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Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif ( Saccharomyces cerevisiae residues 391-415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae . These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.

Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.

Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene ( HTT ), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.

TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

KRAS interacts with the inner leaflet of the plasma membrane (PM) using a hybrid anchor that comprises a lysine-rich polybasic domain (PBD) and a C-terminal farnesyl chain. Electrostatic interactions have been envisaged as the primary determinant of interactions between KRAS and membranes. Here, we integrated molecular dynamics (MD) simulations and superresolution spatial analysis in mammalian cells and systematically compared four equally charged KRAS anchors: the wild-type farnesyl hexa-lysine and engineered mutants comprising farnesyl hexa-arginine, geranylgeranyl hexa-lysine, and geranylgeranyl hexa-arginine. MD simulations show that these equally charged KRAS mutant anchors exhibit distinct interactions and packing patterns with different phosphatidylserine (PtdSer) species, indicating that prenylated PBD-bilayer interactions extend beyond electrostatics. Similar observations were apparent in intact cells, where each anchor exhibited binding specificities for PtdSer species with distinct acyl chain compositions. Acyl chain composition determined responsiveness of the spatial organization of different PtdSer species to diverse PM perturbations, including transmembrane potential, cholesterol depletion, and PM curvature. In consequence, the spatial organization and PM binding of each KRAS anchor precisely reflected the behavior of its preferred PtdSer ligand to these same PM perturbations. Taken together these results show that small GTPase PBD-prenyl anchors, such as that of KRAS, have the capacity to encode binding specificity for specific acyl chains as well as lipid headgroups, which allow differential responses to biophysical perturbations that may have biological and signaling consequences for the anchored GTPase., Competing Interests: The authors declare no competing interest.

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele ( xiamen [ xm ]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3 xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3 xm displayed a large compensatory alteration in switch I, which adopted a β-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3 xm , strongly associated with and stabilized GPR89, and an N -ethyl- N -nitrosourea (ENU)-induced mutation ( explorer ) in Gpr89 phenocopied Rabl3 xm ., Competing Interests: The authors declare no competing interest.

Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Accumulative aggregation of mutant Huntingtin (Htt) is a primary neuropathological hallmark of Huntington's disease (HD). Currently, mechanistic understanding of the cytotoxicity of mutant Htt aggregates remains limited, and neuroprotective strategies combating mutant Htt-induced neurodegeneration are lacking. Here, we show that in Drosophila models of HD, neuronal compartment-specific accumulation of mutant Htt aggregates causes neurodegenerative phenotypes. In addition to the increase in the number and size, we discovered an age-dependent acquisition of thioflavin S + , amyloid-like adhesive properties of mutant Htt aggregates and a concomitant progressive clustering of aggregates with mitochondria and synaptic proteins, indicating that the amyloid-like adhesive property underlies the neurotoxicity of mutant Htt aggregation. Importantly, nicotinamide mononucleotide adenylyltransferase (NMNAT), an evolutionarily conserved nicotinamide adenine dinucleotide (NAD + ) synthase and neuroprotective factor, significantly mitigates mutant Htt-induced neurodegeneration by reducing mutant Htt aggregation through promoting autophagic clearance. Additionally, Nmnat overexpression reduces progressive accumulation of amyloid-like Htt aggregates, neutralizes adhesiveness, and inhibits the clustering of mutant Htt with mitochondria and synaptic proteins, thereby restoring neuronal function. Conversely, partial loss of endogenous Nmnat exacerbates mutant Htt-induced neurodegeneration through enhancing mutant Htt aggregation and adhesive property. Finally, conditional expression of Nmnat after the onset of degenerative phenotypes significantly delays the progression of neurodegeneration, revealing the therapeutic potential of Nmnat-mediated neuroprotection at advanced stages of HD. Our study uncovers essential mechanistic insights to the neurotoxicity of mutant Htt aggregation and describes the molecular basis of Nmnat-mediated neuroprotection in HD., Competing Interests: The authors declare no conflict of interest.

