Biomedical subjects

Gleb P Bourenkov

Publications and source records attributed to Gleb P Bourenkov.

13 recordsLinked to original sources

PubMed2006

Mechanism of phosphoryl transfer catalyzed by shikimate kinase from Mycobacterium tuberculosis.

The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate.

Adenosine Diphosphate↗

PubMed2005

A quantitative approach to data-collection strategies.

Statistical descriptors of the X-ray diffraction data set for a macromolecular crystal can be modelled using the information present in the initial diffraction images. Quantitative relationships between the crystal quality, beam apertures, oscillation width, resolution limit, redundancy and the data statistics are presented. They are analysed in terms of the radiation-dose requirements based on modelling in program the BEST. The influence of radiation damage on the data statistics is discussed.

Anions↗

PubMed2005

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes.

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.

Amino Acid Sequence↗

PubMed2005

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.

Calcium↗

PubMed2005

The NC1 dimer of human placental basement membrane collagen IV: does a covalent crosslink exist?

Triple-helical collagen IV protomers associate through their N- and C-termini, forming a three-dimensional network that provides basement membranes with mechanical strength. Within this network, the C-terminal non-collagenous (NC1) domains form tight dimeric junctions. Crystallographic analyses of isolated NC1 domains show two trimeric cap-like structures interacting via a large interface. Previously, for NC1 from human placenta type-IV collagen we described covalent alpha1-alpha1 and alpha2-alpha2 crosslinks between Met93 and Lys211 of opposing alpha1(IV) and alpha2(IV) NC1-chains, which further stabilize this interface and explain the occurrence of reduction-insensitive NC1-chain dimers. However, their existence was recently questioned, and we therefore analyzed NC1-domain dimers in more detail by biochemical and protein crystallographic methods. Short-exposure diffraction data show a clear electron density cross-connecting the respective residues, which gradually disappears with prolonged crystal irradiation. Sequence analyses of isolated tryptic peptides derived from denatured NC1 monomers and dimers indicate that only the dimers, but not the monomers, yield these chemically labile cross-linked peptides. These data clearly demonstrate the presence of reduction-resistant, but chemically and radiation-sensitive covalent crosslinks between the side chains of Met93 and Lys211 in human placenta type-IV collagen.

Amino Acid Sequence↗

PubMed2004

Structural basis for the interaction of Escherichia coli NusA with protein N of phage lambda.

The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase alpha subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the p14/MP1 scaffolding complex: how a twin couple attaches mitogen-activated protein kinase signaling to late endosomes.

Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (K(d) = 12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-A crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded beta-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by beta-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite "cytoplasmic" face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.

Animals↗

PubMed2003

Choice of data-collection parameters based on statistic modelling.

A new method and the software program BEST for optimal planning of X-ray data collection from protein crystals using the rotation method are presented. From one or a few initial diffraction images, BEST estimates the statistical characteristics of the data set for different combinations of data-collection parameters and suggests the most optimal ones. The anisotropy in diffraction and the permitted width of oscillation without spatially overlapping reflections are taken into account. According to the option chosen, the optimal set of parameters provides a given average signal-to-noise ratio at a given resolution either in the shortest time or with the minimum total radiation dose. BEST has been successfully used at the protein crystallography beamlines at DORIS (DESY). The software proved to be extremely useful in using the available data-collection time in the most efficient way.

Animals↗

PubMed2003

The zinc finger-associated domain of the Drosophila transcription factor grauzone is a novel zinc-coordinating protein-protein interaction module.

About one-third of the more than 300 C2H2 zinc finger proteins of Drosophila contain a conserved sequence motif, the zinc finger-associated domain (ZAD). Genes that encode ZAD proteins are specific for and expanded in the genomes of insects. Only three ZAD-encoding gene functions are established, and the role of ZAD is unknown. Here we present the crystal structure of the ZAD of Grauzone (ZAD(Grau)), a Drosophila transcription factor that specifically controls the maternal Cdc20-like APC subunit Cortex. ZAD forms an atypical treble-clef-like zinc-coordinating fold. Head-to-tail arrangement of two ZAD(Grau) molecules in the crystals suggests dimer formation, an observation supported by crosslinking and dynamic light scattering. The results indicate that ZAD provides a novel protein-protein interaction module that characterizes a large family of insect transcription factors.

Amino Acid Motifs↗

PubMed2003

Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

Amino Acid Sequence↗

PubMed2003

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 A 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 furin's stringent requirement for arginine at P1 and P4, and lysine at P2 sites by highly charge-complementary pockets. The structure also explains furin's preference for basic residues at P3, P5 and P6 sites. This structure will aid in the rational design of antiviral and antibacterial drugs.

Amino Acid Sequence↗

PubMed2002

The 1.9-A 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-A 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 alpha 1 fragments and one alpha 2 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 beta-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 alpha 1 and alpha 2 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.

Amino Acid Sequence↗

PubMed2001

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.

Binding Sites↗