ABSTRACT
CPT-11 is a potent antitumor agent that is activated by carboxylesterases (CE) and intracellular expression of CEs that can activate the drug results in increased cytotoxicity to the drug. As activation of CPT-11 (irinotecan-7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) by human CEs is relatively inefficient, we have developed enzyme/prodrug therapy approaches based on the CE/CPT-11 combination using a rabbit liver CE (rCE). However, the in vivo application of this technology may be hampered by the development of an immune response to rCE. Therefore, we have developed a mutant human CE (hCE1m6), based on the human liver CE hCE1, that can activate CPT-11 approximately 70-fold more efficiently than the wild-type protein and can be expressed at high levels in mammalian cells. Indeed, adenoviral-mediated delivery of hCE1m6 with human tumor cells resulted in up to a 670-fold reduction in the IC(50) value for CPT-11, as compared to cells transduced with vector control virus. Furthermore, xenograft studies with human tumors expressing hCE1m6 confirm the ability of this enzyme to activate CPT-11 in vivo and induce antitumor activity. We propose that this enzyme should likely be less immunogenic than rCE and would be suitable for the in vivo application of CE/CPT-11 enzyme/prodrug therapy.
Subject(s)
Camptothecin/analogs & derivatives , Carboxylesterase/genetics , Prodrugs/therapeutic use , Xenograft Model Antitumor Assays , Adenoviridae/genetics , Amino Acid Sequence , Animals , Antineoplastic Agents, Phytogenic/metabolism , Antineoplastic Agents, Phytogenic/therapeutic use , Blotting, Western , COS Cells , Camptothecin/metabolism , Camptothecin/therapeutic use , Carboxylesterase/chemistry , Carboxylesterase/metabolism , Cell Proliferation/drug effects , Chlorocebus aethiops , Combined Modality Therapy , Crystallography, X-Ray , Genetic Therapy/methods , Humans , Irinotecan , Mice , Mice, SCID , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Prodrugs/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , TransfectionABSTRACT
Individual variation in drug metabolism is a major cause of unpredictable side effects during therapy. Drug metabolism is controlled by a class of orphan nuclear receptors (NRs), which regulate expression of genes such as CYP (cytochrome)3A4 and MDR-1 (multi-drug resistance-1), that are involved in this process. We have found that xenobiotic-mediated induction of CYP3A4 and MDR-1 gene transcription was inhibited by ketoconazole, a commonly used antifungal drug. Ketoconazole mediated its effect by inhibiting the activation of NRs, human pregnenolone X receptor and constitutive androstene receptor, involved in regulation of CYP3A4 and MDR-1. The effect of ketoconazole was specific to the group of NRs that control xenobiotic metabolism. Ketoconazole disrupted the interaction of the xenobiotic receptor PXR with the co-activator steroid receptor co-activator-1. Ketoconazole treatment resulted in delayed metabolism of tribromoethanol anesthetic in mice, which was correlated to the inhibition of PXR activation and downmodulation of cyp3a11 and mdr-1 genes and proteins. These studies demonstrate for the first time that ketoconazole represses the coordinated activation of genes involved in drug metabolism, by blocking activation of a specific subset of NRs. Our results suggest that ketoconazole can be used as a pan-antagonist of NRs involved in xenobiotic metabolism in vivo, which may lead to novel strategies that improve drug effect and tolerance.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antifungal Agents/pharmacology , Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation/drug effects , Ketoconazole/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Blotting, Western , Constitutive Androstane Receptor , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , DNA-Binding Proteins/antagonists & inhibitors , Ethanol/analogs & derivatives , Ethanol/metabolism , Female , Hepatocytes/metabolism , Histone Acetyltransferases/antagonists & inhibitors , Humans , Liver X Receptors , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Receptor Coactivator 1 , Orphan Nuclear Receptors , Pregnane X Receptor , RNA, Messenger/metabolism , Receptors, Steroid/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/antagonists & inhibitors , Tumor Cells, CulturedABSTRACT
Human carboxylesterase 1 (hCE1) is a serine esterase involved in both drug metabolism and activation, as well as other biological processes. hCE1 catalyses the hydrolysis of heroin and cocaine, and the transesterification of cocaine in the presence of ethanol to the toxic metabolite cocaethylene. We have determined the crystal structures of hCE1 in complex with either the cocaine analogue homatropine or the heroin analogue naloxone. These are the first structures of a human carboxylesterase, and they provide details about narcotic metabolism in humans. hCE1's active site contains rigid and flexible pockets, explaining the enzyme's ability to act both specifically and promiscuously. hCE1 has also been reported to contain cholesteryl ester hydrolase, fatty acyl-CoA hydrolase and acyl-CoA:cholesterol acyltransferase activities, and thus appears to be involved in cholesterol metabolism. Since the enzyme may be useful as a treatment for cocaine overdose, and may afford protection against chemical weapons like Sarin, Soman and VX gas, hCE1 could serve as both a drug and a drug target. Selective hCE1 inhibitors targeted to several sites on the enzyme may also pave the way for novel clinical tools to manage cholesterol homoeostasis in humans.
