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. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel ß-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.

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. Archival J99 survived significantly better under acidic conditions than recent J99 strains. H. pylori arsRS encodes a 2-component system critical for stress responses; recent J99 isolates harbored 2 nonsynonymous arsS mutations, and arsS inactivation abolished acid survival. In vivo, acid-resistant archival, but not recent J99, successfully colonized high-acid-secreting rodents. Thus, genetic evolution of arsS may influence progression to hypochlorhydia and gastric cancer.

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. Here, we analyze these critical sequences of NS1/2 at the structural level. Using solution nuclear magnetic resonance methods, we determined folded NS1/2 domain structures from a nonpersistent murine norovirus strain CW3, a persistent strain CR6, and a persistent mutant strain CW3(D94E). We found an unstructured PEST-like domain followed by a novel folded domain in the N-terminus of NS1/2. All three forms of the domain are stable and monomeric in solution. Residue 94, critical for determining persistence, is located in a reverse turn following an α-helix in the folded domain. The longer side chain of glutamate, but not aspartate, allows interaction with the indole group of the nearby tryptophan, reshaping the surface of the domain. The discrimination between glutamyl and aspartyl residue is imposed by the stable tertiary conformation. These structural requirements correlate with the in vivo function of NS1/2 in persistence, a key element of norovirus biology and infection.

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. Here, we report on new small-molecule antagonists of the ERCC1/XPA protein-protein interaction (PPI) discovered using a high-throughput competitive fluorescence polarization binding assay. We discovered a unique structural class of thiopyridine-3-carbonitrile PPI antagonists that block a truncated XPA polypeptide from binding to ERCC1. Preliminary hit-to-lead studies from compound 1 reveal structure-activity relationships (SAR) and identify lead compound 27 o with an EC50 of 4.7 µM. Furthermore, chemical shift perturbation mapping by NMR confirms that 1 binds within the same site as the truncated XPA67-80 peptide. These novel ERCC1 antagonists are useful chemical biology tools for investigating DNA damage repair pathways and provide a good starting point for medicinal chemistry optimization as therapeutics for sensitizing tumors to DNA damaging agents and overcoming resistance to platinum-based chemotherapy.

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. To overcome these difficulties, we recently developed a strategy to stabilize the folded state of membrane proteins by restraining their two termini with a self-assembling protein coupler. The termini-restrained membrane proteins from distinct functional families retain their activities and show increased stability and yield. This strategy enables their structure determination at near-atomic resolution by facilitating the entire pipeline from crystallization, crystal identification, diffraction enhancement and phase determination, to electron density improvement. Furthermore, stabilization of membrane proteins enables their biochemical and biophysical characterization. Here we present the protocol of membrane protein engineering (2 weeks), quality assessment (1-2 weeks), protein production (1-6 weeks), crystallization (1-2 weeks), diffraction improvement (1-3 months) and crystallographic data analysis (1 week). This protocol is intended not only for structural biologists, but also for biochemists, biophysicists and pharmaceutical scientists whose research focuses on membrane proteins.

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. Binding and catalysis are facilitated by hydrogen-bonding interactions in a hydrophobic pocket. The antagonists bind specifically to the same hydrogen-bonding residues and induce a similar closed conformation. Thus, vitamin K antagonists act through mimicking the key interactions and conformational changes required for the VKOR catalytic cycle.

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 5.0 and also when they were cultured at pH 7.0. Genes encoding 14 of these proteins were directly regulated by the ArsRS system, based on observed binding of ArsR to the relevant promoter regions. The ArsRS-regulated proteins identified in this study contribute to acid resistance (urease and amidase), acetone metabolism (acetone carboxylase), resistance to oxidative stress (thioredoxin reductase), quorum sensing (Pfs), and several other functions. These results provide further definition of the ArsRS regulon and underscore the importance of the ArsRS system in regulating expression of H. pylori proteins during bacterial growth at both neutral pH and acidic pH.

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. Mechanistically, Dchs1b interacts via a conserved motif in its intracellular domain with the tetratricopeptide motifs of Ttc28 and regulates its subcellular distribution. Excess Ttc28 impairs cleavages and decreases microtubule turnover, while ttc28 inactivation increases turnover. Moreover, ttc28 deficiency in dchs1b mutants suppresses the microtubule dynamics and midzone microtubule assembly defects. Dchs1b also binds to Aurora B, a known regulator of cleavages and microtubules. Embryonic cleavages in MZdchs1b mutants exhibit increased, and in MZttc28 mutants decreased, sensitivity to Aurora B inhibition. Thus, Dchs1b regulates microtubule dynamics and embryonic cleavages by interacting with Ttc28 and Aurora B.

