Small Molecule Antagonists of the DNA Repair ERCC1/XPA Protein-Protein Interaction.

The DNA excision repair protein ERCC1 and the DNA damage sensor protein, XPA are highly overexpressed in patient samples of cisplatin-resistant solid tumors including lung, bladder, ovarian, and testicular cancer. The repair of cisplatin-DNA crosslinks is dependent upon nucleotide excision repair (NER) that is modulated by protein-protein binding interactions of ERCC1, the endonuclease, XPF, and XPA. Thus, inhibition of their function is a potential therapeutic strategy for the selective sensitization of tumors to DNA-damaging platinum-based cancer therapy.

Stabilization and structure determination of integral membrane proteins by termini restraining.

Integral membrane proteins isolated from cellular environment often lose activity and native conformation required for functional analyses and structural studies. Even in their native state, they lack sufficient surfaces to form crystal contacts. Furthermore, most of them are too small for cryogenic electron microscopy detection and too big for solution NMR.

Structural basis of antagonizing the vitamin K catalytic cycle for anticoagulation.

Vitamin K antagonists are widely used anticoagulants that target vitamin K epoxide reductases (VKOR), a family of integral membrane enzymes. To elucidate their catalytic cycle and inhibitory mechanism, we report 11 x-ray crystal structures of human VKOR and pufferfish VKOR-like, with substrates and antagonists in different redox states. Substrates entering the active site in a partially oxidized state form cysteine adducts that induce an open-to-closed conformational change, triggering reduction.

Atypical Cadherin Dachsous1b Interacts with Ttc28 and Aurora B to Control Microtubule Dynamics in Embryonic Cleavages.

Atypical cadherin Dachsous (Dchs) is a conserved regulator of planar cell polarity, morphogenesis, and tissue growth during animal development. Dchs functions in part by regulating microtubules by unknown molecular mechanisms. Here we show that maternal zygotic (MZ) dchs1b zebrafish mutants exhibit cleavage furrow progression defects and impaired midzone microtubule assembly associated with decreased microtubule turnover.

MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A.

Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that MFN2 activity can be determined by Met and His interactions with Asp and Leu and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion.

Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

The genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation.

Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG.

Genetic Evolution of a Helicobacter pylori Acid-Sensing Histidine Kinase and Gastric Disease.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, which develops within a hypochlorhydric environment. We sequentially isolated H. pylori (strain J99) from a patient who developed corpus-predominant gastritis and hypochlorhydia over a 6-year interval.

Helicobacter pylori RNA polymerase α-subunit C-terminal domain shows features unique to ɛ-proteobacteria and binds NikR/DNA complexes.

Bacterial RNA polymerase is a large, multi-subunit enzyme responsible for transcription of genomic information. The C-terminal domain of the α subunit of RNA polymerase (αCTD) functions as a DNA and protein recognition element localizing the polymerase on certain promoter sequences and is essential in all bacteria. Although αCTD is part of RNA polymerase, it is thought to have once been a separate transcription factor, and its primary role is the recruitment of RNA polymerase to various promoters.

Murine norovirus protein NS1/2 aspartate to glutamate mutation, sufficient for persistence, reorients side chain of surface exposed tryptophan within a novel structured domain.

Compact viral genomes such as those found in noroviruses, which cause significant enteric disease in humans, often encode only a few proteins, but affect a wide range of processes in their hosts and ensure efficient propagation of the virus. Both human and mouse noroviruses (MNVs) persistently replicate and are shed in stool, a highly effective strategy for spreading between hosts. For MNV, the presence of a glutamate rather than an aspartate at position 94 of the NS1/2 protein was previously shown to be essential for persistent replication and shedding.

Analysis of protein expression regulated by the Helicobacter pylori ArsRS two-component signal transduction system.

Previous studies have shown that the Helicobacter pylori ArsRS two-component signal transduction system contributes to acid-responsive gene expression. To identify additional members of the ArsRS regulon and further investigate the regulatory role of the ArsRS system, we analyzed protein expression in wild-type and arsS null mutant strains. Numerous proteins were differentially expressed in an arsS mutant strain compared to a wild-type strain when the bacteria were cultured at pH 5.

Structural analysis of the DNA-binding domain of the Helicobacter pylori response regulator ArsR.

The Helicobacter pylori ArsS-ArsR two-component signal transduction system, comprised of a sensor histidine kinase (ArsS) and a response regulator (ArsR), allows the bacteria to regulate gene expression in response to acidic pH. We expressed and purified the full-length ArsR protein and the DNA-binding domain of ArsR (ArsR-DBD), and we analyzed the tertiary structure of the ArsR-DBD using solution nuclear magnetic resonance (NMR) methods. Both the full-length ArsR and the ArsR-DBD behaved as monomers in size exclusion chromatography experiments.

Structural studies of human Naked2: a biologically active intrinsically unstructured protein.

Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-alpha (TGFalpha) and escorts TGFalpha-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble.

Solution structure of the symmetric coiled coil tetramer formed by the oligomerization domain of hnRNP C: implications for biological function.

During active cell division, heterogeneous nuclear ribonucleoprotein (hnRNP) C is one of the most abundant proteins in the nucleus. hnRNP C exists as a stable tetramer that binds about 230 nucleotides of pre-mRNA and functions in vivo to package nascent transcripts and nucleate assembly of the 40 S hnRNP complex. Previous studies have shown that monomers lacking or possessing mutant oligomerization domains bind RNA with low affinity, strongly suggesting a cooperative protomer-RNA binding mode.

Helicobacter pylori protein HP0222 belongs to Arc/MetJ family of transcriptional regulators.

Helicobacter pylori is a widespread human bacterial pathogen responsible for inducing gastric and duodenal ulcers and gastric cancers. To date, only 16 protein structures from this organism have been determined, and more than 30% of its 1500 protein functions remain unknown. We report the biochemical characterization, the tertiary structure determined by solution nuclear magnetic resonance (NMR) methods and the putative function of the previously uncharacterized protein HP0222 (JHP0208) from H.

Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae.

The SWA2/AUX1 gene has been proposed to encode the Saccharomyces cerevisiae ortholog of mammalian auxilin. Swa2p is required for clathrin assembly/dissassembly in vivo, thereby implicating it in intracellular protein and lipid trafficking. While investigating the 287-residue N-terminal region of Swa2p, we found a single stably folded domain between residues 140 and 180.

Solution structure of the chick TGFbeta type II receptor ligand-binding domain.

The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods.