Biomedical subjects

D E McRee

Publications and source records attributed to D E McRee.

At least 37 records · Page 2Linked to original sources

PubMed1996

Crystal structure of dUTP pyrophosphatase from feline immunodeficiency virus.

We have determined the crystal structure of dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) at 1.9 A resolution. The structure has been solved by the multiple isomorphous replacement (MIR) method using a P6(3) crystal form. The results show that the enzyme is a trimer of 14.3 kDa subunits with marked structural similarity to E. coli dUTPase. In both enzymes the C-terminal strand of an anti-parallel beta-barrel participates in the beta-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. In the P6(3) crystal form one trimer packs on the 6(3) screw-axis and another on the threefold axis so that there are two independent monomers per asymmetric unit. A Mg2+ ion is coordinated by three asparate residues on the threefold axis of each trimer. Alignment of 17 viral, prokaryotic, and eukaryotic dUTPase sequences reveals five conserved motifs. Four of these map onto the interface between pairs of subunits, defining a putative active site region; the fifth resides in the C-terminal 16 residues, which is disordered in the crystals. Conserved motifs from all three subunits are required to create a given active site. With respect to viral protein expression, it is particularly interesting that the gene for dUTPase (DU) resides in the middle of the Pol gene, the enzyme cassette of the retroviral genome. Other enzymes encoded in the Pol polyprotein, including protease (PR), reverse transcriptase (RT), and most likely integrase (IN), are dimeric enzymes, which implies that the stoichiometry of expression of active trimeric dUTPase is distinct from the other Pol-encoded enzymes. Additionally, due to structural constraints, it is unlikely that dUTPase can attain an active form prior to cleavage from the polyprotein.

Amino Acid Sequence↗

PubMed1996

A ligand-gated, hinged loop rearrangement opens a channel to a buried artificial protein cavity.

Conformational changes that gate the access of substrates or ligands to an active site are important features of enzyme function. In this report, we describe an unusual example of a structural rearrangement near a buried artificial cavity in cytochrome c peroxidase that occurs on binding protonated benzimidazole. A hinged main-chain rotation at two residues (Pro 190 and Asn 195) results in a surface loop rearrangement that opens a large solvent-accessible channel for the entry of ligands to an otherwise inaccessible binding site. The trapping of this alternate conformational state provides a unique view of the extent to which protein dynamics can allow small molecule penetration into buried protein cavities.

Crystallography, X-Ray↗

PubMed1996

Crystal structure of Aplysia ADP ribosyl cyclase, a homologue of the bifunctional ectozyme CD38.

ADP ribosyl cyclase synthesizes the novel secondary messenger cyclic ADP ribose (cADPR) utilizing NAD as a substrate. The enzyme shares extensive sequence similarity with two lymphocyte antigens, CD38 and BST-1, which hydrolyse as well as synthesize cADPR. The crystal structure provides a model for these cell surface enzymes. Cyclase contains two spatially separated pockets composed of sequence conserved residues, suggesting that the cyclization reaction may entail use of distinct sites. The enzyme dimer encloses a cavity which may entrap the intermediate, ADP ribose.

ADP-ribosyl Cyclase↗

PubMed1995

The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochrome c peroxidase.

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.

Aspartic Acid↗

PubMed1994

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

The crystal structure of the His-175-->Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (approximately 5%), which, in view of the very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.

Crystallography, X-Ray↗

PubMed1994

Small molecule binding to an artificially created cavity at the active site of cytochrome c peroxidase.

In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.

Amino Acid Sequence↗

PubMed1993

The crystal structure of lysin, a fertilization protein.

Lysin, a protein from abalone sperm, creates a hole in the envelope of the egg, permitting the sperm to pass through the envelope and fuse with the egg. The structure of lysin, refined at 1.9 angstroms resolution, reveals an alpha-helical, amphipathic molecule. The surface of the protein exhibits three features: two tracks of basic residues that span the length of the molecule, a solvent-exposed cluster of aromatic and aliphatic amino acids, and an extended amino-terminal hypervariable domain that is species-specific. The structure suggests possible mechanisms of action.

Amino Acid Sequence↗

PubMed1993

Self-assembling organic nanotubes based on a cyclic peptide architecture.

Hollow tubular structures of molecular dimensions may offer a variety of applications in chemistry, biochemistry and materials science. Concentric carbon nanotubes have attracted a great deal of attention, while the three-dimensional tubular pore structures of molecular sieves have long been exploited industrially. Nanoscale tubes based on organic materials have also been reported previously. Here we report the design, synthesis and characterization of a new class of organic nanotubes based on rationally designed cyclic polypeptides. When protonated, these compounds crystallize into tubular structures hundreds of nanometres long, with internal diameters of 7-8 A. Support for the proposed tubular structures is provided by electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy and molecular modelling. These tubes are open-ended, with uniform shape and internal diameter. We anticipate that they may have possible applications in inclusion chemistry, catalysis, molecular electronics and molecular separation technology.

Amino Acid Sequence↗

PubMed1993

Atomic structure of a cytochrome c' with an unusual ligand-controlled dimer dissociation at 1.8 A resolution.

The crystallographic structure of cytochrome c' from the purple phototrophic bacterium Chromatium vinosum (CVCP) has been determined at 1.8 A resolution using multiple isomorphous replacement. The molecule is a dimer, with each 131-residue chain folding as a four-helical bundle incorporating a covalently bound heme group at the core. This structure is the third of the ubiquitous cytochromes c' to be solved and is similar to the known structures of cytochrome c' from R. molischianum (RMCP) and R. rubrum (RRCP). CVCP is unique in exhibiting ligand-controlled dimer dissociation while RMCP and RRCP do not. The Tyr16 side-chain, which replaced Met16 in RMCP and Leu14 in RRCP, is parallel to the heme plane and located directly above the sixth ligand site of the heme Fe. Any ligand binding to this site, such as CO or CN-, must move the Tyr16 side-chain, which would be expected to cause other conformational changes of helix A, which contributes to the dimer interface, and consequently disrupting the dimer. Thus, the crystallographic structure of CVCP suggests a mechanism for dimer dissociation upon ligand binding. The dimer interface specificity is due to a lock and key shape complementarity of hydrophobic residues and not to any charge complementarity or cross-interface hydrogen bonds as is common in other protein-protein interfaces. The co-ordinates have been deposited in the Brookhaven Data Bank (entry P1BBH).

Amino Acid Sequence↗

PubMed1993

The Asp-His-Fe triad of cytochrome c peroxidase controls the reduction potential, electronic structure, and coupling of the tryptophan free radical to the heme.

The buried charge of Asp-235 in cytochrome c peroxidase (CCP) forms an important hydrogen bond to the histidine ligand of the heme iron. The Asp-His-metal interaction, which is similar to the catalytic triad of serine proteases, is found at the active site of many metalloenzymes and is believed to modulate the character of histidine as a metal ligand. We have examined the influence of this interaction in CCP on the function, redox properties, and iron zero-field splitting in the native ferric state and its effect on the Trp-191 free radical site in the oxidized ES complex. Unlike D235A and D235N, the mutation D235E introduces very little perturbation in the X-ray crystal structure of the enzyme active site, with only minor changes in the geometry of the carboxylate-histidine interaction and no observable change at the Trp-191 free radical site. More significant effects are observed in the position of the helix containing residue Glu-235. However, the small change in hydrogen bond geometry is all that is necessary to (1) increase the reduction potential by 70 mV, (2) alter the anisotropy of the Trp-191 free radical EPR, (3) affect the activity and spin-state equilibrium, and (4) reduce the strength of the iron ligand field as measured by the zero-field splitting. The changes in the redox potential with substitution are correlated with the observed zero-field splitting, suggesting that redox control is exerted through the heme ligand by a combination of electrostatic and ligand field effects. The replacement of Asp-235 with Glu appears to result in a significantly weaker hydrogen bond in which the proton resides essentially with His-175. This hydrogen bond is nevertheless strong enough to prevent the reorientation of Trp-191 and the conversion to one of two low-spin states observed for D235A and D235N. The Asp-His-Fe interaction is therefore as important in defining the redox properties and imidazolate character of His-175 as has been proposed, yet its most important role in peroxidase function may be to correctly orient Trp-191 for efficient coupling of the free radical to the heme and to maintain a high-spin 5-coordinate heme center.