Subject(s)
Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Drug Design , Pharmaceutical Preparations/metabolism , Cocaine/pharmacokinetics , Crystallography, X-Ray , Heroin/pharmacokinetics , Humans , Models, Molecular , Protein Conformation , Substrate SpecificityABSTRACT
The human nuclear pregnane X receptor (hPXR) activates cytochrome P450-3A expression in response to a wide variety of xenobiotics and plays a critical role in mediating dangerous drug-drug interactions. We present the crystal structures of the ligand-binding domain of hPXR both alone and in complex with the cholesterol-lowering drug SR12813 at resolutions of 2.5 and 2.75 angstroms, respectively. The hydrophobic ligand-binding cavity of hPXR contains a small number of polar residues, permitting SR12813 to bind in three distinct orientations. The position and nature of these polar residues were found to be critical for establishing the precise pharmacologic activation profile of PXR. Our findings provide important insights into how hPXR detects xenobiotics and may prove useful in predicting and avoiding drug-drug interactions.
Subject(s)
Diphosphonates/metabolism , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/chemistry , Receptors, Steroid/metabolism , Xenobiotics/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Diphosphonates/chemistry , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Pregnane X Receptor , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Rifampin/metabolismABSTRACT
Human topoisomerase I helps to control the level of DNA supercoiling in cells and is vital for numerous DNA metabolic events, including replication, transcription, and recombination. The 2.6 A crystal structure of human topoisomerase I in noncovalent complex with a DNA duplex containing a cytosine at the -1 position of the scissile strand rather than the favored thymine is reported. The hydrogen bond between the O2 position of this -1 base and the epsilon-amino of the conserved Lys-532 residue, the only base-specific contact observed previously in the human topoisomerase I-DNA interaction, is maintained in this complex. Several unique features of this structure, however, have implications for the DNA-binding and active-site mechanisms of the enzyme. First, the ends of the DNA duplex were observed to shift by up to 5.4 A perpendicular to the DNA helical axis relative to structures reported previously, suggesting a novel degree of plasticity in the interaction between human topoisomerase I and its DNA substrate. Second, 12 additional residues at the NH(2) terminus of the protein (Trp-203-Gly-214) could be built in this structure, and they were found to pack against the putative hinge region implicated in the clamping of the enzyme around duplex DNA. Third, a water molecule was observed adjacent to the scissile phosphate and the active-site residues; the potential specific base character of this solvent molecule in the active-site mechanism of the enzyme is discussed. Fourth, the scissile phosphate group was found to be rotated by 75 degrees, bringing Lys-532 into hydrogen-bonding distance of one of the nonbridging phosphate oxygens. This orientation of the scissile phosphate group implicates Lys-532 as a fifth active-site residue, and also mimics the orientation observed for the 3'-phosphotyrosine linkage in the covalent human topoisomerase I-DNA complex structure. The implications of these structural features for the mechanism of the enzyme are discussed, including the potential requirement for a rotation of the scissile phosphate group during DNA strand cleavage and covalent attachment.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA/chemistry , Binding Sites , Crystallography, X-Ray , DNA Topoisomerases, Type I/genetics , DNA-Binding Proteins/chemistry , Humans , Hydrogen Bonding , Models, Molecular , Mutation , Nucleic Acid Conformation , Nucleoproteins/chemistry , Protein ConformationABSTRACT
