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Biomedical subjects

Dimitar B Nikolov

Publications and source records attributed to Dimitar B Nikolov.

At least 19 recordsLinked to original sources
PubMed2006

Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells.

The advent of the multiwavelength anomalous diffraction phasing method has significantly accelerated crystal structure determination and has become the norm in protein crystallography. This method allows researchers to take advantage of the anomalous signal from diverse atoms, but the dominant method for derivative preparation is selenomethionine substitution. Several generally applicable, high-efficiency labeling protocols have been developed for use in the bacterial, yeast, and baculovirus/insect cell expression systems but not for mammalian tissue culture. As a large number of proteins of biomedical importance can only be produced in yields sufficient for X-ray diffraction experiments in mammalian expression systems, it becomes all the more important to develop such protocols. We therefore evaluated several variables that play roles in determining incorporation levels and report here a simple protocol for selenomethionine modification of proteins in mammalian cells routinely yielding >90% labeling efficiency.

Amino Acid Substitution↗
PubMed2006

Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex.

The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.

Amino Acid Sequence↗
PubMed2005

Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans.

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

ADAM Proteins↗
PubMed2005

Crystal structure of the ephrin-B1 ectodomain: implications for receptor recognition and signaling.

Eph receptors and their ephrin ligands are involved in various aspects of cell-cell communication during development, including axonal pathfinding in the nervous system and cell-cell interactions of the vascular endothelial cells. Recent structural studies revealed unique molecular features, not previously seen in any other receptor-ligand families, and explained many of the biochemical and signaling properties of Ephs and ephrins. However, unresolved questions remain regarding the potential oligomerization and clustering of these important signaling molecules. In this study, the biophysical properties and receptor-binding preferences of the extracellular domain of ephrin-B1 were investigated and its crystal structure was determined at 2.65 A resolution. Ephrin-B1 is a monomer both in solution and in the crystals, while it was previously shown that the closely related ephrin-B2 forms homodimers. The main structural difference between ephrin-B1 and ephrin-B2 is the conformation of the receptor-binding G-H loop and the partially disordered N-terminal tetramerization region of ephrin-B1. The G-H loop is structurally rigid in ephrin-B2 and adopts the same conformation in both the receptor-bound and unbound ligand, where it mediates receptor-independent homodimerization. In the ephrin-B1 structure, on the other hand, the G-H loop is not involved in any homotypic interactions and adopts a new, distinct conformation. The implications of the ephrin-B1 structure, in context of available ephrin-B1 mutagenesis data, for the mechanism of Eph-ephrin recognition and signaling initiation are discussed.

Amino Acid Sequence↗
PubMed2005

Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3.

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2.ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B-C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.

Amino Acid Motifs↗
PubMed2005

Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2 recognition.

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.

Amino Acid Sequence↗
PubMed2004

Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling.

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

Alkaline Phosphatase↗
PubMed2003

Structure of the semaphorin-3A receptor binding module.

The semaphorins are a large group of extracellular proteins involved in a variety of processes during development, including neuronal migration and axon guidance. Their distinctive feature is a conserved 500 amino acid semaphorin domain, a ligand-receptor interaction module also present in plexins and scatter-factor receptors. We report the crystal structure of a secreted 65 kDa form of Semaphorin-3A (Sema3A), containing the full semaphorin domain. Unexpectedly, the semaphorin fold is a variation of the beta propeller topology. Analysis of the Sema3A structure and structure-based mutagenesis data identify the neuropilin binding site and suggest a potential plexin interaction site. Based on the structure, we present a model for the initiation of semaphorin signaling and discuss potential similarities with the signaling mechanisms of other beta propeller cell surface receptors, such as integrins and the LDL receptor.

Amino Acid Sequence↗
PubMed2003

Structure and axon outgrowth inhibitor binding of the Nogo-66 receptor and related proteins.

The myelin-derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell-surface proteins signal through multi-subunit neuronal receptors that contain a common ligand-binding glycosylphosphatidylinositol-anchored subunit termed the Nogo-66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell-surface NgR requires the entire leucine-rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi-ligand binding, we determined the crystal structure of the biologically active ligand-binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N-terminal cap and a small C-terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.

Amino Acid Sequence↗
PubMed2003

Substrate specificity of NovM: implications for novobiocin biosynthesis and glycorandomization.

[reaction: see text] In an effort to expand the scope of natural product in vitro glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new "unnatural" natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.

Anti-Bacterial Agents↗
PubMed2003

Eph signaling: a structural view.