Aspartic Acid↗

PubMed1993

Purification, crystallization and space group determination of DNA repair enzyme exonuclease III from E. coli.

Escherichia coli exonuclease III possesses multiple catalytic activities: (1) a nucleotidyl hydrolase activity cutting 5' to apurinic/apyrimidinic sites and urea residues in DNA; (2) a 3' to 5' exonuclease activity specific for double-stranded DNA; (3) a RNase H activity preferentially degrading the RNA strand of a DNA.RNA hybrid and (4) an activity that can remove a number of 3' termini from duplex DNA including 3' phosphates, 3' phosphoglycolate residues, 3' phosphoglycolaldehyde residues and 3' trans-4-hydroxy-2-pentenal-5-phosphate residues. These multiple activities make exonuclease III a major enzyme in the base excision repair pathway for DNA damage. We have purified exonuclease III and grown crystals by the vapor diffusion method using polyethylene glycol 4000 as the precipitant. Buffers were found to have profound effects on crystallization with high concentrations of imidazole/malate buffer (0.4 M to 1.0 M) yielding larger crystals with less twinning. The crystals belong to the space group P3(1)21 or its enantiomorph P3(2)21 with unit cell dimensions of a = b = 107.8 A, c = 42.2 A, alpha = beta = 90 degrees, gamma = 120 degrees, have one 31 kDa monomer per asymmetric unit and diffract to 1.6 A. These crystals are stable to X-rays and suitable for high resolution structure determination.

Crystallization↗

PubMed1993

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

Amino Acid Sequence↗

PubMed1992

Atomic structure of the DNA repair [4Fe-4S] enzyme endonuclease III.

The crystal structure of the DNA repair enzyme endonuclease III, which recognizes and cleaves DNA at damaged bases, has been solved to 2.0 angstrom resolution with an R factor of 0.185. This iron-sulfur [4Fe-4S] enzyme is elongated and bilobal with a deep cleft separating two similarly sized domains: a novel, sequence-continuous, six-helix domain (residues 22 to 132) and a Greek-key, four-helix domain formed by the amino-terminal and three carboxyl-terminal helices (residues 1 to 21 and 133 to 211) together with the [4Fe-4S] cluster. The cluster is bound entirely within the carboxyl-terminal loop with a ligation pattern (Cys-X6-Cys-X2-Cys-X5-Cys) distinct from all other known [4Fe-4S] proteins. Sequence conservation and the positive electrostatic potential of conserved regions identify a surface suitable for binding duplex B-DNA across the long axis of the enzyme, matching a 46 angstrom length of protected DNA. The primary role of the [4Fe-4S] cluster appears to involve positioning conserved basic residues for interaction with the DNA phosphate backbone. The crystallographically identified inhibitor binding region, which recognizes the damaged base thymine glycol, is a seven-residue beta-hairpin (residues 113 to 119). Location and side chain orientation at the base of the inhibitor binding site implicate Glu112 in the N-glycosylase mechanism and Lys120 in the beta-elimination mechanism. Overall, the structure reveals an unusual fold and a new biological function for [4Fe-4S] clusters and provides a structural basis for studying recognition of damaged DNA and the N-glycosylase and apurinic/apyrimidinic-lyase mechanisms.

Bacterial Proteins↗

PubMed1992

Crystallization and crystallographic characterization of the iron-sulfur-containing DNA-repair enzyme endonuclease III from Escherichia coli.

Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities. It could serve to repair damaged thymine residues in DNA via base excision-repair. We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals. Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A. A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence. The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme. The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.

Bacterial Proteins↗

PubMed1991

Refined crystal structure of Cd, Zn metallothionein at 2.0 A resolution.

