Search PubMed⌕ Search

Biomedical subjects

D C Rees

Publications and source records attributed to D C Rees.

At least 19 recordsLinked to original sources
PubMed2006

An inward-facing conformation of a putative metal-chelate-type ABC transporter.

The crystal structure of a putative metal-chelate-type adenosine triphosphate (ATP)-binding cassette (ABC) transporter encoded by genes HI1470 and HI1471 of Haemophilus influenzae has been solved at 2.4 angstrom resolution. The permeation pathway exhibits an inward-facing conformation, in contrast to the outward-facing state previously observed for the homologous vitamin B12 importer BtuCD. Although the structures of both HI1470/1 and BtuCD have been solved in nucleotide-free states, the pairs of ABC subunits in these two structures differ by a translational shift in the plane of the membrane that coincides with a repositioning of the membrane-spanning subunits. The differences observed between these ABC transporters involve relatively modest rearrangements and may serve as structural models for inward- and outward-facing conformations relevant to the alternating access mechanism of substrate translocation.

ATP-Binding Cassette Transporters↗
PubMed2004

The genetic basis of the interaction between pyrimidine 5' nucleotidase I deficiency and hemoglobin E.

We have previously described a family in which the interaction between pyrimidine 5' nucleotidase I (P5N-I) deficiency and hemoglobin E resulted in severe haemolytic anaemia. In this study we explored the genetic basis of the severe clinical phenotype and look for evidence of the interaction between these conditions. A P5N-I gene mutation (IVS8 + 1-2delGT) was found in the family, confirming that the severe phenotype results from the interaction between two genetic diseases.

5'-Nucleotidase↗
PubMed2004

Diagnosis of plasma cell leukaemia: findings of the UK NEQAS for Leucocyte Immunophenotyping scheme.

The diagnosis of plasma cell leukaemia, a rare disorder with an aggressive clinical course and poor prognosis, is not always straightforward and may be dependent on the results of immunophenotyping. Samples from two cases of plasma cell leukaemia have been issued by the UK NEQAS for Leucocyte Immunophenotyping Scheme during the last 5 years and on each occasion a significant number of laboratories failed to make the correct diagnosis. The details of the two samples issued and the results of both surveys are presented. The data highlights the need to adhere to guidelines for immunophenotyping, with respect to using the correct antibody panels, the importance of data interpretation in conjunction with morphological appearance as well as the need to participate in external quality assurance schemes.

Antigens, Surface↗
PubMed2003

Bone transplantation.

Bony deficiency, particularly loss of bone stock associated with failed joint replacements or tumours, is a challenging problem in orthopaedic surgery. Bone transplantation techniques provide solutions that can be tailored to the clinical problem. However, the risks of bone transplantation are well documented and the biology of allograft incorporation remains unpredictable and poorly understood.

Bone Morphogenetic Protein 7↗
PubMed2001

Genetic basis of hemolytic anemia caused by pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase (P5'N-1) deficiency is an autosomal recessive condition causing hemolytic anemia characterized by marked basophilic stippling and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. It is implicated in the anemia of lead poisoning and is possibly associated with learning difficulties. Recently, a protein with P5'N-1 activity was analyzed and a provisional complementary DNA (cDNA) sequence published. This sequence was used to study 3 families with P5'N-1 deficiency. This approach generated a genomic DNA sequence that was used to search GenBank and identify the gene for P5'N-1. It is found on chromosome 7, consists of 10 exons with alternative splicing of exon 2, and produces proteins 286 and 297 amino acids long. Three homozygous mutations were identified in this gene in 4 subjects with P5'N-1 deficiency: codon 98 GAT-->GTT, Asp-->Val (linked to a silent polymorphism codon 92, TAC-->TAT), codon 177, CAA-->TAA, Gln-->termination, and IVS9-1, G-->T. The latter mutation results in the loss of exon 9 (201 bp) from the cDNA. None of these mutations was found in 100 normal controls. The DNA analysis was complicated by P5'N-1 pseudogenes found on chromosomes 4 and 7. This study is the first description of the structure and location of the P5'N-1 gene, and 3 mutations have been identified in affected patients from separate kindreds. (Blood. 2001;97:3327-3332)

5'-Nucleotidase↗
PubMed2001

Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+.

