Reiner Kiefersauer

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.

The Crystal Structure of Dipeptidyl Peptidase IV (CD26) Reveals its Functional Regulation and Enzymatic Mechanism

The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 Å crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated β-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed β-propeller with open Velcro topology and the C-terminal α/β-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the β-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu–Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P2-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.

Catalysis at a dinuclear [CuSMo(O)OH] cluster in a CO dehydrogenase resolved at 1.1-Å resolution

The CO dehydrogenase of the eubacterium Oligotropha carboxidovorans is a 277-kDa Mo- and Cu-containing iron-sulfur flavoprotein. Here, the enzymes active site in the oxidized or reduced state, after inactivation with potassium cyanide or with n-butylisocyanide bound to the active site, has been reinvestigated by multiple wavelength anomalous dispersion measurements at atomic resolution, electron spin resonance spectroscopy, and chemical analyses. We present evidence for a dinuclear heterometal [CuSMo(O)OH] cluster in the active site of the oxidized or reduced enzyme, which is prone to cyanolysis. The cluster is coordinated through interactions of the Mo with the dithiolate pyran ring of molybdopterin cytosine dinucleotide and of the Cu with the Sγ of Cys-388, which is part of the active-site loop VAYRC388SFR. The previously reported active-site structure [Dobbek, H., Gremer, L., Meyer, O. & Huber, R. (1999) Proc. Natl. Acad. Sci. USA 96, 8884–8889] of an Mo with three oxygen ligands and an SeH-group bound to the Sγ atom of Cys-388 could not be confirmed. The structure of CO dehydrogenase with the inhibitor n-butylisocyanide bound has led to a model for the catalytic mechanism of CO oxidation which involves a thiocarbonate-like intermediate state. The dinuclear [CuSMo(O)OH] cluster of CO dehydrogenase establishes a previously uncharacterized class of dinuclear molybdoenzymes containing the pterin cofactor.

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane‐embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl‐pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD‐binding domain of the p‐hydroxybenzoate‐hydrolase fold and a substrate‐binding domain that enclose a narrow active site cavity beneath the FAD and an α‐helical membrane‐binding domain. The active site architecture suggests a specific substrate‐binding mode compatible with the unusual six‐electron oxidation. The membrane‐binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.

Inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.

Crystal structure of the caspase activator human granzyme B, a proteinase highly specific for an Asp-P1 residue.

Abstract Granzyme B is the prototypic member of the granzymes, a family of trypsin-like serine proteinases localized in the dense cytoplasmic granules of activated natural killer cells and cytotoxic T lymphocytes. Granzyme B directly triggers apoptosis in target cells by activating the caspase pathway, and has been implicated in the etiology of rheumatoid arthritis. Human granzyme B expressed in a baculovirus system has been crystallized without inhibitor and its structure has been determined to 3.1 Å resolution, after considerably improving the diffraction power of the crystals by controlled humidity changes. The granzyme B structure reveals an overall fold similar to that found in cathepsin G and human chymase. The guanidinium group of Arg226, anchored at the back of the S1-specificity pocket, can form a salt bridge with the P1-Asp side chain of a bound peptide substrate. The architecture of the substrate binding site of granzyme B appears to be designed to accommodate and cleave hexapeptides such as the sequence Ile-Glu-Thr-Asp↓ -Ser-Gly present in the activation site of pro-caspase-3, a proven physiological substrate of granzyme B. These granzyme B crystals, with fully accessible active sites, are well suited for soaking with small synthetic inhibitors that might be used for a treatment of chronic inflammatory disorders.

Protein-Crystal Density by Volume Measurement and Amino-Acid Analysis

The densities of protein crystals have been determined by crystal-volume measurement and amino-acid analysis. For this purpose, protein crystals were freely mounted under humidity control by means of airstream-crystal-regulated humidity. With a video system (charge-coupled-device camera) the two-dimensional shadow projections of crystals are recorded and by the method of back projection the crystal shape is reconstructed and the volume determined. It is shown that the method is independent of the crystal morphology by the consistency of the results from a number of different crystals and materials.

Key Factors for Successful Generation of Protein–Fragment Structures: Requirement on Protein, Crystals, and Technology

In the past two decades, fragment-based approaches have evolved as a predominant strategy in lead discovery. The availability of structural information on the interaction geometries of binding fragments is key to successful structure-guided fragment-to-lead evolution. In this chapter, we illustrate methodological advances for protein-fragment crystal structure generation in order to offer general lessons on the importance of fragment properties and the most appropriate crystallographic setup to evaluate them. We analyze elaborate protocols, methods, and clues applied to challenging complex formation projects. The results should assist medicinal chemists to select the most promising targets and strategies for fragment-based crystallography as well as provide a tutorial to structural biologists who attempt to determine protein-fragment structures.

IR laser-induced protein crystal transformation

A novel method and the associated instrumentation for improving crystalline order (higher resolution of X-ray diffraction and reduced mosaicity) of protein crystals by precisely controlled heating is demonstrated. Crystal transformation is optically controlled by a video system.

Determination of hemihedral twinning and initial structural analysis of crystals of the procarboxypeptidase A ternary complex

The initial structural analysis of the ternary complex of procarboxypeptidase A from hemihedrally twinned crystals diffracting up to 2.8 A is described. Detection of twinning by different techniques is presented, including biochemical and intensity statistics approaches. The structure was initially solved using Patterson-search techniques, and the three positioned search models were used to effectively deconvolute the twinned data.