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K + , water, K + -ion-bound configuration) of the K + channel's selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type-like ion selectivity and apparent K + -binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K + channel's selectivity filter, which fully agrees with the water-K + -ion-coupled transport detected by streaming potential measurements., Competing Interests: The authors declare no conflict of interest.

Mutant huntingtin (mHTT), the causative protein in Huntington's disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases., Competing Interests: Conflict of interest statement: R.M.F. is on the board of NeuBase Therapeutics. No funding for this work was received from NeuBase Therapeutics.

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila , only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions., Competing Interests: The authors declare no conflict of interest.

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion., Competing Interests: The authors declare no conflict of interest.

Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans , DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel's outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation., Competing Interests: The authors declare no conflict of interest.

The β-cell-enriched MAFA transcription factor plays a central role in regulating glucose-stimulated insulin secretion while also demonstrating oncogenic transformation potential in vitro. No disease-causing MAFA variants have been previously described. We investigated a large pedigree with autosomal dominant inheritance of diabetes mellitus or insulinomatosis, an adult-onset condition of recurrent hyperinsulinemic hypoglycemia caused by multiple insulin-secreting neuroendocrine tumors of the pancreas. Using exome sequencing, we identified a missense MAFA mutation (p.Ser64Phe, c.191C>T) segregating with both phenotypes of insulinomatosis and diabetes. This mutation was also found in a second unrelated family with the same clinical phenotype, while no germline or somatic MAFA mutations were identified in nine patients with sporadic insulinomatosis. In the two families, insulinomatosis presented more frequently in females (eight females/two males) and diabetes more often in males (12 males/four females). Four patients from the index family, including two homozygotes, had a history of congenital cataract and/or glaucoma. The p.Ser64Phe mutation was found to impair phosphorylation within the transactivation domain of MAFA and profoundly increased MAFA protein stability under both high and low glucose concentrations in β-cell lines. In addition, the transactivation potential of p.Ser64Phe MAFA in β-cell lines was enhanced compared with wild-type MAFA. In summary, the p.Ser64Phe missense MAFA mutation leads to familial insulinomatosis or diabetes by impacting MAFA protein stability and transactivation ability. The human phenotypes associated with the p.Ser64Phe MAFA missense mutation reflect both the oncogenic capacity of MAFA and its key role in islet β-cell activity., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

The 20S proteasome core particle (20S CP) plays an integral role in cellular homeostasis by degrading proteins no longer required for function. The process is, in part, controlled via gating residues localized to the ends of the heptameric barrel-like CP structure that occlude substrate entry pores, preventing unregulated degradation of substrates that might otherwise enter the proteasome. Previously, we showed that the N-terminal residues of the α-subunits of the CP from the archaeon Thermoplasma acidophilum are arranged such that, on average, two of the seven termini are localized inside the lumen of the proteasome, thereby plugging the entry pore and functioning as a gate. However, the mechanism of gating remains unclear. Using solution NMR and a labeling procedure in which a series of mixed proteasome rings are prepared such that the percentage of gate-containing subunits is varied, we address the energetics of gating and establish whether gating is a cooperative process involving the concerted action of residues from more than a single protomer. Our results establish that the intrinsic probability of a gate entering the lumen favors the in state by close to 20-fold, that entry of each gate is noncooperative, with the number of gates that can be accommodated inside the lumen a function of the substrate entry pore size and the bulkiness of the gating residues. Insight into the origin of the high affinity for the in state is obtained from spin-relaxation experiments. More generally, our approach provides an avenue for dissecting interactions of individual protomers in homo-oligomeric complexes., Competing Interests: The authors declare no conflict of interest.

Nucleotidyl cyclases, including membrane-integral and soluble adenylyl and guanylyl cyclases, are central components in a wide range of signaling pathways. These proteins are architecturally diverse, yet many of them share a conserved feature, a helical region that precedes the catalytic cyclase domain. The role of this region in cyclase dimerization has been a subject of debate. Although mutations within this region in various cyclases have been linked to genetic diseases, the molecular details of their effects on the enzymes remain unknown. Here, we report an X-ray structure of the cytosolic portion of the membrane-integral adenylyl cyclase Cya from Mycobacterium intracellulare in a nucleotide-bound state. The helical domains of each Cya monomer form a tight hairpin, bringing the two catalytic domains into an active dimerized state. Mutations in the helical domain of Cya mimic the disease-related mutations in human proteins, recapitulating the profiles of the corresponding mutated enzymes, adenylyl cyclase-5 and retinal guanylyl cyclase-1. Our experiments with full-length Cya and its cytosolic domain link the mutations to protein stability, and the ability to induce an active dimeric conformation of the catalytic domains. Sequence conservation indicates that this domain is an integral part of cyclase machinery across protein families and species. Our study provides evidence for a role of the helical domain in establishing a catalytically competent dimeric cyclase conformation. Our results also suggest that the disease-associated mutations in the corresponding regions of human nucleotidyl cyclases disrupt the normal helical domain structure., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Deregulated extracellular signal-regulated kinase (ERK) signaling drives cancer growth. Normally, ERK activity is self-limiting by the rapid inactivation of upstream kinases and delayed induction of dual-specificity MAP kinase phosphatases (MKPs/DUSPs). However, interactions between these feedback mechanisms are unclear. Here we show that, although the MKP DUSP5 both inactivates and anchors ERK in the nucleus, it paradoxically increases and prolongs cytoplasmic ERK activity. The latter effect is caused, at least in part, by the relief of ERK-mediated RAF inhibition. The importance of this spatiotemporal interaction between these distinct feedback mechanisms is illustrated by the fact that expression of oncogenic BRAF V600E , a feedback-insensitive mutant RAF kinase, reprograms DUSP5 into a cell-wide ERK inhibitor that facilitates cell proliferation and transformation. In contrast, DUSP5 deletion causes BRAF V600E -induced ERK hyperactivation and cellular senescence. Thus, feedback interactions within the ERK pathway can regulate cell proliferation and transformation, and suggest oncogene-specific roles for DUSP5 in controlling ERK signaling and cell fate., Competing Interests: The authors declare no conflict of interest.

Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes., Competing Interests: The authors declare no conflict of interest.

Mumps virus (MuV) remains an important pathogen worldwide, causing epidemic parotitis, orchitis, meningitis, and encephalitis. Here we show that MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid in unbranched sugar chains as a receptor. Crystal structures of the MuV attachment protein hemagglutinin-neuraminidase (MuV-HN) alone and in complex with the α2,3-sialylated trisaccharide revealed that in addition to the interaction between the MuV-HN active site residues and sialic acid, other residues, including an aromatic residue, stabilize the third sugar of the trisaccharide. The importance of the aromatic residue and the third sugar in the MuV-HN-receptor interaction was confirmed by computational energy calculations, isothermal titration calorimetry studies, and glycan-binding assays. Furthermore, MuV-HN was found to bind more efficiently to unbranched α2,3-sialylated sugar chains compared with branched ones. Importantly, the strategically located aromatic residue is conserved among the HN proteins of sialic acid-using paramyxoviruses, and alanine substitution compromised their ability to support cell-cell fusion. These results suggest that not only the terminal sialic acid but also the adjacent sugar moiety contribute to receptor function for mumps and these paramyxoviruses. The distribution of structurally different sialylated glycans in tissues and organs may explain in part MuV's distinct tropism to glandular tissues and the central nervous system. In the crystal structure, the epitopes for neutralizing antibodies are located around the α-helices of MuV-HN that are not well conserved in amino acid sequences among different genotypes of MuV. This may explain the fact that MuV reinfection sometimes occurs., Competing Interests: The authors declare no conflict of interest.

We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK-ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV-Gαi complexes and activates GIV's GEF function; then a second phosphomodification terminates GIV's GEF function, triggers the assembly of GIV-Gαs complexes, and activates GIV's GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK-ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses., Competing Interests: The authors declare no conflict of interest.