BACKGROUND: Studies performed within the last decade have indicated that microbial reduction of Fe(III) to Fe(II) is a biologically significant process. The ferric reductase (FeR) from Archaeoglobus fulgidus is the first reported archaeal ferric reductase and it catalyzes the flavin-mediated reduction of ferric iron complexes using NAD(P)H as the electron donor. Based on its catalytic activity, the A. fulgidus FeR resembles the bacterial and eukaryotic assimilatory type of ferric reductases. However, the high cellular abundance of the A. fulgidus FeR (approximately 0.75% of the total soluble protein) suggests a catabolic role for this enzyme as the terminal electron acceptor in a ferric iron-based respiratory pathway [1]. RESULTS: The crystal structure of recombinant A. fulgidus FeR containing a bound FMN has been solved at 1.5 A resolution by multiple isomorphous replacement/ anomalous diffraction (MIRAS) phasing methods, and the NADP+- bound complex of FeR was subsequently determined at 1.65 A resolution. FeR consists of a dimer of two identical subunits, although only one subunit has been observed to bind the redox cofactors. Each subunit is organized around a six-stranded antiparallel beta barrel that is homologous to the FMN binding protein from Desulfovibrio vulgaris. This fold has been shown to be related to a circularly permuted version of the flavin binding domain of the ferredoxin reductase superfamily. The A. fulgidus ferric reductase is further distinguished from the ferredoxin reductase superfamily by the absence of a Rossmann fold domain that is used to bind the NAD(P)H. Instead, FeR uses its single domain to provide both the flavin and the NAD(P)H binding sites. Potential binding sites for ferric iron complexes are identified near the cofactor binding sites. CONCLUSIONS: The work described here details the structures of the enzyme-FMN, enzyme-FMN-NADP+, and possibly the enzyme-FMN-iron intermediates that are present during the reaction mechanism. This structural information helps identify roles for specific residues during the reduction of ferric iron complexes by the A. fulgidus FeR.

Archaeoglobus fulgidus↗
PubMed2001

Crystal structure of the Acidaminococcus fermentans 2-hydroxyglutaryl-CoA dehydratase component A.

Acidaminococcus fermentans degrades glutamate via the hydroxyglutarate pathway, which involves the syn-elimination of water from (R)-2-hydroxyglutaryl-CoA in a key reaction of the pathway. This anaerobic process is catalyzed by 2-hydroxyglutaryl-CoA dehydratase, an enzyme with two components (A and D) that reversibly associate during reaction cycles. Component A (CompA), a homodimeric protein of 2x27 kDa, contains a single, bridging [4Fe-4S] cluster and uses the hydrolysis of ATP to deliver an electron to the dehydratase component (CompD), where the electron is used catalytically. The structure of the extremely oxygen-sensitive CompA protein was solved by X-ray crystallography to 3 A resolution. The protein was found to be a member of the actin fold family, revealing a similar architecture and nucleotide-binding site. The key differences between CompA and other members of the actin fold family are: (i) the presence of a cluster binding segment, the "cluster helix"; (ii) the [4Fe-4S] cluster; and (iii) the location of the homodimer interface, which involves the bridging cluster. Possible reaction mechanisms are discussed in light of the close structural similarity to members of the actin-fold family and the functional similarity to the nitrogenase Fe- protein.

Actins↗
PubMed2001

MgATP-Bound and nucleotide-free structures of a nitrogenase protein complex between the Leu 127 Delta-Fe-protein and the MoFe-protein.

A mutant form of the nitrogenase iron protein with a deletion of residue Leu 127, located in the switch II region of the nucleotide binding site, forms a tight, inactive complex with the nitrogenase molybdenum iron (MoFe) protein in the absence of nucleotide. The structure of this complex generated with proteins from Azotobacter vinelandii (designated the L127Delta-Av2-Av1 complex) has been crystallographically determined in the absence of nucleotide at 2.2 A resolution and with bound MgATP (introduced by soaking) at 3.0 A resolution. As observed in the structure of the complex between the wild-type A. vinelandii nitrogenase proteins stabilized with ADP.AlF(4-), the most significant conformational changes in the L127Delta complex occur in the Fe-protein component. While the interactions at the interface between the MoFe-protein and Fe-proteins are conserved in the two complexes, significant differences are evident at the subunit-subunit interface of the dimeric Fe-proteins, with the L127Delta-Av2 structure having a more open conformation than the wild-type Av2 in the complex stabilized by ADP.AlF(4-). Addition of MgATP to the L127Delta-Av2-Av1 complex results in a further increase in the separation between Fe-protein subunits so that the structure more closely resembles that of the wild-type, nucleotide-free, uncomplexed Fe-protein, rather than the Fe-protein conformation in the ADP.AlF(4-) complex. The L127Delta mutation precludes key interactions between the Fe-protein and nucleotide, especially, but not exclusively, in the region corresponding to the switch II region of G-proteins, where the deletion constrains Gly 128 and Asp 129 from forming hydrogen bonds to the gamma-phosphate and activating water for attack on this group, respectively. These alterations account for the inability of this mutant to support mechanistically productive ATP hydrolysis. The ability of the L127Delta-Av2-Av1 complex to bind MgATP demonstrates that dissociation of the nitrogenase complex is not required for nucleotide binding.

Adenosine Triphosphate↗
PubMed2001

Crystal structure of the all-ferrous [4Fe-4S]0 form of the nitrogenase iron protein from Azotobacter vinelandii.

The structure of the nitrogenase iron protein from Azotobacter vinelandii in the all-ferrous [4Fe-4S](0) form has been determined to 2.25 A resolution by using the multiwavelength anomalous diffraction (MAD) phasing technique. The structure demonstrates that major conformational changes are not necessary either in the iron protein or in the cluster to accommodate cluster reduction to the [4Fe-4S](0) oxidation state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands in proteins of known structure reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the ability of the iron protein to exist in three oxidation states.

Amides↗
PubMed2001

High-resolution structures of the oxidized and reduced states of cytochrome c554 from Nitrosomonas europaea.

Cytochrome c554 (cyt c554) is a tetra-heme cytochrome involved in the oxidation of NH3 by Nitrosomonas europaea. The X-ray crystal structures of both the oxidized and dithionite-reduced states of cyt c554 in a new, rhombohedral crystal form have been solved by molecular replacement, at 1.6 A and 1.8 A resolution, respectively. Upon reduction, the conformation of the polypeptide chain changes between residues 175 and 179, which are adjacent to hemes III and IV. Cyt c554 displays conserved heme-packing motifs that are present in other heme-containing proteins. Comparisons to hydroxylamine oxidoreductase, the electron donor to cyt c554, and cytochrome c nitrite reductase, an enzyme involved in nitrite ammonification, reveal substantial structural similarity in the polypeptide chain surrounding the heme core environment. The structural determinants of these heme-packing motifs extend to the buried water molecules that hydrogen bond to the histidine ligands to the heme iron. In the original structure determination of a tetragonal crystal form, a cis peptide bond between His129 and Phe130 was identified that appeared to be stabilized by crystal contacts. In the rhombohedral crystal form used in the present high-resolution structure determination, this peptide bond adopts the trans conformation, but with disallowed angles of phi and psi.

Amino Acid Motifs↗
PubMed2001

Factor V Leiden mutation screened by PCR and detected with lanthanide-labeled probes.

The Factor V Leiden mutation is an important human polymorphism, responsible for increased risk of venous thrombosis in heterozygotes as well as homozygotes. Therefore, screening is a useful possibility, and many detection systems have been described for PCR products. We have developed a simplified and robust assay using oligonucleotide probes for normal and mutant sequences, labeled with europium and samarium, respectively, and measured by time-resolved fluorescence. Populations consisting of 233 Welsh and 148 Irish subjects were examined by both restriction fragment length polymorphism (RFLP) analysis and our assay. The allele frequency was 14/466 in the Welsh and 5/296 in the Irish population, in line with other surveys of European populations. Results were not obtained in 2/381 samples by RFLP, compared with 1/381 with our method. We conclude that our method represents an improved system capable of considerable throughput at reasonable cost.

Factor V↗
PubMed2001

Some thermodynamic implications for the thermostability of proteins.

An analysis of the thermodynamics of protein stability reveals a general tendency for proteins that denature at higher temperatures to have greater free energies of maximal stability. To a reasonable approximation, the temperature of maximal stability for the set of globular, water-soluble proteins surveyed by Robertson and Murphy occurs at T* approximately 283K, independent of the heat denaturation temperature, T(m). This observation indicates, at least for these proteins, that thermostability tends to be achieved through elevation of the stability curve rather than by broadening or through a horizontal shift to higher temperatures. The relationship between the free energy of maximal stability and the temperature of heat denaturation is such that an increase in maximal stability of approximately 0.008 kJ/mole/residue is, on average, associated with a 1 degrees C increase in T(m). An estimate of the energetic consequences of thermal expansion suggests that these effects may contribute significantly to the destabilization of the native state of proteins with increasing temperature.

Hot Temperature↗
PubMed2000

Non-depolarizing neuromuscular blocking activity of bisquaternary amino di- and tripeptide derivatives.

Herein we describe the synthesis of novel di- and tripeptide derivatives with two quaternary nitrogen groups attached and the biological testing of these compounds for neuromuscular blocking (NMB) activity in vitro and in vivo. The short peptide scaffold was selected because it offers potential for desired distance between the two pharmacophoric quaternary nitrogen groups, short duration of action, straightforward synthesis, and compatibility with an injectable formulation. From a small series of compounds 20c,e are identified as effective non-depolarizing NMB agents in vitro and in vivo in anesthetized cats and Rhesus monkeys with potencies similar to those of the clinical reference compounds rocuronium (4) and suxamethonium (2) (monkey ED(90) = 0.68, 0.23, 0.16, 5.04 micromol/kg, respectively). These new peptide derivatives 20c,e have similar potency and onset time but longer duration and slower recovery than the clinically used reference compounds. The structure-activity relationships described for this chemical series lead to the conclusion that the di- or tripeptide fragment can be regarded as an alternative template to the steroid or aliphatic ester of previously reported NMBs and within this tripeptide-derived series clog P correlates well with in vitro NMB activity.

Amines↗
PubMed2000

Crystal structure of the "cab"-type beta class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum.

The structure of the "cab"-type beta class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) has been determined to 2.1-A resolution using the multiwavelength anomalous diffraction phasing technique. Cab exists as a dimer with a subunit fold similar to that observed in "plant"-type beta class carbonic anhydrases. The active site zinc is coordinated by protein ligands Cys(32), His(87), and Cys(90), with the tetrahedral coordination completed by a water molecule. The major difference between plant- and cab-type beta class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes buffer molecule bound 8 A away from the active site zinc, which suggests a possible proton transfer pathway from the active site to the solvent.

Amino Acid Sequence↗
PubMed2000

A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila.

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.

Archaeal Proteins↗