The crystal structure of Cd5,Zn2-metallothionein from rat liver has been refined at 2.0 A resolution of a R-value of 0.176 for all observed data. The five Cd positions in the asymmetric unit of the crystal create a pseudo-centrosymmetric constellation about a crystallographic 2-fold axis. Consequently, the distribution of anomalous differences is almost ideally centrosymmetric. Therefore, the previously reported metal positions and the protein model derived therefrom are incorrect. Direct methods were applied to the protein amplitudes to locate the Cd positions. The new positions were used to calculate a new electron density map based on the Cd anomalous scattering and partial structure to model the metal clusters and the protein. Phases calculated from this model predict the positions of three sites in a (NH4)2WS4 derivative. Single isomorphous replacement phases calculated with these tungsten sites confirm the positions of the Cd sites from the new direct methods calculations. The refined metallothionein structure has a root-mean-square deviation of 0.016 A from ideality of bonds and normal stereochemistry of phi, phi and chi torsion angles. The metallothionein crystal structure is in agreement with the structures for the alpha and beta domains in solution derived by nuclear magnetic resonance methods. The overall chain folds and all metal to cysteine bonds are the same in the two structure determinations. The handedness of a short helix in the alpha-domain (residues 41 to 45) is the same in both structures. The crystal structure provides information concerning the metal cluster geometry and cysteine solvent accessibility and side-chain stereochemistry. Short cysteine peptide sequences repeated in the structure adopt restricted conformations which favor the formation of amide to sulfur hydrogen bonds. The crystal packing reveals intimate association of molecules about the diagonal 2-fold axes and trapped ions of crystallization (modeled as phosphate and sodium). Variation in the chemical and structural environments of the metal sites is in accord with data for metal exchange reactions in metallothioneins.

Amino Acid Sequence↗

PubMed1991

Mechanism and atomic structure of superoxide dismutase.

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases, and the interpretation of the electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu,Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.

Animals↗

PubMed1990

Changes in crystallographic structure and thermostability of a Cu,Zn superoxide dismutase mutant resulting from the removal of a buried cysteine.

In principle, protein thermostability depends on efficient interior packing of apolar residues and on avoidance of irreversible denaturation in the unfolded state. To study these effects, the single free cysteine in the highly stable enzyme bovine Cu,Zn superoxide dismutase was mutated to alanine (Cys6----Ala), and the recombinant protein was expressed in yeast, purified, characterized for reversible and irreversible denaturation, crystallized isomorphously to the wild-type enzyme, and used to determine the atomic structure. Removal of the chemically reactive thiol significantly decreased the rate of irreversible denaturation (as monitored by thermal inactivation at 70 degrees C), but the observed energetic cost (delta delta G of 0.7-1.3 kcal/mol as determined by differential scanning calorimetry) was much less than predicted from either the change in hydrophobicity or packing due to removal of the interior sulfur atom. X-ray diffraction data were collected to 2.1-A resolution using an area detector, and the atomic model for the mutant enzyme was determined by fitting to electron density difference maps, followed by reciprocal space refinement both with stereochemical restraints using PROLSQ and with molecular dynamics using X-PLOR. The refined 2.1-A resolution crystallographic structure suggests that small concerted and compensating shifts (less than 0.5 A) of the surrounding side chains and of the adjacent N- and C-terminal beta-strands significantly reduced the energetic cost of the interior mutation by improving packing and stereochemistry in the mutant enzyme. Taken together, these results differentiate between the effects of reversible and irreversible processes as they impact the design of thermostable proteins and suggest that relatively subtle concerted shifts can significantly reduce the energetic cost of evolutionary variation in internal residues of proteins with Greek key beta-barrel folds.

Alanine↗

PubMed1990

Crystallographic characterization of a Cu,Zn superoxide dismutase from Photobacterium leiognathi.

Crystals of a copper-zinc superoxide dismutase from Photobacterium leiognathi, a luminescent marine bacterium that is the species-specific symbiont of the ponyfish, have been obtained from 2-methyl-2,4-pentanediol solutions. The space group was determined using screenless small-angle precession photographs, and was confirmed by analyzing area detector diffraction data with the XENGEN programs for indexing and refinement. The crystals are monoclinic, space group C2 (a = 126.4 A, b = 87.0 A, c = 44.4 A, beta = 92.8 A), and have two 32,000 Mr dimers per asymmetric unit. The crystals diffract to at least 2.7 A resolution, are resistant to radiation damage, and are suitable for determination of the structure by X-ray diffraction.

Photobacterium↗