Human topoisomerase I plays a critical role in chromosomal stability by relaxing the DNA superhelical tension that arises from a variety of nuclear processes, including replication, transcription, and chromatin remodeling. Human topoisomerase I is a approximately 91 kDa enzyme composed of four major domains: a 24 kDa N-terminal domain, a56 kDa core domain, a7 kDa linker domain, and a6 kDa C-terminal domain containing the active-site Tyr723 residue. A monoclinic crystal structure of a 70 kDa N-terminally truncated form of human topoisomerase I in non-covalent complex with a 22 bp DNA duplex exhibited remarkable crystal-to-crystal non-isomorphism; shifts in cell constants of up to 9 A in the b -axis length and up to 8.5 degrees in the beta-angle were observed. Eight crystal structures of human topoisomerase I - DNA complexes from this crystal form were determined to between 2.8 and 3.25 A resolution. These structures revealed both dramatic shifts in crystal packing and functionally suggestive regions of conformational flexibility in the structure of the enzyme. Crystal packing shifts of up to 20.5 A combined with rotations of up to 11.5 degrees were observed, helping to explain the variability in cell constants. When the core subdomain III regions of the eight structures are superimposed, the "cap" (core subdomains I and II) of the molecule is observed to rotate by up to 4.6 degrees and to shift by up to 3.6 A. The linker domain shows the greatest degree of conformational flexibility, rotating and shifting by up to 2.5 degrees and 4.6 A, respectively, in five of eight structures, and becoming disordered altogether in the remaining three. These observed regions of conformational flexibility in the cap and the linker domain are consistent with the structural flexibility invoked in the "controled rotation" mechanism proposed for the relaxation of DNA superhelical tension by human topoisomerase I.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA/chemistry , Crystallization , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Protein ConformationABSTRACT
A significant portion of new protein structures contain folds that are related to those seen before. During the development of a computer program that can accurately position, in electron density maps, large protein domains with large structural deviations, it became apparent that the redundancy in protein folds could be used in a non trivial manner during a protein structure determination. As a result a computational procedure, Database Assisted Density Interpretation (DADI), was developed and tested to aid in the building of models in protein crystallography and to assist in interpreting electron density maps. The initial tests of the DADI procedure using a small database of protein domains are described. The philosophy is to first work with entire domains then with the secondary structure elements of these domains and finally with individual residues of the secondary structure elements via Monte Carlo, "chopping" and "clipping" procedures, respectively. The first test case was a traceable 3.2 A multiple isomorphous replacement with anomalous scattering (MIRAS) electron density map of a human topoisomerase I-DNA complex. The second test case uses poor electron density for the third domain of the diphtheria toxin repressor resulting from a molecular replacement solution with the first two domains. Despite the fact that a fairly small database was employed in these test cases, the DADI procedure was able to find a large portion of the protein backbone with very few errors. In the first case nearly 45% of the backbone and more than 80% of the secondary structure was placed automatically. In the second test case nearly 50% of the third domain was automatically detected. A particular encouraging result was that in both cases more than 75% of the beta sheet secondary structure was found automatically by the DADI procedure. Clearly, the procedures employed are promising avenues to exploit the current explosion of protein structures for the determination of future structures. Proteins 1999;36:526-541.
Subject(s)
Computer Simulation , Crystallography, X-Ray , Databases, Factual , Models, Molecular , Proteins/chemistry , Algorithms , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Computational Biology , Crystallography, X-Ray/methods , DNA/chemistry , DNA/metabolism , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Electrons , Homeodomain Proteins/chemistry , Humans , Integrases/chemistry , Monte Carlo Method , Protein Folding , Protein Structure, Secondary , Software , src Homology DomainsABSTRACT
A Monte Carlo procedure, encoded in the program Blob, has been developed and tested for the purpose of positioning large molecular fragments or small flexible molecules in electron density maps. The search performed by the algorithm appears to be sufficiently thorough to accurately position a small flexible ligand in well-defined density while remaining sufficiently random to offer interesting alternate suggestions for density representing disordered binding modes of a ligand. Furthermore, the algorithm is shown to be efficient enough to accurately position large rigid molecular fragments. In the first of the test cases with large molecular fragments, Blob was surprisingly effective in positioning a poly-alanine model of a 53-residue domain in poor electron density resulting from molecular replacement with a partial model. At 3.0 A resolution the domain was positioned consistently within 0.2 A of its experimentally determined position. Even at 6.0 A resolution Blob could consistently position the domain to within 0.75 A of its actual position. A second set of tests with large molecular fragments revealed that Blob could correctly position large molecular fragments with quite significant deviations from the actual structure. In this test case, fragments ranging from a 170-residue protein domain with a 3.8 A rms deviation from the actual structure to a 22-base pair ideal B-form DNA duplex were positioned accurately in a 3.2 A electron density map derived from multiple isomorphous replacement methods. Even when decreasing the quality of the maps, from a figure of merit of 0.57 to as low as 0. 35, Blob could still effectively position the large protein domain and the DNA duplex. Since it is efficient, can handle large molecular fragments, and works in poor and low resolution maps, Blob could be a useful tool for interpreting electron density maps in de novo structure determinations and in molecular replacement studies. Proteins 1999;36:512-525.
Subject(s)
Algorithms , Crystallography, X-Ray/methods , Electrons , Models, Molecular , Nucleic Acids/chemistry , Peptide Fragments/chemistry , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , DNA/chemistry , DNA/metabolism , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Homeodomain Proteins/chemistry , Humans , Integrases/chemistry , Ligands , Molecular Structure , Molecular Weight , Monte Carlo Method , Nucleic Acids/metabolism , Peptide Fragments/metabolism , Software , Time FactorsABSTRACT
Topoisomerases relax the DNA superhelical tension that arises in cells as a result of several nuclear processes, including transcription, replication and recombination. Recently determined crystal structures of human topoisomerase I in complex with DNA and of the 27 kDa catalytic domain of the vaccinia virus topoisomerase have advanced our understanding of the eukaryotic type IB topoisomerases. These recent structural results provide insights into functional aspects of these topoisomerases, including their DNA binding, strand cleavage and religation activities, as well as the mechanism that these enzymes use to relax DNA superhelical tension. In addition, two proposed models of the anticancer drug camptothecin bound to a covalent complex of human topoisomerase I and DNA suggest a structural basis for the mode of action of the drug.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/chemistry , Camptothecin/metabolism , Camptothecin/pharmacology , DNA Topoisomerases, Type I/classification , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Substrate Specificity , Vaccinia virus/enzymologyABSTRACT
Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA-Binding Proteins , DNA/metabolism , Protein Conformation , Antineoplastic Agents, Phytogenic/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Binding Sites , Camptothecin/analogs & derivatives , Camptothecin/metabolism , Camptothecin/pharmacology , Crystallography, X-Ray , DNA/chemistry , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , Homeodomain Proteins/chemistry , Host Cell Factor C1 , Humans , Hydrogen Bonding , Integrases/chemistry , Models, Molecular , Mutation , Nucleic Acid Conformation , Octamer Transcription Factor-1 , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Protein Structure, Secondary , Recombinant Proteins/chemistry , Transcription Factors/chemistry , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
The three-dimensional structure of a 70-kilodalton amino terminally truncated form of human topoisomerase I in complex with a 22-base pair duplex oligonucleotide, determined to a resolution of 2.8 angstroms, reveals all of the structural elements of the enzyme that contact DNA. The linker region that connects the central core of the enzyme to the carboxyl-terminal domain assumes a coiled-coil configuration and protrudes away from the remainder of the enzyme. The positively charged DNA-proximal surface of the linker makes only a few contacts with the DNA downstream of the cleavage site. In combination with the crystal structures of the reconstituted human topoisomerase I before and after DNA cleavage, this information suggests which amino acid residues are involved in catalyzing phosphodiester bond breakage and religation. The structures also lead to the proposal that the topoisomerization step occurs by a mechanism termed "controlled rotation."
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , DNA/metabolism , Models, Chemical , Protein Conformation , Amino Acid Sequence , Arginine/chemistry , Arginine/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , DNA/chemistry , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Protein Structure, Secondary , Tyrosine/chemistry , Tyrosine/metabolismABSTRACT
Adenylosuccinate lyase (ASL) from Bacillus subtilis has been crystallized and structural analysis by X-ray diffraction is in progress. ASL is a 200-kDa homotetramer that catalyzes two distinct steps of de novo purine biosynthesis leading to the formation of AMP and IMP; both steps involve the beta-elimination of fumarate. A single point mutation in the human ASL gene has been linked to mental retardation with autistic features. In addition, ASL plays an important role in the bioprocessing of anti-HIV therapeutics. B subtilis ASL, which shares 30% sequence identity and 70% sequence similarity with human ASL, has been crystallized and data to 3.3 A have been collected at 100 K. The space group is P6(1)22 or P6(5)22 with a = b = 129.4 A; the length of the c-axis varies between 275 and 290 A, depending on the crystal. An analysis of solvent content indicates a dimer in the asymmetric unit, although a self-rotation function and an analysis of native Pattersons failed to identify unambiguously the location of any noncrystallographic symmetry axes. Structure determination by isomorphous replacement is in progress.
Subject(s)
Adenylosuccinate Lyase/chemistry , Autistic Disorder/enzymology , Bacillus subtilis/enzymology , Crystallization , Crystallography, X-Ray , Humans , Purines/biosynthesisABSTRACT
Plastocyanin is one of the best characterized of the photosynthetic electron transfer proteins. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 A resolution. Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between the Chlamydomonas and poplar proteins), the three-dimensional structures are remarkably conserved (e.g., 0.76 A rms deviation in the C alpha positions between the Chlamydomonas and poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel beta-barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochrome f and Photosystem I, and validated the model of two functionally significant electron transfer paths in plastocyanin. One putative electron transfer path is relatively short (approximately 4 A) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (approximately 12-15 A) and involves the nearly conserved residue Tyr-83 in the negative patch.
Subject(s)
Plastocyanin/chemistry , Amino Acid Sequence , Molecular Sequence Data , Plastocyanin/metabolism , Protein Conformation , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.
Subject(s)
Chlamydomonas reinhardtii/metabolism , Plastocyanin/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Animals , Chlorophyta/metabolism , Chromatography, Gel , Chromatography, Ion Exchange , Copper/metabolism , Crystallization , Models, Molecular , Molecular Sequence Data , Plastocyanin/isolation & purification , Plastocyanin/metabolism , Software , X-Ray Diffraction/methodsABSTRACT
The structure determination of a macromolecule from a hemihedrally twinned crystal specimen with a twinning fraction of one-half is described. Twinning was detected by analysis of crystal-packing density and intensity statistics. The structure was solved using molecular replacement, and the positioned search model was used to overcome the twinning by a novel method of 'detwinning' the observed data. Estimates of the unobservable crystallographic intensities from each of the twin domains were obtained and used to refine the model. The structure of a new algal plastocyanin from Chlamydomonas reinhardtii was determined by this method to 1.6 A resolution with a 'twinned' R factor of 15.6%. Additional data from a crystal specimen with a low twinning fraction were used to establish the accuracy of the structure solution from the perfectly twinned data, and to finalize the refinement to 1.5 A resolution and a true R factor of 16.8%. Methods for detecting twinning and obtaining a molecular-replacement solution in the presence of twinning are discussed.