Biomedical subjects

R Raag

Publications and source records attributed to R Raag.

17 recordsLinked to original sources

PubMed1997

Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases.

Tyrosine hydroxylase (TyrOH) catalyzes the conversion of tyrosine to L-DOPA, the rate-limiting step in the biosynthesis of the catecholamines dopamine, adrenaline, and noradrenaline. TyrOH is highly homologous in terms of both protein sequence and catalytic mechanism to phenylalanine hydroxylase (PheOH) and tryptophan hydroxylase (TrpOH). The crystal structure of the catalytic and tetramerization domains of TyrOH reveals a novel alpha-helical basket holding the catalytic iron and a 40 A long anti-parallel coiled coil which forms the core of the tetramer. The catalytic iron is located 10 A below the enzyme surface in a 17 A deep active site pocket and is coordinated by the conserved residues His 331, His 336 and Glu 376. The structure provides a rationale for the effect of point mutations in TyrOH that cause L-DOPA responsive parkinsonism and Segawa's syndrome. The location of 112 different point mutations in PheOH that lead to phenylketonuria (PKU) are predicted based on the TyrOH structure.

Amino Acid Sequence↗

PubMed1995

Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase.

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.

Amino Acid Sequence↗

PubMed1995

Single-chain Fvs.

Single-chain Fvs (sFvs) are recombinant antibody fragments consisting of only the variable light chain (VL) and variable heavy chain (VH) domains covalently connected to one another by a polypeptide linker. Due to their small size, sFvs have rapid pharmacokinetics and tumor penetration in vivo. Single-chain Fvs also show a concentration-dependent tendency to oligomerize. Bivalent sFvs are formed when the variable domains of a sFv disassociate from one another and reassociate with the variable domains of a second sFv. Similar rearrangement and reassociation of variable domains from different sFvs can result in the formation of trimers or higher multimeric oligomers. Each Fv in a bivalent or multivalent Fv is composed of the VL domain from one sFv and the VH domain from a second sFv. Modifying linker length or the inclusion of antigen may stabilize the VL/VH interface against rearrangement such that specific multimeric or monomeric forms of sFvs may be isolated. Nuclear magnetic resonance studies have shown that McPC603-derived Fv and sFvs have similar structures, and that the sFv linker is a rapidly moving, highly flexible peptide with a random coil-like structure. In X-ray crystallographic investigations of three different sFvs, linkers have also been found to be disordered. Indirect evidence suggests that a monomeric sFv has been crystallized in one case, and dimeric sFvs in the other two.

Amino Acid Sequence↗

PubMed1993

Crystallization of single-chain Fv proteins.

Single-chain Fv (sFv) proteins consist of the variable heavy chain (VH) and variable light chain (VL) domains of an antibody, covalently joined by an engineered polypeptide linker. We report the crystallization of single-chain Fv's with specificities for fluorescein (4-4-20 sFv) and the TAG-72 pan-carcinoma glycoprotein antigen (CC49 sFv). Concentration of these proteins, preliminary to crystallization, results in a monomer-multimer equilibrium, causing aggregation which interferes with crystallization. Aggregation has been observed to depend primarily on an intact linker between VL and VH domains, although other factors are likely to modulate this phenomenon as well, including the specific identity of Fv and ligand, presence or absence of the ligand, linker length and possibly sequence. We have found two methods to overcome sFv aggregation, both of which yield X-ray diffraction quality crystals. The first, discovered serendipitously, is by introducing a proteolytic clip into the linker region (effectively yielding an Fv fragment). The second is the purification of the sFv dimer form, with linker regions intact, from an equilibrium mixture of aggregates. The sFv molecular association in a dimer is believed to be unusual in that each VL/VH interface may not be formed by the two linker-connected VL and VH domains, but rather by interaction of VL and VH domains from two distinct sFv monomers. Structure determination of the CC49 sFv dimer, with the 14-residue linker designated 212, is underway to test this model. Increasing linker length, to relieve steric strain on the monomer, and inclusion of the appropriate antigen, to slow transitions between monomeric and multimeric forms, may prove valuable strategies with sFv proteins less amenable to crystallization.

Amino Acid Sequence↗

PubMed1993

Inhibitor-induced conformational change in cytochrome P-450CAM.