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 Mfn2 cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that MFN2 activity can be determined by Met376 and His380 interactions with Asp725 and Leu727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser378 Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg94→Gln94 and MFN2 Thr105→Met105, as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr105→Met105 mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.

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 Norovirus 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. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein.

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. In order to understand the role of the oligomerization domain in defining the biological functions and structure of hnRNP C tetramers, we have determined the high-resolution NMR structure of the oligomerization interface that is formed at the core of the complex, examining specific molecular interactions that drive assembly and contribute to the structural integrity of the tetramer. The determined structure reveals an antiparallel four-helix coiled coil, where classically described knobs-into-holes packing interactions at interhelical contact surfaces are optimized so that side-chains interdigitate to create an even distribution of hydrophobic surfaces along the core. While the stoichiometry of the complex appears to be primarily specified by occlusion of hydrophobic surfaces, particularly the interfacial residue L198, from solvent, helix orientation is primarily determined by electrostatic attractions across helix interfaces. The creation of potential interaction surfaces for other hnRNP C domains along the coiled coil exterior and the assembly of oligomerization interfaces in an antiparallel orientation shape the tertiary fold of full-length monomers and juxtapose RNA-binding elements at distal surfaces of the tetrameric complex in the quaternary assembly. In addition, we discuss the specific challenges encountered in structure determination of this symmetric oligomer by NMR methods, specifically in sorting ambiguous interatomic distance constraints into classes that define different elements of the coiled coil structure.

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. pylori. Recombinant HP0222 behaves as a dimer in crosslinking and size exclusion chromatography experiments. The structure consists of a ribbon-helix-helix fold characteristic of transcription factors of the Arc/MetJ family, which all bind DNA as higher order oligomers. Electrophoretic mobility shift assays reveal that HP0222 binds to double-stranded DNA. Previous studies have shown significant increases in transcription levels of HP0222 in response to acid shock and adherence to gastric epithelial cells. To assess possible involvement of HP0222 in acid resistance, we constructed and assayed an H. pylori HP0222 null mutant. We propose that HP0222 is a novel transcriptional regulator in H. pylori.

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. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.

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. Using binding assays and structural analysis, we established this to be a ubiquitin-associated (UBA) domain, unidentified by bioinformatics of the yeast genome. We determined the solution structure of this Swa2p domain and found a characteristic three-helix UBA fold. Comparisons of structures of known UBA folds reveal that the position of the third helix is quite variable. This helix in Swa2p UBA contains a bulkier tyrosine in place of smaller residues found in other UBAs and cannot pack as close to the second helix. The molecular surface of Swa2p UBA has a mostly negative potential, with a single hydrophobic surface patch found also in the UBA domains of human protein, HHR23A. The presence of a UBA domain implicates Swa2p in novel roles involving ubiquitin and ubiquitinated substrates. We propose that Swa2p is a multifunctional protein capable of recognizing several proteins through its protein-protein recognition domains.

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. Despite the conservation of the subunits of RNA polymerase among bacteria, the mechanisms of regulation of transcription vary significantly. We have determined the tertiary structure of Helicobacter pylori αCTD. It is larger than other structurally determined αCTDs due to an extra, highly amphipathic helix near the C-terminal end. Residues within this helix are highly conserved among ɛ-proteobacteria. The surface of the domain that binds A/T rich DNA sequences is conserved and showed binding to DNA similar to αCTDs of other bacteria. Using several NikR dependent promoter sequences, we observed cooperative binding of H. pylori αCTD to NikR:DNA complexes. We also produced αCTD lacking the 19 C-terminal residues, which showed greatly decreased stability, but maintained the core domain structure and binding affinity to NikR:DNA at low temperatures. The modeling of H. pylori αCTD into the context of transcriptional complexes suggests that the additional amphipathic helix mediates interactions with transcriptional regulators.

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. The structure of ArsR-DBD consists of an N-terminal four-stranded beta-sheet, a helical core, and a C-terminal beta-hairpin. The overall tertiary fold of the ArsR-DBD is most closely related to DBD structures of the OmpR/PhoB subfamily of bacterial response regulators. However, the orientation of the N-terminal beta-sheet with respect to the rest of the DNA-binding domain is substantially different in ArsR compared with the orientation in related response regulators. Molecular modeling of an ArsR-DBD-DNA complex permits identification of protein elements that are predicted to bind target DNA sequences and thereby regulate gene transcription in H. pylori.

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. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein.