John P. Rose

The first structure of an aldehyde dehydrogenase reveals novel interactions between NAD and the Rossmann fold.

The first structure of an aldehyde dehydrogenase (ALDH) is described at 2.6 Å resolution. Each subunit of the dimeric enzyme contains an NAD-binding domain, a catalytic domain and a bridging domain. At the interface of these domains is a 15 Å long funnel-shaped passage with a 6 × 12 Å opening leading to a putative catalytic pocket. A new mode of NAD binding, which differs substantially from the classic β-α-β binding mode associated with the ‘Rossmann fold’, is observed which we term the β-α,β mode. Sequence comparisons of the class 3 ALDH with other ALDHs indicate a similar polypeptide fold, novel NAD-binding mode and catalytic site for this family. A mechanism for enzymatic specificity and activity is postulated.

The 2.0 Å structure of human ferrochelatase, the terminal enzyme of heme biosynthesis

Human ferrochelatase (E.C. 4.99.1.1) is a homodimeric (86 kDa) mitochondrial membrane-associated enzyme that catalyzes the insertion of ferrous iron into protoporphyrin to form heme. We have determined the 2.0 Å structure from the single wavelength iron anomalous scattering signal. The enzyme contains two NO-sensitive and uniquely coordinated [2Fe-2S] clusters. Its membrane association is mediated in part by a 12-residue hydrophobic lip that also forms the entrance to the active site pocket. The positioning of highly conserved residues in the active site in conjunction with previous biochemical studies support a catalytic model that may have significance in explaining the enzymatic defects that lead to the human inherited disease erythropoietic protoporphyria.

Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters

Abstract. Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.

The crystal structure of augmenter of liver regeneration: A mammalian FAD-dependent sulfhydryl oxidase

The crystal structure of recombinant rat augmenter of liver regeneration (ALRp) has been determined to 1.8 Å. The protein is a homodimer, stabilized by extensive noncovalent interactions and a network of hydrogen bonds, and possesses a noncovalently bound FAD in a motif previously found only in the related protein ERV2p. ALRp functions in vitro as a disulfide oxidase using dithiothreitol as reductant. Reduction of the flavin by DTT occurs under aerobic conditions resulting in a spectrum characteristic of a neutral semiquinone. This semiquinone is stable and is only fully reduced by addition of dithionite. Mutation of either of two cysteine residues that are located adjacent to the FAD results in inactivation of the oxidase activity. A comparison of ALRp with ERV2p is made that reveals a number of significant structural differences, which are related to the in vivo functions of these two proteins. Possible physiological roles of ALR are examined and a hypothesis that it may serve multiple roles is proposed.

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 Å resolution. The hormone-binding site is located at the end of a 310-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.

All three Ca2+-binding loops of photoproteins bind calcium ions: The crystal structures of calcium-loaded apo-aequorin and apo-obelin.

The crystal structures of calcium‐loaded apoaequorin and apo‐obelin have been determined at resolutions 1.7 Å and 2.2 Å, respectively. A calcium ion is observed in each of the three EF‐hand loops that have the canonical calcium‐binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium‐loaded apo‐proteins retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2‐hydroperoxycoelenterazine, and also the same as the Ca2+‐discharged obelin bound with the product, coelenteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these subtle shifts are the basis of the ability of these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.

Fast native-SAD phasing for routine macromolecular structure determination

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Characterization of a corrinoid protein involved in the C1 metabolism of strict anaerobic bacterium Moorella thermoacetica

The strict anaerobic, thermophilic bacterium Moorella thermoacetica metabolizes C1 compounds for example CO2/H2, CO, formate, and methanol into acetate via the Wood/Ljungdahl pathway. Some of the key steps in this pathway include the metabolism of the C1 compounds into the methyl group of methylenetetrahydrofolate (MTHF) and the transfer of the methyl group from MTHF to the methyl group of acetyl‐CoA catalyzed by methyltransferase, corrinoid protein and CO dehydrogenase/acetyl CoA synthase. Recently, we reported the crystallization of a 25 kDa methanol‐induced corrinoid protein from M. thermoacetica (Zhou et al., Acta Crystallogr F 2005; 61:537–540). In this study we analyzed the crystal structure of the 25 kDa protein and provide genetic and biochemical evidences supporting its role in the methanol metabolism of M. thermoacetia. The 25 kDa protein was encoded by orf1948 of contig 303 in the M. thermoacetica genome. It resembles similarity to MtaC the corrinoid protein of the methanol:CoM methyltransferase system of methane producing archaea. The latter enzyme system also contains two additional enzymes MtaA and MtaB. Homologs of MtaA and MtaB were found to be encoded by orf2632 of contig 303 and orf1949 of contig 309, respectively, in the M. thermoacetica genome. The orf1948 and orf1949 were co‐transcribed from a single polycistronic operon. Metal analysis and spectroscopic data confirmed the presence of cobalt and the corrinoid in the purified 25 kDa protein. High resolution X‐ray crystal structure of the purified 25 kDa protein revealed corrinoid as methylcobalamin with the imidazole of histidine as the α‐axial ligand replacing benziimidazole, suggesting base‐off configuration for the corrinoid. Methanol significantly activated the expression of the 25 kDa protein. Cyanide and nitrate inhibited methanol metabolism and suppressed the level of the 25 kDa protein. The results suggest a role of the 25 kDa protein in the methanol metabolism of M. thermoacetica. Proteins 2007.

Structural studies of several clinically important oncology drugs in complex with human serum albumin.

BACKGROUND Serum albumin is a major pharmacokinetic effector of drugs. To gain further insight into albumin binding chemistry, the crystal structures of six oncology agents were determined in complex with human serum albumin at resolutions of 2.8 to 2.0Å: camptothecin, 9-amino-camptothecin, etoposide, teniposide, bicalutamide and idarubicin. METHODS Protein crystal growth and low temperature X-ray crystallography RESULTS These large, complex drugs are all bound within the subdomain IB binding region which can be described as a hydrophobic groove formed by α-helices h7, h8 and h9 covered by the extended polypeptide L1. L1 creates a binding cavity with two access sites, one between loop L1 and α-helices h7 and h8 (distal site: IBd) and the other between L1 and α-helix h9 (proximal site: IBp). Camptothecin (2.4Å) and 9 amino camptothecin (2.0Å) are clearly bound as the open lactone form (IBp). Idarubicin (2.8Å) binds in a DNA like dimer complex via an intermolecular π stacking arrangement in IBd. Bicalutamide (2.4Å) is bound in a folded intramolecular π stacking arrangement between two aromatic rings in IBd similar to idarubicin. Teniposide (2.7Å) and etoposide (2.7Å), despite small chemical differences, are bound in two distinctly different sites at or near IB. Teniposide is internalized via primarily hydrophobic interactions and spans through both openings (IBp-d). Etoposide is bound between the exterior of IB and IIA and exhibits an extensive hydrogen bonding network. CONCLUSIONS Subdomain IB is a major binding site for complex heterocyclic molecules. GENERAL SIGNIFICANCE The structures have important implications for drug design and development. This article is part of a Special Issue entitled Serum Albumin.

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.