The X-ray crystal structures of cytochrome P-450CAM complexed with both enantiomers of a chiral, multifunctional inhibitor have been refined to R-factors of 21.0% [(+)-enantiomer] and 19.6% [(-)-enantiomer] at approximately 2.1-A resolution. Binding of either enantiomer, both considerably larger than the natural substrate camphor, results in similar, dramatic structural changes in the enzyme. In contrast to all previous P-450CAM crystallographic structures, the Tyr96 side chain is not pointing "down" toward the heme but is rather directed "up" into the proposed substrate access channel. This conformational change is accompanied by the displacement of the Phe193 side chain out into the solvent at the enzyme surface. These changes are consistent with the assignment of this region of the enzyme as the access channel [Poulos et al. (1986) Biochemistry 25, 5314-5322] and suggest that several aromatic residues lining the channel may be involved in substrate recognition and channeling to the active site. The cation usually observed coordinated to the Tyr96 carbonyl oxygen is missing in the presence of the (+)-enantiomer but is present with the (-)-enantiomer. The Phe87 side chain, located near the inhibitor binding site, adopts different orientations depending upon which enantiomer is bound. Finally, electron density reveals that although the inhibitor enantiomers were dichlorinated as provided, when bound to P-450CAM the chlorine atoms are present at only 0-20% occupancy, probably reflecting selective binding of impurities in the samples. Coordinates of these inhibited P-450CAM complexes have been deposited in the Brookhaven Protein Data Bank [Bernstein et al. (1977) J. Mol. Biol. 112, 535-542].

Binding Sites↗

PubMed1993

Crystal structure of lignin peroxidase.

The crystal structure of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium has been determined to 2.6 A resolution by usine multiple isomorphous replacement methods and simulated annealing refinement. Of the 343 residues, residues 3-335 have been accounted for in the electron density map, including four disulfide bonds. The overall three-dimensional structure is very similar to the only other peroxidase in this group for which a high-resolution crystal structure is available, cytochrome c peroxidase, despite the fact that the sequence identity is only approximately 20%, LiP has four disulfide bonds, while cytochrome c peroxidase has none, and LiP is larger (343 vs. 294 residues). The basic helical fold and connectivity defined by 11 helical segments with the heme sandwiched between the distal and proximal helices found in cytochrome c peroxidase is maintained in LiP. Both enzymes have a histidine as a proximal heme ligand, which is hydrogen bonded to a buried aspartic acid side chain. The distal or peroxide binding pocket also is similar, including the distal arginine and histidine. The most striking difference is that, whereas cytochrome c peroxidase has tryptophans contacting the distal and proximal heme surfaces, LiP has phenylalanines. This in part explains why, in the reaction with peroxides, cytochrome c peroxidase forms an amino acid-centered free radical, whereas LiP forms a porphyrin pi cation radical.

Journal Article↗

PubMed1992

Cytochrome P450cam: crystallography, oxygen activation, and electron transfer.

Several crystal structures of various substrate and inhibited complexes of the camphor monoxygenase, cytochrome P450cam from Pseudomonas putida, are now available. These structures, together with mutagenesis, biochemical, and biophysical studies, have allowed for a detailed penetration into the problem of how P450s activate molecular oxygen, control stereoselectivity, and transfer electrons. This review will provide a summary of the crystallographic work in light of what these structures have taught us about P450 function.

Amino Acid Sequence↗

PubMed1992

Functional implications of interleukin-1 beta based on the three-dimensional structure.

The molecular structure of interleukin-1 beta, a hormone-like cytokine with roles in several disease processes, has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. The framework of this molecule consists of 12 antiparallel beta-strands exhibiting pseudo-3-fold symmetry. Six of the strands make up a beta-barrel with polar residues concentrated at either end. Analysis of the three-dimensional structure, together with results from site-directed mutagenesis and biochemical and immunological studies, suggest that the core of the beta-barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL-1 interacts with its receptor.

Humans↗

PubMed1991

Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.

The crystal structure of a cytochrome P-450CAM site-directed mutant in which the active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252 is a key active site residue, forming part of the dioxygen-binding site. Mutation of the active site threonine to alanine produces an enzyme in which substrate hydroxylation is uncoupled from electron transfer. Specifically, hydrogen peroxide and "excess" water are produced instead of the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local distortion in the distal helix between Gly248 and Thr252 becomes even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the native enzyme is positioned in the dioxygen-binding region of the mutant enzyme active site. In this location, the solvent molecule could sterically interfere with and destabilize dioxygen binding. In addition, the active site solvent molecule is connected, via a network of hydrogen bonds, with an internal solvent channel which links distal helix residues to a buried Glu side chain. Thus, solvent protons appear to be much more accessible to dioxygen in the mutant than in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or water production instead of substrate hydroxylation. On the basis of crystallographic and mutagenesis data, a proton delivery pathway involving residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM. Coordinates of structures discussed in this paper have been submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).

