Manuel E. Than

Structure and mechanism of the aberrant ba(3)-cytochrome c oxidase from thermus thermophilus.

Cytochrome c oxidase is a respiratory enzyme catalysing the energy‐conserving reduction of molecular oxygen to water. The crystal structure of the ba3‐cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 Å resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure‐based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from CuA to CuB. Specific features of the ba3‐oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O2−, OH− or H2O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified.

The crystal structure of the proprotein processing proteinase furin explains its stringent specificity

In eukaryotes, many essential secreted proteins and peptide hormones are excised from larger precursors by members of a class of calcium-dependent endoproteinases, the prohormone-proprotein convertases (PCs). Furin, the best-characterized member of the mammalian PC family, has essential functions in embryogenesis and homeostasis but is also implicated in various pathologies such as tumor metastasis, neurodegeneration and various bacterial and viral diseases caused by such pathogens as anthrax and pathogenic Ebola virus strains. Furin cleaves protein precursors with narrow specificity following basic Arg-Xaa-Lys/Arg-Arg-like motifs. The 2.6 Å crystal structure of the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk)–inhibited mouse furin ectodomain, the first PC structure, reveals an eight-stranded jelly-roll P domain associated with the catalytic domain. Contoured surface loops shape the active site by cleft, thus explaining furins stringent requirement for arginine at P1 and P4, and lysine at P2 sites by highly charge-complementary pockets. The structure also explains furins preference for basic residues at P3, P5 and P6 sites. This structure will aid in the rational design of antiviral and antibacterial drugs.

Crystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods

BACKGROUND The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes

A novel device for capillary-free mounting of protein crystals is described. A controlled stream of air allows an accurate adjustment of the humidity at the crystal. The crystal is held on the tip of a micropipette. With a video system (CCD camera), the two-dimensional shadow projections of crystals can be recorded for optical analysis. Instead of the micropipette, a standard loop can also be used. Experiments and results for different crystal systems demonstrate the use of this method, also in combination with shock-freezing, to improve crystal order. Working with oxygen-free gases offers the possibility of crystal measurements under anaerobic conditions. Furthermore, the controlled application of arbitrary volatile substances with the gas stream is practicable.

The 1.9-Å crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link

Triple-helical collagen IV protomers associate through their N- and C-termini forming a three-dimensional network, which provides basement membranes with an anchoring scaffold and mechanical strength. The noncollagenous (NC1) domain of the C-terminal junction between two adjacent collagen IV protomers from human placenta was crystallized and its 1.9-Å structure was solved by multiple anomalous diffraction (MAD) phasing. This hexameric NC1 particle is composed of two trimeric caps, which interact through a large planar interface. Each cap is formed by two α1 fragments and one α2 fragment with a similar previously uncharacterized fold, segmentally arranged around an axial tunnel. Each monomer chain folds into two structurally very similar subdomains, which each contain a finger-like hairpin loop that inserts into a six-stranded β-sheet of the neighboring subdomain of the same or the adjacent chain. Thus each trimer forms a quite regular, but nonclassical, sixfold propeller. The trimer–trimer interaction is further stabilized by a previously uncharacterized type of covalent cross-link between the side chains of a Met and a Lys residue of the α1 and α2 chains from opposite trimers, explaining previous findings of nonreducible cross-links in NC1. This structure provides insights into NC1-related diseases such as Goodpasture and Alport syndromes.

Structure and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein

The amyloid precursor protein (APP) is the key player in Alzheimer’s disease pathology, yet APP and its analogues are also essential for neuronal development and cell homeostasis in mammals. We have determined the crystal structure of the entire N-terminal APP-E1 domain consisting of the growth factor like and the copper binding domains at 2.7-Å resolution and show that E1 functions as a rigid functional entity. The two subdomains interact tightly in a pH-dependent manner via an evolutionarily conserved interface area. Two E1 entities dimerize upon their interaction with heparin, requiring 8–12 sugar rings to form the heparin-bridged APP-E1 dimer in an endothermic and pH-dependent process that is characterized by a low micromolar dissociation constant. Limited proteolysis confirms that the heparin-bridged E1 dimers obtained in solution correspond to a dimer contact in our crystal, enabling us to model this heparin-[APP-E1]2 complex. Correspondingly, the APP-based signal transduction, cell–cell- and/or cell–ECM interaction should depend on dimerization induced by heparin, as well as on pH, arguing that APP could fulfill different functions depending on its (sub)cellular localization.

Metal Binding Dictates Conformation and Function of the Amyloid Precursor Protein (APP) E2 Domain.

The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimers disease and appear essential for neuronal development and cell homeostasis in mammals. Proteolytic processing of APP is influenced by metal ions, protein ligands and its oligomerization state. However, the structural basis and functional mechanism of APP regulation are hitherto largely unknown. Here we identified a metal-dependent molecular switch located within the E2 domain of APP containing four evolutionary highly conserved histidine residues. Three X-ray structures of the metal-bound molecule were solved at 2.6-2.0 Å resolution. Using protein crystallographic and biochemical methods, we characterized this novel high-affinity binding site within the E2 domain that binds competitively to copper and zinc at physiological concentrations. Metal-specific coordination spheres induce large conformational changes and enforce distinct structural states, most likely regulating the physiological function of APP and its processing in Alzheimers disease.

Potent inhibitors of furin and furin-like proprotein convertases containing decarboxylated P1 arginine mimetics

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer, and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporating decarboxylated arginine mimetics in the P1 position of substrate analogue peptidic inhibitors, we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K(i) value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells, inhibitor 15 inhibited proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long-term infection test. Molecular modeling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

X-ray Structures of Human Furin in Complex with Competitive Inhibitors

Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacological use require a detailed knowledge of the protease’s substrate and inhibitor binding properties. Here we present a novel preparation of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of additional inhibitors and substrate analogues. Thus, our work provides a basis for the rational design of furin inhibitors.

The endoproteinase furin contains two essential Ca2+ ions stabilizing its N-terminus and the unique S1 specificity pocket

The mammalian prohormone/proprotein convertase (PC) furin is responsible for the maturation of a great variety of homeostatic but also many pathogenic proteins within the secretory pathway and the endosomal pathway and at the cell surface. Similar to other members of the PC family, furin requires calcium for catalytic activity. In a previous paper, the structural association of the catalytic and the P-domain of furin was shown and data were presented indicating two or three calcium-binding sites. The exact number and the three-dimensional localization of the essential calcium sites within furin have now been determined by collecting X-ray diffraction data on either side of the Ca K absorption edge and by calculating a novel type of double difference map from these anomalous scattering data. Two calcium ions were unambiguously identified: the purely structural Ca-1 also conserved in the bacterial digestive subtilisins and the Ca-2 site specific to PCs and essential for the formation of the P1 specificity-determining S1-binding pocket. In addition, these anomalous diffraction data show that no tightly bound K(+) sites exist in furin.