Structural Basis for Cooperative Binding of EBNA1 to the Epstein-Barr Virus Dyad Symmetry Minimal Origin of Replication.

Epstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised oriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to the oriP Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the oriP Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.IMPORTANCE Epstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element in oriP is the essential minimal replicator of oriP Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

BACKGROUND: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. METHODS: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. RESULTS: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. CONCLUSIONS: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

Docking to large allosteric binding sites on protein surfaces.

The inactive porphobilinogen synthase (PBGS) hexamer has an oligomer-specific and phylogenetically variable surface cavity that is not present in the active octamer. The octamer and hexamer are components of a dynamic quaternary structure equilibrium characteristic of morpheeins. Small molecules that bind to the hexamer-specific surface cavity, which is at the interface of three subunits, are predicted to act as allosteric inhibitors that function by drawing the oligomeric equilibrium toward the hexamer. We used GLIDE as a tool to enrich a 250,000 molecule library for molecules with enhanced probability of acting as hexamer-stabilizing allosteric inhibitors of PBGS from Yersinia enterocolitica. Eighty-six compounds were tested in vitro and five showed hexamer stabilization. We discuss the application of computational docking to surface cavities as an approach to find allosteric modulators of protein function with specific reference to morpheeins that function as an equilibrium of non-additive quaternary structure assemblies.

Pseudomonas aeruginosa porphobilinogen synthase assembly state regulators: hit discovery and initial SAR studies.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of the essential heme, chlorophyll and vitamin B(12) heme pigments. PBGS activity is regulated by assembly state, with certain oligomers exhibiting biological activity and others either partially or completely inactive, affording an innovative means of allosteric drug action. Pseudomonas aeruginosa PBGS is functionally active as an octamer, and inactive as a dimer. We have identified a series of compounds that stabilize the inactive P. aeruginosa dimer by a computational prescreen followed by native PAGE gel mobility shift analysis. From those results, we have prepared related thiadiazoles and evaluated their ability to regulate P. aeruginosa PBGS assembly state.

Allosteric inhibition of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329-337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of approximately 111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90-100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.

Nucleotide-binding flexibility in ultrahigh-resolution structures of the SRP GTPase Ffh.

Two structures of the nucleotide-bound NG domain of Ffh, the GTPase subunit of the bacterial signal recognition particle (SRP), have been determined at ultrahigh resolution in similar crystal forms. One is GDP-bound and one is GMPPCP-bound. The asymmetric unit of each structure contains two protein monomers, each of which exhibits differences in nucleotide-binding conformation and occupancy. The GDP-bound Ffh NG exhibits two binding conformations in one monomer but not the other and the GMPPCP-bound protein exhibits full occupancy of the nucleotide in one monomer but only partial occupancy in the other. Thus, under the same solution conditions, each crystal reveals multiple binding states that suggest that even when nucleotide is bound its position in the Ffh NG active site is dynamic. Some differences in the positioning of the bound nucleotide may arise from differences in the crystal-packing environment and specific factors that have been identified include the relative positions of the N and G domains, small conformational changes in the P-loop, the positions of waters buried within the active site and shifts in the closing loop that packs against the guanine base. However, ;loose' binding may have biological significance in promoting facile nucleotide exchange and providing a mechanism for priming the SRP GTPase prior to its activation in its complex with the SRP receptor.

Shape shifting leads to small-molecule allosteric drug discovery.

Enzymes that regulate their activity by modulating an equilibrium of alternate, nonadditive, functionally distinct oligomeric assemblies (morpheeins) constitute a recently described mode of allostery. The oligomeric equilibrium for porphobilinogen synthase (PBGS) consists of high-activity octamers, low-activity hexamers, and two dimer conformations. A phylogenetically diverse allosteric site specific to hexamers is proposed as an inhibitor binding site. Inhibitor binding is predicted to draw the oligomeric equilibrium toward the low-activity hexamer. In silico docking enriched a selection from a small-molecule library for compounds predicted to bind to this allosteric site. In vitro testing of selected compounds identified one compound whose inhibition mechanism is species-specific conversion of PBGS octamers to hexamers. We propose that this strategy for inhibitor discovery can be applied to other proteins that use the morpheein model for allosteric regulation.

Analysis of protein hydration in ultrahigh-resolution structures of the SRP GTPase Ffh.

Two new structures of the SRP GTPase Ffh have been determined at 1.1 A resolution and provide the basis for comparative examination of the extensive water structure of the apo conformation of these GTPases. A set of well defined water-binding positions have been identified in the active site of the two-domain ;NG' GTPase, as well as at two functionally important interfaces. The water hydrogen-bonding network accommodates alternate conformations of the protein side chains by undergoing local rearrangements and, in one case, illustrates binding of a solute molecule within the active site by displacement of water molecules without further disruption of the water-interaction network. A subset of the water positions are well defined in several lower resolution structures, including those of different nucleotide-binding states; these appear to function in maintaining the protein structure. Consistent arrangements of surface water between three different ultrahigh-resolution structures provide a framework for beginning to understand how local water structure contributes to protein-ligand and protein-protein binding in the SRP GTPases.

Novel protein and Mg2+ configurations in the Mg2+GDP complex of the SRP GTPase ffh.

Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium-dependent and magnesium-independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg(2+)GDP reveals two new binding conformations-in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the gamma-phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDP-bound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis.

Structural basis for mobility in the 1.1 A crystal structure of the NG domain of Thermus aquaticus Ffh.

The NG domain of the prokaryotic signal recognition protein Ffh is a two-domain GTPase that comprises part of the prokaryotic signal recognition particle (SRP) that functions in co-translational targeting of proteins to the membrane. The interface between the N and G domains includes two highly conserved sequence motifs and is adjacent in sequence and structure to one of the conserved GTPase signature motifs. Previous structural studies have shown that the relative orientation of the two domains is dynamic. The N domain of Ffh has been proposed to function in regulating the nucleotide-binding interactions of the G domain. However, biochemical studies suggest a more complex role for the domain in integrating communication between signal sequence recognition and interaction with receptor. Here, we report the structure of the apo NG GTPase of Ffh from Thermus aquaticus refined at 1.10 A resolution. Although the G domain is very well ordered in this structure, the N domain is less well ordered, reflecting the dynamic relationship between the two domains previously inferred. We demonstrate that the anisotropic displacement parameters directly visualize the underlying mobility between the two domains, and present a detailed structural analysis of the packing of the residues, including the critical alpha4 helix, that comprise the interface. Our data allows us to propose a structural explanation for the functional significance of sequence elements conserved at the N/G interface.