Alanine↗

PubMed1991

Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation.

X-ray crystal structures have been determined for complexes of cytochrome P-450CAM with the substrates camphane, adamantane, and thiocamphor. Unlike the natural substrate camphor, which hydrogen bonds to Tyr96 and is metabolized to a single product, camphane, adamantane and thiocamphor do not hydrogen bond to the enzyme and all are hydroxylated at multiple positions. Evidently the lack of a substrate-enzyme hydrogen bond allows substrates greater mobility in the active site, explaining this lower regiospecificity of metabolism as well as the inability of these substrates to displace the distal ligand to the heme iron. Tyr96 is a ligand, via its carbonyl oxygen atom, to a cation that is thought to stabilize the camphor-P-450CAM complex [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. The occupancy and temperature factor of the cationic site are lower and higher, respectively, in the presence of the non-hydrogen-bonding substrates investigated here than in the presence of camphor, underscoring the relationship between cation and substrate binding. Thiocamphor gave the most unexpected orientation in the active site of any of the substrates we have investigated to date. The orientation of thiocamphor is quite different from that of camphor. That is, carbons 5 and 6, at which thiocamphor is primarily hydroxylated [Atkins, W. M., & Sligar, S. G. (1988) J. Biol. Chem. 263, 18842-18849], are positioned near Tyr96 rather than near the heme iron. Therefore, the crystallographically observed thiocamphor-P-450CAM structure may correspond to a nonproductive complex. Disordered solvent has been identified in the active site in the presence of uncoupling substrates that channel reducing equivalents away from substrate hydroxylation toward hydrogen peroxide and/or "excess" water production. A buried solvent molecule has also been identified, which may promote uncoupling by moving from an internal location to the active site in the presence of highly mobile substrates.

Adamantane↗

PubMed1990

Formation, crystal structure, and rearrangement of a cytochrome P-450cam iron-phenyl complex.

Cytochrome P-450cam reacts with phenyldiazene (PhN = NH), or less efficiently with phenylhydrazine, to give a catalytically inactive complex with an absorption maximum at 474 nm. The prosthetic group extracted anaerobically from the inactivated protein has the spectroscopic properties of a sigma phenyl-iron complex and rearranges, on exposure to air and acid, to an approximately equal mixture of the four N-phenylprotoporphyrin IX regioisomers. The crystal structure of the intact protein complex, refined at 1.9-A resolution to an R factor of 20%, confirms that the phenyl group is directly bonded through one of its carbons to the iron atom. The phenyl ring is tilted from the heme normal by about 10 degrees in the opposite direction from that in which carbon monoxide tilts when bound to P-450cam. Camphor, the natural substrate for P-450cam, is larger than a phenyl group and hydrogen bonds to Tyr 96, the only hydrophilic residue near the active site. Electron density in the active site in addition to that contributed by the phenyl group suggests that two water molecules occupy part of the camphor binding site but are not within hydrogen-bonding distance of Tyr 96. As observed in a previous crystallographic study of inhibitor-P-450cam complexes [Poulos, T.L., & Howard, A.J. (1987) Biochemistry 26, 8165-8174], there are large changes in both the atomic positions and mobilities of the residues in the proposed substrate access channel region of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1989

Crystal structure of the carbon monoxide-substrate-cytochrome P-450CAM ternary complex.

The crystal structure of the ternary complex formed between carbon monoxide (CO), camphor, and ferrous cytochrome P-450CAM has been refined to an R value of 17.9% at 1.9-A resolution. To accommodate the CO molecule, the substrate, camphor, moves about 0.8 A while at the same time remaining in nonbonded contact with CO. The average temperature factor of the camphor atoms is about 50% higher in the CO complex, suggesting that the camphor is more loosely bound in this ternary complex. The Fe-C-O angle is about 166 degrees, and thus, CO appears to be bent from the heme normal, as it is in various CO-globin complexes, due to steric interactions with active site groups. The oxygen atom of the CO molecule is nestled into a groove formed by an unusual helical hydrogen bond in the distal helix between the highly conserved Thr 252 and Gly 248 residues. In the transition from the ferric camphor-bound binary complex to the ferrous CO-camphor-bound ternary complex, the heme iron atom moves into the plane defined by the pyrrole nitrogens by about 0.41 A. Although the axial Cys ligand also moves toward the heme, the S-Fe bond stretches from about 2.20 A in the absence of CO to about 2.41 A once CO has bound.

