JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase.

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor.

The respiratory syncytial virus polymerase complex, consisting of the polymerase (L) and phosphoprotein (P), catalyzes nucleotide polymerization, cap addition, and cap methylation via the RNA dependent RNA polymerase, capping, and Methyltransferase domains on L. Several nucleoside and non-nucleoside inhibitors have been reported to inhibit this polymerase complex, but the structural details of the exact inhibitor-polymerase interactions have been lacking. Here, we report a non-nucleoside inhibitor JNJ-8003 with sub-nanomolar inhibition potency in both antiviral and polymerase assays. Our 2.9 Å resolution cryo-EM structure revealed that JNJ-8003 binds to an induced-fit pocket on the capping domain, with multiple interactions consistent with its tight binding and resistance mutation profile. The minigenome and gel-based de novo RNA synthesis and primer extension assays demonstrated that JNJ-8003 inhibited nucleotide polymerization at the early stages of RNA transcription and replication. Our results support that JNJ-8003 binding modulates a functional interplay between the capping and RdRp domains, and this molecular insight could accelerate the design of broad-spectrum antiviral drugs.

Structure of human spermine oxidase in complex with a highly selective allosteric inhibitor.

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer.

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.

Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter.

Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein-protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional (1)H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the "rocking bundle" hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.

Interplay between base excision repair activity and toxicity of 3-methyladenine DNA glycosylases in an E. coli complementation system.

DNA glycosylases carry out the first step of base excision repair by removing damaged bases from DNA. The N3-methyladenine (3MeA) DNA glycosylases specialize in alkylation repair and are either constitutively expressed or induced by exposure to alkylating agents. To study the functional and evolutionary significance of constitutive versus inducible expression, we expressed two closely related yeast 3MeA DNA glycosylases - inducible Saccharomyces cerevisiae MAG and constitutive S. pombe Mag1 - in a glycosylase-deficient Escherichia coli strain. In both cases, constitutive expression conferred resistance to alkylating agent exposure. However, in the absence of exogenous alkylation, high levels of expression of both glycosylases were deleterious. We attribute this toxicity to excessive glycosylase activity, since suppressing spMag1 expression correlated with improved growth in liquid culture, and spMag1 mutants exhibiting decreased glycosylase activity showed improved growth and viability. Selection of a random spMag1 mutant library for increased survival in the presence of exogenous alkylation resulted in the selection of hypomorphic mutants, providing evidence for the presence of a genetic barrier to the evolution of enhanced glycosylase activity when constitutively expressed. We also show that low levels of 3MeA glycosylase expression improve fitness in our glycosylase-deficient host, implying that 3MeA glycosylase activity is likely necessary for repair of endogenous lesions. These findings suggest that 3MeA glycosylase activity is evolutionarily conserved for repair of endogenously produced alkyl lesions, and that inducible expression represents a common strategy to rectify deleterious effects of excessive 3MeA activity in the absence of exogenous alkylation challenge.

Mcm10 self-association is mediated by an N-terminal coiled-coil domain.

Minichromosome maintenance protein 10 (Mcm10) is an essential eukaryotic DNA-binding replication factor thought to serve as a scaffold to coordinate enzymatic activities within the replisome. Mcm10 appears to function as an oligomer rather than in its monomeric form (or rather than as a monomer). However, various orthologs have been found to contain 1, 2, 3, 4, or 6 subunits and thus, this issue has remained controversial. Here, we show that self-association of Xenopus laevis Mcm10 is mediated by a conserved coiled-coil (CC) motif within the N-terminal domain (NTD). Crystallographic analysis of the CC at 2.4 Å resolution revealed a three-helix bundle, consistent with the formation of both dimeric and trimeric Mcm10 CCs in solution. Mutation of the side chains at the subunit interface disrupted in vitro dimerization of both the CC and the NTD as monitored by analytical ultracentrifugation. In addition, the same mutations also impeded self-interaction of the full-length protein in vivo, as measured by yeast-two hybrid assays. We conclude that Mcm10 likely forms dimers or trimers to promote its diverse functions during DNA replication.

Non-productive DNA damage binding by DNA glycosylase-like protein Mag2 from Schizosaccharomyces pombe.

Schizosaccharomyces pombe contains two paralogous proteins, Mag1 and Mag2, related to the helix-hairpin-helix (HhH) superfamily of alkylpurine DNA glycosylases from yeast and bacteria. Phylogenetic analysis of related proteins from four Schizosaccharomyces and other fungal species shows that the Mag1/Mag2 duplication is unique to the genus Schizosaccharomyces and most likely occurred in its ancestor. Mag1 excises N3- and N7-alkylguanines and 1,N(6)-ethenoadenine from DNA, whereas Mag2 has been reported to have no detectible alkylpurine base excision activity despite high sequence and active site similarity to Mag1. To understand this discrepancy we determined the crystal structure of Mag2 bound to abasic DNA and compared it to our previously determined Mag1-DNA structure. In contrast to Mag1, Mag2 does not flip the abasic moiety into the active site or stabilize the DNA strand 5' to the lesion, suggesting that it is incapable of forming a catalytically competent protein-DNA complex. Subtle differences in Mag1 and Mag2 interactions with the DNA duplex illustrate how Mag2 can stall at damage sites without fully engaging the lesion. We tested our structural predictions by mutational analysis of base excision and found a single amino acid responsible at least in part for Mag2's lack of activity. Substitution of Mag2 Asp56, which caps the helix at the base of the DNA intercalation loop, with the corresponding serine residue in Mag1 endows Mag2 with ɛA excision activity comparable to Mag1. This work provides novel insight into the chemical and physical determinants by which the HhH glycosylases engage DNA in a catalytically productive manner.

Recent advances in the structural mechanisms of DNA glycosylases.

DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities.

Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase.

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N(6)-ethenoadenine (ɛA) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their ɛA activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

Helix dipole movement and conformational variability contribute to allosteric GDP release in Galphai subunits.

Heterotrimeric G proteins (Galphabetagamma) transmit signals from activated G protein-coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal alpha5 helix of Galpha subunits participates in Galpha-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the alpha5 helix of the Galpha subunit [Oldham, W. M., et al. (2006) Nat. Struct. Mol. Biol. 13, 772-777]. On the basis of this result, an engineered disulfide bond was designed to constrain the alpha5 helix of Galpha(i1) into its EPR-measured receptor-associated conformation through the introduction of cysteines at position 56 in the alpha1 helix and position 333 in the alpha5 helix (I56C/Q333C Galpha(i1)). A functional mimetic of the EPR-measured alpha5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino terminus of this helix, D328R Galpha(i1). Both proteins exhibit a dramatically elevated level of basal nucleotide exchange. The 2.9 A resolution crystal structure of I56C/Q333C Galpha(i1) in complex with GDP-AlF(4)(-) reveals the shift of the alpha5 helix toward the guanine nucleotide binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Galpha(i1) subunit further revealed altered positions for the switch regions and throughout the Galpha(i1) subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the alpha5 helix dipole and overall conformational variability during nucleotide release.