oxidation in monomeric sarcosine oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an approximately 10(4)- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted ( approximately 4-5 A) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a "flipped-out" Arg265 and exhibit negligible oxygen activation, similar to Lys265Met. The 400-fold higher oxygen reactivity observed with Lys265Arg is attributed to a minor (<1%) "flipped-in" Arg265 conformer whose oxygen reactivity is similar to that of wild-type MSOX. A structural water (WAT1), found above the si-face of the flavin ring in all previously determined MSOX structures, is part of an apparent proton relay system that extends from FAD N(5) to bulk solvent. WAT1 is strikingly absent in Lys265Met and Lys265Arg, a feature that may account for the apparent kinetic stabilization of a reductive half-reaction intermediate that is detectable with the mutants but not wild-type MSOX.

Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity.

The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

Factors that affect oxygen activation and coupling of the two redox cycles in the aromatization reaction catalyzed by NikD, an unusual amino acid oxidase.

NikD is a flavoprotein oxidase that catalyzes the oxidation of piperideine-2-carboxylate (P2C) to picolinate in a remarkable aromatization reaction comprising two redox cycles and at least one isomerization step. Tyr258 forms part of an "aromatic cage" that surrounds the ring in picolinate and its precursors. Mutation of Tyr258 to Phe does not perturb the structure of nikD but does affect the coupling of the two redox cycles and causes a 10-fold decrease in turnover rate. Tyr258Phe catalyzes a quantitative two-electron oxidation of P2C, but only 60% of the resulting dihydropicolinate intermediate undergoes a second redox cycle to produce picolinate. The mutation does not affect product yield with an alternate substrate (3,4-dehydro-L-proline) that is aromatized in a single two-electron oxidation step. Wild-type and mutant enzymes exhibit identical rate constants for oxidation of P2C to dihydropicolinate and isomerization of a reduced enzyme.dihydropicolinate complex. The observed rates are 200- and 10-fold faster, respectively, than the mutant turnover rate. Release of picolinate from Tyr258Phe is 100-fold faster than turnover. The presence of a bound substrate or product is a key factor in oxygen activation by wild-type nikD, as judged by the 10-75-fold faster rates observed for complexes of the reduced enzyme with picolinate, benzoate, or 1-cyclohexenoate, a 1-deaza-P2C analogue. The reduced Tyr258Phe x 1-cyclohexenoate complex is 25-fold less reactive with oxygen than the wild-type complex. We postulate that mutation of Tyr258 causes subtle changes in active site dynamics that promote release of the reactive dihydropicolinate intermediate and disrupt the efficient synchronization of oxygen activation observed with wild-type nikD.

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful. However, the G81A mutants of MDH and of MDH-GOX2 displayed approximately 100-fold lower reactivity with substrate and a modestly higher reactivity towards molecular oxygen. In order to understand the effect of the mutation and to identify the mode of substrate binding in MDH-GOX2, a crystallographic investigation of the G81A mutant of the MDH-GOX2 enzyme was initiated. The structures of ligand-free G81A mutant MDH-GOX2 and of its complexes with the substrates 2-hydroxyoctanoate and 2-hydroxy-3-indolelactate were determined at 1.6, 2.5 and 2.2 A resolution, respectively. In the ligand-free G81A mutant protein, a sulfate anion previously found at the active site is displaced by the alanine side chain introduced by the mutation. 2-Hydroxyoctanoate binds in an apparently productive mode for subsequent reaction, while 2-hydroxy-3-indolelactate is bound to the enzyme in an apparently unproductive mode. The results of this investigation suggest that a lowering of the polarity of the flavin environment resulting from the displacement of nearby water molecules caused by the glycine-to-alanine mutation may account for the lowered catalytic activity of the mutant enzyme, which is consistent with the 30 mV lower flavin redox potential. Furthermore, the altered binding mode of the indolelactate substrate may account for its reduced activity compared with octanoate, as observed in the crystalline state.