Camphor↗

PubMed1989

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin.

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.

Amino Acid Sequence↗

PubMed1989

The structural basis for substrate-induced changes in redox potential and spin equilibrium in cytochrome P-450CAM.

The crystal structures of cytochrome P-450CAM complexed with the alternative substrates norcamphor and adamantanone have been refined at 2.0-A resolution and compared with the native, camphor-bound form of the enzyme. Norcamphor lacks the 8-, 9-, and 10-methyl groups of camphor. Thus, specific interactions between these groups and phenylalanine 87 and valines 247 and 295 are missing in the norcamphor complex. As a result, norcamphor binds about 0.9 A further from the oxygen-binding site than does camphor, which allows sufficient room for a water molecule or hydroxide ion to remain coordinated with the heme iron atom. The larger adamantanone occupies a position closer to that of camphor and, as in the camphor-bound enzyme, the heme iron remains pentacoordinate with no solvent molecule coordinated as a sixth ligand. A comparison of crystallographic temperature factors indicates that norcamphor is more "loosely" bound than are either camphor or adamantanone, as might be expected from the relative sizes of the different substrates. The looser fit of norcamphor in the active-site pocket results in a less specific pattern of hydroxylation. The presence of an aqua ligand is the likely structural basis for the norcamphor-P-450CAM complex having both a lower redox potential and higher percentage of low-spin heme than do either the camphor-P-450CAM or adamantanone-P-450CAM complexes.

Adamantane↗

PubMed1988

Structure of the lamprey yolk lipid-protein complex lipovitellin-phosvitin at 2.8 A resolution.

The X-ray crystallographic structure of the lipid-protein complex lipovitellin-phosvitin has been determined with the multiple isomorphous replacement method using four heavy-atom derivatives. Lamprey yolk lipovitellin-phosvitin is a dimeric molecule of molecular weight 352,000. The monomer consists of three polypeptide chains. The smallest is known as phosvitin and has an extremely high phosphoserine content. The monomeric unit also contains about 16% (w/w) of non-covalently bound lipid, probably in a monolayer or bilayer-like configuration. Within each monomer is a "cavity" or region of low electron density. The cavity has a volume of about 68,000 A3 and is believed to contain the lipid in a presumably disordered state. The cavity is roughly conical in shape and is lined on two sides by seven and eight-stranded antiparallel beta-sheets. The base of the cavity opens away from the intersubunit interface, but appears partially closed off from solvent regions by additional antiparallel beta-sheet structure. The beta-sheets lining the sides of the cavity are surrounded by a shell of two curved layers of 16 interconnected helices. The helices in either layer of the shell are all roughly parallel to each other and antiparallel to all of the helices of the other layer. The connectivity of the helices resembles a "superhelix" and is different from the connectivities seen in proteins containing four-helix bundles. There are an estimated 1300 amino acids in lamprey lipovitellin-phosvitin and almost 1000 alanine residues have been modeled into electron density. The remaining residues are assumed to be disordered.

Animals↗

PubMed1986

A comparison of negatively stained electron micrographs and projections obtained from single crystal X-ray studies: the lipovitellin complex from lamprey.

The analysis of single crystals of the lipovitellin complex from lamprey has made it possible to compare electron density projections derived from X-ray diffraction and density modification methods with previously published electron micrographs. The close correlation between the images obtained by the two methods demonstrates that the fidelity of images obtained by electron microscopy is excellent despite the loss of specimen order. These correlations also attest to the ability of density modification methods, currently used in macromolecular crystallography, to estimate the phases of small angle X-ray reflections.

Animals↗

PubMed1984

Preparation of single crystals of a yolk lipoprotein.

Single crystals of a yolk lipoprotein from two species of lamprey oocytes have been prepared and shown to belong to the monoclinic space group C2. The unit cell dimensions combined with molecular weight determinations support the idea that the lipoprotein is a symmetrical dimer centered on a crystallographic 2-fold symmetry axis. The lipid content is estimated to be about 15% (w/w) and the crystals are suitable for structural studies by single crystal X-ray analysis. The X-ray results are correlated with those obtained by electron diffraction.

Animals↗