Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe-2S] cluster. Gel filtration chromatography and dynamic light scattering measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453+/-10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a=93.5 A, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.

Crystallographic analysis of the neurophysin-oxytocin complex. A preliminary report.

Single crystals of a bovine neurophysin II-oxytocin complex have been obtained using (NH4)2SO4 as the precipitating agent. The crystals diffract to at least 2.7 A resolution, belong to Laue group 4/mmm and exhibit systematic absences consistent with either space group P4(1)2(1)2 or P4(3)2(1)2. The cell dimensions are a = b = 69.07 A and c = 113.26 A. The crystals contain one neurophysin-oxytocin dimer per asymmetric unit. Based on a Vm of 2.9 A3/Da, the solvent content is calculated to be 58%. Chromatographic analysis of the dissolved crystals suggests the presence of three oxytocin molecules per neurophysin dimer.

Single crystals of a winter flounder antifreeze polypeptide.

Component 6 of the winter flounder's antifreeze polypeptides has been crystallized. The space group is P21, with cell parameters of a = 38.14 A, b = 37.19 A, c = 21.82 A, beta = 101.5 degrees. There are two molecules of 3300 Mr per asymmetric unit.

Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 A resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2-position. The protein-coelenterazine 2-oxy complex observed in the crystals is photo-active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the second de novo protein structure determined using the anomalous scattering signal of the sulfur substructure in the crystal. The method used here is theoretically different from that used for crambin in 1981 (4.72 kDa) and represents a significant advancement in protein crystal structure determination.

Structures of an unliganded neurophysin and its vasopressin complex: implications for binding and allosteric mechanisms.

The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 A and 2.3 A, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of beta-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone alpha-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed.

Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription.

Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined. In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution. The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices. Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC'. This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter. Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site.

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions.

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.

Warfarin sensitivity after mechanical heart valve replacement.

We performed a retrospective chart review of 60 patients after mechanical heart valve replacement to assess warfarin sensitivity. The overall international normalized ratio (INR) on day 3 of therapy was 4.1+/-3.9 (range 1.1-17.1). In a control group of 100 patients who received anticoagulation for atrial fibrillation, pulmonary embolism, and deep vein thrombosis, the overall mean INR at day 3 was 1.9+/-0.7 (range 1.0-4.9). The difference between groups was statistically significant (p<0.05). We conclude that patients receiving warfarin after mechanical heart valve replacement are more sensitive to the drug than those receiving it for other indications, and reduced dosages may be necessary during the first 3 days after valve replacement.

Coronary thrombosis and platelet/fibrin microemboli in death associated with acute myocardial infarction.

The frequency and clinical significance of platelet/fibrin microemboli in the microcirculation were investigated in 24 patients whose deaths (before and during hospital admission) were associated with acute myocardial infarction. An acute coronary thrombus was present in all the hearts. In nine hearts an acute thrombus was found in more than one major epicardial coronary artery. A total of 35 acute thrombi were found in the 24 hearts. Platelet/fibrin microemboli were found in 19 (79%) hearts. Eighteen patients died in hospital. The hearts of 16 of these cases showed microemboli; 16 had important arrhythmias or various forms of heart block; 13 showed acute pathological changes in the conduction system. Fourteen of the deaths in hospital were primarily the result of cardiogenic shock and four were primarily caused by arrhythmia. Six of the deaths that occurred before admission to hospital were regarded as being arrhythmic in origin. Three of these showed microemboli and the other three had acute pathological changes in the conduction system. Microemboli were found in two (24%) of 12 control hearts. Coronary thrombosis was found in most deaths caused by acute myocardial infarction and platelet/fibrin microemboli were present in the majority of such hearts. These may arise from the coronary thrombus in the larger upstream vessel supplying the microcirculation.

Long-term follow-up of patients having cardiac catheterization and cardiac operation. Small community hospital results before and after surgery program.

We followed, for a mean period of 67 months, 710 unselected consecutive cases of cardiac catheterization. Catheterizations were done on 298 patients without an in-house cardiac surgery team. When a cardiac operation was required, patients in this group were referred to a distant university medical center and were followed up after 49 and again at 103 months. After the community hospital's surgical team was established, 412 patients were catheterized and follow-up carried out for 45 months. Results show that patients in a community hospital without an in-house cardiac surgery team can be catheterized with low risk, then transferred safely to a distant center for surgical treatment without interim mortality and with good long-term results.

Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution.

The crystal structure of T7 RNA polymerase reveals a molecule organized around a cleft that can accommodate a double-stranded DNA template. A portion (approximately 45%) of the molecule displays extensive structural homology to the polymerase domain of Klenow fragment and more limited homology to the human immunodeficiency virus HIV-1 reverse transcriptase. A comparison of the structures and sequences of these polymerases identifies structural elements that may be responsible for discriminating between ribonucleotide and deoxyribonucleotide substrates, and RNA and DNA templates. The relative locations of the catalytic site and a specific promoter recognition residue allow the orientation of the polymerase on the template to be defined.

Single crystals of bacteriophage T7 RNA polymerase.

Single crystals of T7 RNA polymerase have been grown to a maximum size of 1.8 x 0.3 x 0.3 mm. The crystals are composed of fully intact T7 RNA polymerase which is enzymatically active upon dissolution. These crystals belong to the monoclinic space group P2(1) and have unit cell parameters a = 114.5 A, b = 139.6 A, c = 125.7 A, and beta = 98.1 degrees. Self-rotation function studies indicate that there are three molecules per asymmetric unit. The crystals diffract to at least 3.0 A resolution. These are the first crystals of a DNA-dependent RNA polymerase suitable for high-resolution X-ray structure determination.

Crystallization and preliminary X-ray diffraction analysis of the mitochondrial transcription factor sc-mtTFB from Saccharomyces cerevisiae.

Eukaryotic mitochondria contain a distinct mini-chromosome. In yeast, transcription of the mitochondrial genome is mediated by a nuclear-encoded RNA polymerase consisting of a single polypeptide core enzyme and a specificity factor termed sc-mtTFB which bears some similarity to bacterial sigma-factors. sc-mtTFB from Saccharomyces cerevisiae has been cloned, expressed, purified and crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 89.7, b = 44.6, c = 98.9 A, beta = 110 degrees. Based on one molecule per asymmetric unit, the solvent content is estimated to be 48%. Small crystals of dimensions 0.01 x 0.05 x 0.13 mm diffract to at least 2.7 A resolution on a rotating-anode X-ray source.

Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase.

NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.

Crystals of ligand-free bovine neurophysin II.

A modified neurophysin, des 1-6 bovine neurophysin II, has been crystallized in the absence of bound hormone or hormone analogue. These crystals represent the first crystals of ligand-free neurophysin, and are essential for understanding neurophysin-hormone recognition as well as hormone-induced neurophysin dimerization. The crystals diffract to beyond 1.8 A resolution, belong to space group P3(1)21 (or P3(2)21) with a = 48.86, c = 78.61 A, and contain one molecule per asymmetric unit.

Linking topography of its potential surface with the dynamics of folding of a protein model.

The "3-color, 46-bead" model of a folding polypeptide is the vehicle for adapting to proteins a mode of analysis used heretofore for atomic clusters, to relate the topography of the potential surface to the dynamics that lead to formation of selected structures. The analysis is based on sequences of stationary points-successive minima, joined by saddles-that rise monotonically in energy from basin bottoms. Like structure-seeking clusters, the potential surface of the model studied here is staircase-like, rather than sawtooth-like, with highly collective motions required for passage from one minimum to the next. The surface has several deep basins whose minima correspond to very similar structures, but which are separated by high energy barriers.

The xenograft antigen in complex with GS-1-B4 lectin: crystallization and preliminary X-ray analysis.

The implantation of animal organs is one approach to overcoming the shortage of human donor organs for medical transplantation. Although readily available, non-primate tissues are subject to hyperacute rejection wherein human anti-Galalpha(1-3)Gal antibodies react with haptens present on the transplanted cells' surfaces. The understanding of this interaction on a molecular level will further the development of a strategy for the prevention of hyperacute rejection in xenotransplantation. The Galalpha(1-3)Gal hapten ('xenograft antigen') has been cocrystallized with the Gal-specific B(4) isolectin of Griffonia simplicifolia lectin-1. Crystals were analyzed by cryocrystallography and were found to diffract to moderately high resolution on a rotating-anode X-ray source. They belong to the P2(1)2(1)2 space group, with unit-cell parameters a = 111.0, b = 51.3, c = 76.9 A, and contain two molecules per asymmetric unit.

Crystal structure of the neurophysin-oxytocin complex.

The first crystal structure of the pituitary hormone oxytocin complexed with its carrier protein neurophysin has been determined and refined to 3.0 A resolution. The hormone-binding site is located at the end of a 3(10)-helix and involves residues from both domains of each monomer. Hormone residues Tyr 2, which is buried deep in the binding pocket, and Cys 1 have been confirmed as the key residues involved in neurophysin-hormone recognition. We have compared the bound oxytocin observed in the neurophysin-oxytocin complex, the X-ray structures of unbound oxytocin analogues and the NMR-derived structure for bound oxytocin. We find that while our structure is in agreement with the previous crystallographic findings, it differs from the NMR result with regard to how Tyr 2 of the hormone is recognized by neurophysin.