NikD, an unusual amino acid oxidase essential for nikkomycin biosynthesis: structures of closed and open forms at 1.15 and 1.90 A resolution.

NikD is an unusual amino-acid-oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate, and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 A, respectively. The two forms differ by an 11 degrees rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form, but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop.

Identification of a stable flavin-thiolate adduct in heterotetrameric sarcosine oxidase.

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing>or=90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.

Heterotetrameric sarcosine oxidase: structure of a diflavin metalloenzyme at 1.85 A resolution.

The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.

Crystal structure of thrombin in complex with fibrinogen gamma' peptide.

Elevated levels of heterodimeric gamma(A)/gamma' fibrinogen 2 have been associated with an increased incidence of coronary artery disease, whereas a lowered content of gamma' chains is associated with an increased risk of venous thrombosis. Both situations may be related to the unique features of thrombin binding to variant gamma' chains. The gamma' peptide is an anionic fragment that binds thrombin with high affinity without interfering directly with substrate binding. Here we report the crystal structure of thrombin bound to the gamma' peptide, solved at 2.4 A resolution. The complex reveals extensive interactions between thrombin and the gamma' peptide mediated by electrostatic contacts with residues of exosite II and hydrophobic interactions with a pocket in close proximity to the Na(+) binding site. In its binding mode, the gamma' peptide completely overlaps with heparin bound to exosite II. These findings are consistent with functional data and broaden our understanding of how thrombin interacts with fibrinogen at the molecular level.

Molecular cloning and structural analysis of quinohemoprotein alcohol dehydrogenase ADH-IIG from Pseudomonas putida HK5.

Depending on the alcohols used as growth substrates, Pseudomonas putida HK5 produces two distinct quinohemoprotein alcohol dehydrogenases, ADH-IIB and ADH-IIG, both of which contain pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups but show different substrate specificities, especially for diol substrates. Molecular cloning of the gene of ADH-IIB and its crystal structure are already reported. Here, molecular cloning of the gene, qgdA, and solution of the three-dimensional structure of ADH-IIG are reported. The enzyme consists of 718 amino acid residues including a signal sequence of 29 amino acid residues. The PQQ domain is highly homologous to other quinoproteins, especially to quinohemoproteins. The crystal structure of ADH-IIG, determined at 2.2A resolution, shows that the overall structure and the amino acid residues involved in PQQ binding are quite similar to ADH-IIB and to another quinohemoprotein ADH, qhEDH from Comamonas testosteroni. However, the lengths of the linker regions connecting the PQQ and the cytochrome domains are different from each other, leading to a significant difference in orientation of the cytochrome domain with respect to the PQQ domain. Apart from ADH-IIB and qhEDH, ADH-IIG has an extra 12-residue helix within loop 3 in the PQQ domain and an extra 3(10) helix in the C terminus of the cytochrome domain, and both helices appear parallel and linked by a hydrogen bond. The amino acid residues contacting substrate/product in the crystal structures are also different among them. In the crystal structure of ADH-IIG with 1,2-propanediol, one of the hydroxyl groups of the substrate forms a hydrogen bond with O5 of PQQ and OD1 of Asp300, and the other interacts with a water molecule and with NE2 of Trp386, the corresponding residue of which is not found in ADH-IIB and qhEDH, and might be the residue responsible for making ADH-IIG prefer diol substrates.

High resolution crystal structures of free thrombin in the presence of K(+) reveal the molecular basis of monovalent cation selectivity and an inactive slow form.

Structural biology has recently advanced our understanding of the molecular mechanisms of activation and selectivity in monovalent cation activated enzymes. Here we report a 1.9 Angstrom resolution crystal structure of free thrombin, a Na(+) selective enzyme, in the presence of KCl. There are two molecules in the asymmetric unit, one with the cation site bound to K(+) and the other with this site free. The K(+)-bound form shows key differences compared with the Na(+)-bound structure that explain the different kinetics of activation. The cation-free form, on the other hand, assumes a conformation where the monovalent cation binding site is completely disordered, the S1 pocket is inaccessible to substrate and binding to exosite I is compromised by an unprecedented >20 Angstrom shift in the position of the autolysis loop. This form, named S(*), corresponds to the inactive Na(+)-free slow form identified by early kinetic studies. A simple model of thrombin allostery that incorporates the contribution of S(*) is proposed.

Structure at 1.9 A resolution of a quinohemoprotein alcohol dehydrogenase from Pseudomonas putida HK5.

The type II quinohemoprotein alcohol dehydrogenase of Pseudomonas putida is a periplasmic enzyme that oxidizes substrate alcohols to the aldehyde and transfers electrons first to pyrroloquinoline quinone (PQQ) and then to an internal heme group. The 1.9 A resolution crystal structure reveals that the enzyme contains a large N-terminal eight-stranded beta propeller domain (approximately 60 kDa) similar to methanol dehydrogenase and a small C-terminal c-type cytochrome domain (approximately 10 kDa) similar to the cytochrome subunit of p-cresol methylhydoxylase. The PQQ is bound near the axis of the propeller domain about 14 A from the heme. A molecule of acetone, the product of the oxidation of isopropanol present during crystallization, appears to be bound in the active site cavity.

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design.

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent l-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b(2) (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC(50) in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 Å resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the ∼100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest.

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations.

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.

Correlation of rhombic distortion of the type 1 copper site of M98Q amicyanin with increased electron transfer reorganization energy.

Mutation of the axial Met ligand of the type 1 copper site of amicyanin to Ala or Gln yielded M98A amicyanin, which exhibits typical axial type 1 ligation geometry but with a water molecule providing the axial ligand, and M98Q amicyanin, which exhibits significant rhombic distortion of the type 1 site (Carrell, C. J., Ma, J. K., Antholine, W. E., Hosler, J. P., Mathews, F. S., and Davidson, V. L. (2007) Biochemistry 46, 1900-1912). Despite the change of the axial ligand, the M98Q and M98A mutations had little effect on the redox potential of copper. The true electron transfer (ET) reactions from O-quinol methylamine dehydrogenase to oxidized native and mutant amicyanins revealed that the M98A mutation had little effect on kET, but the M98Q mutation reduced kET 45-fold. Thermodynamic analysis of the latter showed that the decrease in kET was due to an increase of 0.4 eV in the reorganization energy (lambda) associated with the ET reaction to M98Q amicyanin. No change in the experimentally determined electronic coupling or ET distance was observed, confirming that the mutation had not altered the rate-determining step for ET and that this was still a true ET reaction. The basis for the increased lambda is not the nature of the atom that provides the axial ligand because each uses an oxygen from Gln in M98Q amicyanin and from water in M98A amicyanin. Comparisons of the distance of the axial copper ligand from the equatorial plane that is formed by the other three copper ligands in isomorphous crystals of native and mutant amicyanins at atomic resolution indicate an increase in distance from 0.20 A in the native to 0.42 A in M98Q amicyanin and a slight decrease in distance for M98A amicyanin. This correlates with the rhombic distortion caused by the M98Q mutation that is clearly evident in the EPR and visible absorption spectra of the protein and suggests that the extent of rhombicity of the type 1 copper site influences the magnitude of lambda.

Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery.

Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an approximately 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-A resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl, as revealed by the 1.8-A resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation.

Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4.

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 ((38)SFNGGPQNTFEEFPLSDIE(56)) and PAR4 ((51)KSSDKPNPR downward arrow GYPGKFCANDSDTLELPASSQA(81), downward arrow = site of cleavage) have been solved at 2.0 and 3.5 A resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment (47)FEEFPLSDIE(56). Occupancy of exosite I by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d approximately 10 A with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite I and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop.