Biomedical subjects

Douglas C Rees

Publications and source records attributed to Douglas C Rees.

28 records · Page 2Linked to original sources

PubMed2002

The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter.

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes.

ATP-Binding Cassette Transporters↗

PubMed2002

Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel.

The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution demonstrates that Escherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements.

Amino Acid Sequence↗

PubMed2002

Nitrogenase MoFe-protein at 1.16 A resolution: a central ligand in the FeMo-cofactor.

A high-resolution crystallographic analysis of the nitrogenase MoFe-protein reveals a previously unrecognized ligand coordinated to six iron atoms in the center of the catalytically essential FeMo-cofactor. The electron density for this ligand is masked in structures with resolutions lower than 1.55 angstroms, owing to Fourier series termination ripples from the surrounding iron and sulfur atoms in the cofactor. The central atom completes an approximate tetrahedral coordination for the six iron atoms, instead of the trigonal coordination proposed on the basis of lower resolution structures. The crystallographic refinement at 1.16 angstrom resolution is consistent with this newly detected component being a light element, most plausibly nitrogen. The presence of a nitrogen atom in the cofactor would have important implications for the mechanism of dinitrogen reduction by nitrogenase.

Azotobacter vinelandii↗

PubMed2002

High resolution crystal structures of the wild type and Cys-55-->Ser and Cys-59-->Ser variants of the thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus.

The [2Fe-2S] ferredoxin (Fd4) from Aquifex aeolicus adopts a thioredoxin-like polypeptide fold that is distinct from other [2Fe-2S] ferredoxins. Crystal structures of the Cys-55 --> Ser (C55S) and Cys-59 --> Ser (C59S) variants of this protein have been determined to 1.25 A and 1.05 A resolution, respectively, whereas the resolution of the wild type (WT) has been extended to 1.5 A. The improved WT structure provides a detailed description of the [2Fe-2S] cluster, including two features that have not been noted previously in any [2Fe-2S] cluster-containing protein, namely, pronounced distortions in the cysteine coordination to the cluster and a Calpha-H-Sgamma hydrogen bond between cluster ligands Cys-55 and Cys-9. These features may contribute to the unusual electronic and magnetic properties of the [2Fe-2S] clusters in WT and variants of this ferredoxin. The structures of the two variants of Fd4, in which single cysteine ligands to the [2Fe-2S] cluster are replaced by serine, establish the metric details of serine-ligated Fe-S active sites with unprecedented accuracy. Both the cluster and its surrounding protein matrix change in subtle ways to accommodate this ligand substitution, particularly in terms of distortions of the Fe(2)S(2) inorganic core from planarity and displacements of the polypeptide chain. These high resolution structures illustrate how the interactions between polypeptide chains and Fe-S active sites reflect combinations of flexibility and rigidity on the part of both partners; these themes are also evident in more complex systems, as exemplified by changes associated with serine ligation of the nitrogenase P cluster.

Bacterial Proteins↗

PubMed2002

X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides.

In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.

Cations↗

PubMed2002

The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism.

The ABC transporters are ubiquitous membrane proteins that couple adenosine triphosphate (ATP) hydrolysis to the translocation of diverse substrates across cell membranes. Clinically relevant examples are associated with cystic fibrosis and with multidrug resistance of pathogenic bacteria and cancer cells. Here, we report the crystal structure at 3.2 angstrom resolution of the Escherichia coli BtuCD protein, an ABC transporter mediating vitamin B12 uptake. The two ATP-binding cassettes (BtuD) are in close contact with each other, as are the two membrane-spanning subunits (BtuC); this arrangement is distinct from that observed for the E. coli lipid flippase MsbA. The BtuC subunits provide 20 transmembrane helices grouped around a translocation pathway that is closed to the cytoplasm by a gate region whereas the dimer arrangement of the BtuD subunits resembles the ATP-bound form of the Rad50 DNA repair enzyme. A prominent cytoplasmic loop of BtuC forms the contact region with the ATP-binding cassette and appears to represent a conserved motif among the ABC transporters.

ATP-Binding Cassette Transporters↗

PubMed2002

Structure of a cofactor-deficient nitrogenase MoFe protein.

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.

Amino Acid Sequence↗

PubMed2002

Crystallographic studies of the Escherichia coli quinol-fumarate reductase with inhibitors bound to the quinol-binding site.

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integral-membrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH(2)) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH(2), bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q(P) and Q(D), indicating their positions proximal (Q(P)) or distal (Q(D)) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH(2) at the Q(P) site. In the structures with the inhibitor bound at Q(P), no density is observed at Q(D), which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q(D). A comparison of the Q(P) site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.

Amino Acid Sequence↗

PubMed2001

Great metalloclusters in enzymology.

Metallocluster-containing enzymes catalyze some of the most basic redox transformations in the biosphere. The reactions catalyzed by these enzymes typically involve small molecules such as N2, CO, and H2 that are used to generate both chemical building blocks and energy for metabolic purposes. During the past decade, structures have been established for the iron-sulfur-based metalloclusters present in the molybdenum nitrogenase, the iron-only hydrogenase, and the nickel-carbon monoxide dehydrogenase, and for the copper-sulfide-based cluster in nitrous oxide reductase. Although these clusters are built from interactions observed in simpler metalloproteins, they contain novel features that may be relevant for their catalytic function. The mechanisms of metallocluster-containing enzymes are still poorly defined, and represent substantial and continuing challenges to biochemists, biophysicists, and synthetic chemists. These proteins also provide a window into the union of the biological and inorganic worlds that may have been relevant to the early evolution of biochemical catalysis.

Aldehyde Oxidoreductases↗

PubMed2001

The alpha-helix and the organization and gating of channels.

The structures of an increasing number of channels and other alpha-helical membrane proteins have been determined recently, including the KcsA potassium channel, the MscL mechanosensitive channel, and the AQP1 and GlpF members of the aquaporin family. In this chapter, the orientation and packing characteristics of bilayer-spanning helices are surveyed in integral membrane proteins. In the case of channels, alpha-helices create the sealed barrier that separates the hydrocarbon region of the bilayer from the permeation pathway for solutes. The helices surrounding the permeation pathway tend to be rather steeply tilted relative to the membrane normal and are consistently arranged in a right-handed bundle. The helical framework further provides a supporting scaffold for nonmembrane-spanning structures associated with channel selectivity. Although structural details remain scarce, the conformational changes associated with gating transitions between closed and open states of channels are reviewed, emphasizing the potential roles of helix-helix interactions in this process.

Animals↗