Classical cadherin cell-cell adhesion proteins are essential for the formation and maintenance of tissue structures; their primary function is to physically couple neighboring cells and withstand mechanical force. Cadherins from opposing cells bind in two distinct trans conformations: strand-swap dimers and X-dimers. As cadherins convert between these conformations, they form ideal bonds (i.e., adhesive interactions that are insensitive to force). However, the biophysical mechanism for ideal bond formation is unknown. Here, we integrate single-molecule force measurements with coarse-grained and atomistic simulations to resolve the mechanistic basis for cadherin ideal bond formation. Using simulations, we predict the energy landscape for cadherin adhesion, the transition pathways for interconversion between X-dimers and strand-swap dimers, and the cadherin structures that form ideal bonds. Based on these predictions, we engineer cadherin mutants that promote or inhibit ideal bond formation and measure their force-dependent kinetics using single-molecule force-clamp measurements with an atomic force microscope. Our data establish that cadherins adopt an intermediate conformation as they shuttle between X-dimers and strand-swap dimers; pulling on this conformation induces a torsional motion perpendicular to the pulling direction that unbinds the proteins and forms force-independent ideal bonds. Torsional motion is blocked when cadherins associate laterally in a cis orientation, suggesting that ideal bonds may play a role in mechanically regulating cadherin clustering on cell surfaces., Competing Interests: The authors declare no conflict of interest.

The function of the RIG-I-like receptors (RLRs; including RIG-I, MDA5, and LGP2) as key cytoplasmic sensors of viral pathogen-associated molecular patterns (PAMPs) has been subjected to numerous pathogenic challenges and has undergone a dynamic evolution. We found evolutionary evidence that RIG-I was lost in the Chinese tree shrew lineage. Along with the loss of RIG-I, both MDA5 (tMDA5) and LGP2 (tLGP2) have undergone strong positive selection in the tree shrew. tMDA5 or tMDA5/tLGP2 could sense Sendai virus (an RNA virus posed as a RIG-I agonist) for inducing type I IFN, although conventional RIG-I and MDA5 were thought to recognize distinct RNA structures and viruses. tMDA5 interacted with adaptor tMITA (STINGTMEM173/ERIS), which was reported to bind only with RIG-I. The positively selected sites in tMDA5 endowed the substitute function for the lost RIG-I. These findings provided insights into the adaptation and functional diversity of innate antiviral activity in vertebrates., Competing Interests: The authors declare no conflict of interest.

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the α-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis., Competing Interests: The authors declare no conflict of interest.

Glycogen synthase kinase 3 (GSK3)-like kinases play important roles in brassinosteroid (BR), abscisic acid, and auxin signaling to regulate many aspects of plant development and stress responses. The Arabidopsis thaliana GSK3-like kinase BR-INSENSITIVE 2 (BIN2) acts as a key negative regulator in the BR signaling pathway, but the mechanisms regulating BIN2 function remain unclear. Here we report that the histone deacetylase HDA6 can interact with and deacetylate BIN2 to repress its kinase activity. The hda6 mutant showed a BR-repressed phenotype in the dark and was less sensitive to BR biosynthesis inhibitors. Genetic analysis indicated that HDA6 regulates BR signaling through BIN2. Furthermore, we identified K189 of BIN2 as an acetylated site, which can be deacetylated by HDA6 to influence BIN2 activity. Glucose can affect the acetylation level of BIN2 in plants, indicating a connection to cellular energy status. These findings provide significant insights into the regulation of GSK3-like kinases in plant growth and development., Competing Interests: The authors declare no conflict of interest.

Recent studies have identified impairments in neural induction and in striatal and cortical neurogenesis in Huntington's disease (HD) knock-in mouse models and associated embryonic stem cell lines. However, the potential role of these developmental alterations for HD pathogenesis and progression is currently unknown. To address this issue, we used BACHD:CAG-Cre(ERT2) mice, which carry mutant huntingtin (mHtt) modified to harbor a floxed exon 1 containing the pathogenic polyglutamine expansion (Q97). Upon tamoxifen administration at postnatal day 21, the floxed mHtt-exon1 was removed and mHtt expression was terminated (Q97(CRE)). These conditional mice displayed similar profiles of impairments to those mice expressing mHtt throughout life: (i) striatal neurodegeneration, (ii) early vulnerability to NMDA-mediated excitotoxicity, (iii) impairments in motor coordination, (iv) temporally distinct abnormalities in striatal electrophysiological activity, and (v) altered corticostriatal functional connectivity and plasticity. These findings strongly suggest that developmental aberrations may play important roles in HD pathogenesis and progression.

Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease.

p53, known as a tumor suppressor, is a DNA binding protein that regulates cell cycle, activates DNA repair proteins, and triggers apoptosis in multicellular animals. More than 50% of human cancers contain a mutation or deletion of the p53 gene, and p53R175 is one of the hot spots of p53 mutation. Nucleic acid aptamers are short single-stranded oligonucleotides that are able to bind various targets, and they are typically isolated from an experimental procedure called systematic evolution of ligand exponential enrichment (SELEX). Using a previously unidentified strategy of contrast screening with SELEX, we have isolated an RNA aptamer targeting p53R175H. This RNA aptamer (p53R175H-APT) has a significantly stronger affinity to p53R175H than to the wild-type p53 in both in vitro and in vivo assays. p53R175H-APT decreased the growth rate, weakened the migration capability, and triggered apoptosis in human lung cancer cells harboring p53R175H. Further analysis actually indicated that p53R175H-APT might partially rescue or correct the p53R175H to function more like the wild-type p53. In situ injections of p53R175H-APT to the tumor xenografts confirmed the effects of this RNA aptamer on p53R175H mutation in mice.

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

Many receptors display conformational flexibility, in which the binding pocket has an open inactive conformation in the absence of ligand and a tight active conformation when bound to ligand. Here we study the bacterial adhesin FimH to address the role of the inactive conformation of the pocket for initiating binding by comparing two variants: a wild-type FimH variant that is in the inactive state when not bound to its target mannose, and an engineered activated variant that is always in the active state. Not surprisingly, activated FimH has a longer lifetime and higher affinity, and bacteria expressing activated FimH bound better in static conditions. However, bacteria expressing wild-type FimH bound better in flow. Wild-type and activated FimH demonstrated similar mechanical strength, likely because mechanical force induces the active state in wild-type FimH. However, wild-type FimH displayed a faster bond association rate than activated FimH. Moreover, the ability of different FimH variants to mediate adhesion in flow reflected the fraction of FimH in the inactive state. These results demonstrate a new model for ligand-associated conformational changes that we call the kinetic-selection model, in which ligand-binding selects the faster-binding inactive state and then induces the active state. This model predicts that in physiological conditions for cell adhesion, mechanical force will drive a nonequilibrium cycle that uses the fast binding rate of the inactive state and slow unbinding rate of the active state, for a higher effective affinity than is possible at equilibrium.

Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting "Mutation-Associated Neo-Antigens" bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide-HLA complex even when the peptide differs by only one amino acid from the normal, WT form.

Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-L-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-L-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.

Double C2-like domain-containing proteins alpha and beta (Doc2α and Doc2β) are tandem C2-domain proteins proposed to function as Ca(2+) sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca(2+) to the β isoform. The Ca(2+) ligands in the C2A domain were dispensable for Ca(2+)-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca(2+) ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca(2+)/lipid-binding loops of synaptotagmin 1, a Ca(2+) sensor for synchronous release, with corresponding loops from Doc2β, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2β that underlie its function during the slow phase of synaptic transmission.

Four-stranded nucleic acid structures called G-quadruplexes have been associated with important cellular processes, which should require G-quadruplex-protein interaction. However, the structural basis for specific G-quadruplex recognition by proteins has not been understood. The DEAH (Asp-Glu-Ala-His) box RNA helicase associated with AU-rich element (RHAU) (also named DHX36 or G4R1) specifically binds to and resolves parallel-stranded G-quadruplexes. Here we identified an 18-amino acid G-quadruplex-binding domain of RHAU and determined the structure of this peptide bound to a parallel DNA G-quadruplex. Our structure explains how RHAU specifically recognizes parallel G-quadruplexes. The peptide covers a terminal guanine base tetrad (G-tetrad), and clamps the G-quadruplex using three-anchor-point electrostatic interactions between three positively charged amino acids and negatively charged phosphate groups. This binding mode is strikingly similar to that of most ligands selected for specific G-quadruplex targeting. Binding to an exposed G-tetrad represents a simple and efficient way to specifically target G-quadruplex structures.

Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined.

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using (31)P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.

Cyclic nucleotide-gated (CNG) ion channels, despite a significant homology with the highly selective K(+) channels, do not discriminate among monovalent alkali cations and are permeable also to several organic cations. We combined electrophysiology, molecular dynamics (MD) simulations, and X-ray crystallography to demonstrate that the pore of CNG channels is highly flexible. When a CNG mimic is crystallized in the presence of a variety of monovalent cations, including Na(+), Cs(+), and dimethylammonium (DMA(+)), the side chain of Glu66 in the selectivity filter shows multiple conformations and the diameter of the pore changes significantly. MD simulations indicate that Glu66 and the prolines in the outer vestibule undergo large fluctuations, which are modulated by the ionic species and the voltage. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels.

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd(2+) coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd(2+) forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K(+) (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd(2+) binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.

Escherichia coli senses envelope stress using a signaling cascade initiated when DegS cleaves a transmembrane inhibitor of a transcriptional activator for response genes. Each subunit of the DegS trimer contains a protease domain and a PDZ domain. During stress, unassembled outer-membrane proteins (OMPs) accumulate in the periplasm and their C-terminal peptides activate DegS by binding to its PDZ domains. In the absence of stress, autoinhibitory interactions, mediated by the L3 loop, stabilize inactive DegS, but it is not known how this autoinhibition is reversed during activation. Here, we show that OMP peptides initiate a steric clash between the PDZ domain and the L3 loop that results in a structural rearrangement of the loop and breaking of autoinhibitory interactions. Many different L3-loop sequences are compatible with activation but those that relieve the steric clash reduce OMP activation dramatically. Our results provide a compelling molecular mechanism for allosteric activation of DegS by OMP-peptide binding.

The gain-of-function mutant p53 (mtp53) transcriptome has been studied, but, to date, no detailed analysis of the mtp53-associated proteome has been described. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53-driven proteome. Our fractionation data highlight the underappreciated biology that missense mtp53 proteins R273H, R280K, and L194F are tightly associated with chromatin. Using SILAC coupled to tandem MS, we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells up- and down-regulated multiple proteins and metabolic pathways. Here we provide the data set obtained from sequencing 73,154 peptide pairs that then corresponded to 3,010 proteins detected under reciprocal labeling conditions. Importantly, the high impact regulated targets included the previously identified transcriptionally regulated mevalonate pathway proteins but also identified two new levels of mtp53 protein regulation for nontranscriptional targets. Interestingly, mtp53 depletion profoundly influenced poly(ADP ribose) polymerase 1 (PARP1) localization, with increased cytoplasmic and decreased chromatin-associated protein. An enzymatic PARP shift occurred with high mtp53 expression, resulting in increased poly-ADP-ribosylated proteins in the nucleus. Mtp53 increased the level of proliferating cell nuclear antigen (PCNA) and minichromosome maintenance 4 (MCM4) proteins without changing the amount of pcna and mcm4 transcripts. Pathway enrichment analysis ranked the DNA replication pathway above the cholesterol biosynthesis pathway as a R273H mtp53 activated proteomic target. Knowledge of the proteome diversity driven by mtp53 suggests that DNA replication and repair pathways are major targets of mtp53 and highlights consideration of combination chemotherapeutic strategies targeting cholesterol biosynthesis and PARP inhibition.

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4-S5 linker helix. By homology modeling, we identified the corresponding L596-W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4-S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar-nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.

Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.