Eph receptors, the largest subfamily of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape and mobility. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bidirectional signaling cascades, with information transduced in both the receptor-expressing and the ligand-expressing cells. Recent structural and biophysical studies summarized in this review reveal unique molecular features not previously observed in any other receptor-ligand families and explain many of the biochemical and signaling properties of Ephs and ephrins. Of particular importance is the insight into how approximation of ligand-expressing and receptor-expressing cells could lead to the formation and activation of highly ordered signaling centers at cell-cell interfaces.

Animals↗
PubMed2003

Eph receptors and ephrins.

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication regulating cell attachment, shape, and mobility. Eph signaling is crucial for the development of many tissues and organs including the nervous and cardiovascular systems. Both Ephs and ephrins are membrane-bound and their interactions at sites of cell-cell contact initiate unique bi-directional signaling cascades where information is transduced in both the receptor- and the ligand-expressing cells. Recent studies summarized in this review reveal how the signaling process is triggered upon ligand-receptor binding via the formation of a 2:2 circular heterotetramer. This fixes the orientation of the participating molecules and facilitates phosphorylation of their cytoplasmic domains which then interact with downstream signaling factors. The elucidation of the structural details of Eph-ephrin recognition and binding should yield insight into the future development of novel therapeutic agents targeting cardiovascular function, nerve regeneration, and cancer.

Animals↗
PubMed2003

Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC.

The osmotically inducible protein OsmC, like its better-characterized homolog, the organic hydroperoxide protein Ohr, is involved in defense against oxidative stress caused by exposure to organic hydroperoxides. The crystal structure of Escherichia coli OsmC reported here reveals that the protein is a tightly folded domain-swapped dimer with two active sites located at the monomer interface on opposite sides of the molecule. We demonstrate that OsmC preferentially metabolizes organic hydroperoxides over inorganic hydrogen peroxide. On the basis of structural and enzymatic similarities, we propose that the OsmC catalytic mechanism is analogous to that of the Ohr proteins and of the structurally unrelated peroxiredoxins, directly using highly reactive cysteine thiol groups to elicit hydroperoxide reduction.

Amino Acid Sequence↗
PubMed2003

GABARAP: lessons for synaptogenesis.

The clustering of neurotransmitter receptors at the postsynaptic terminals is a critical requirement for efficient neurotransmission and neuronal communication. This process is facilitated by adaptor proteins, which bridge the postsynaptic receptors and the underlying cytoskeleton. One such molecule, the GABAA receptor-associated protein, GABARAP, was identified as a potential linker between GABAA receptors and microtubules. GABARAP belongs to an expanding family of proteins that are implicated in a variety of intracellular transport processes. GABARAP has been shown to interact with myriad binding partners, including the gamma2 subunit of the GABAA receptor, tubulin and microtubules, the N-ethyl maleimide sensitive factor, gephyrin, and the transferin receptor. The recent determination of the GABARAP crystal structure has revealed individual GABARAP domains, motifs, and surface regions involved in specific protein-protein interactions. Currently, a more general role is emerging for GABARAP, including shipping GABAA receptors to and from the cell surface, organizing them into postsynaptic clusters, and regulating the steady-state receptor density.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr.

Bacteria have developed complex strategies to detoxify and repair damage caused by reactive oxygen species. These compounds, produced during bacterial aerobic respiration as well as by the host immune system cells as a defense mechanism against the pathogenic microorganisms, have the ability to damage nucleic acids, proteins and phospholipid membranes. Here we describe the crystal structure of Pseudomonas aeruginosa Ohr, a member of a recently discovered family of organic hydroperoxide resistance proteins. Ohr is a tightly folded homodimer, with a novel alpha/beta fold, and contains two active sites located at the monomer interface on opposite sides of the molecule. Using in vitro assays, we demonstrate that Ohr functions directly as a hydroperoxide reductase, converting both inorganic and organic hydroperoxides to less toxic metabolites. Site-directed mutagenesis confirms that the two conserved cysteines in each active site are essential for catalytic activity. We propose that the Ohr catalytic mechanism is similar to that of the structurally unrelated peroxiredoxins, directly utilizing highly reactive cysteine thiol groups to elicit hydroperoxide reduction.

Amino Acid Sequence↗
PubMed2002

Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering.

In vitro "glycorandomization" is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-d-glucopyranosyl phosphate thymidylyltransferase (E(p)). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three "designed" mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.

Amino Acid Substitution↗