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Bioprocesses converting carbon dioxide with molecular hydrogen to methane (CH4) are currently being developed to enable a transition to a renewable energy production system. In this study, we present a comprehensive physiological and biotechnological examination of 80 methanogenic archaea (methanogens) quantifying growth and CH4 production kinetics at hyperbaric pressures up to 50 bar with regard to media, macro-, and micro-nutrient supply, specific genomic features, and cell envelope architecture. Our analysis aimed to systematically prioritize high-pressure and high-performance methanogens. We found that the hyperthermophilic methanococci Methanotorris igneus and Methanocaldococcoccus jannaschii are high-pressure CH4 cell factories. Furthermore, our analysis revealed that high-performance methanogens are covered with an S-layer, and that they harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase. Thus, high-pressure biological CH4 production in pure culture could provide a purposeful route for the transition to a carbon-neutral bioenergy sector., Mauerhofer et al. examine 80 species of methanogenic archaea at high pressures and evaluate growth and methane production, identifying Methanotorris igneus and Methanocaldococcoccus jannaschii as high-pressure methane cell factories. They find that high-performance methanogens are covered with an S-layer and harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase.

The ATR–dependent DNA damage checkpoint is one of the major checkpoint pathways. The interaction between the Rad17–RFC2-5 and 9–1–1 complexes is central to the ATR–Chk1 pathway. However, little is known about the regulation of the interaction. We recently showed that vertebrate Rad17 proteins share a conserved C-terminal tail and that the C-terminal tails have a conserved amino acid motif named iVERGE that must be intact for the interaction between Rad17 and the 9‒1‒1 complex. In human Rad17, the Y665 and S667 residues are conserved in iVERGE. The Rad17-S667 residue is phosphorylated by CK2, and the phosphorylation is important for the interaction with the 9‒1‒1 complex. Here, we show that a C-terminal threonine residue of Rad17, T670 in human Rad17, is constitutively phosphorylated in vivo. The T670 phosphorylation is important for the S667 phosphorylation, and vice versa. Phosphomimetic mutations in the T670 residue promote the interaction with the 9–1–1 complex. The T670 and Y665 residues show functional redundancy, and their roles are dependent on the S667 phosphorylation. Rad17-T670 is phosphorylated by casein kinase 1δ/e. Our data suggest that iVERGE integrates multiple signaling pathways to regulate the ATR–Chk1 pathway.

BACKGROUND The insect cuticle protects against environmental stresses such as insecticides, physical injury, dehydration and pathogenic microorganisms. Structural cuticular proteins (CPs) are the primary components, and are of interest for physiology-based pest management methods. Anopheles sinensis CPs are poorly characterised at present, therefore we performed whole genome sequence analysis and re-analysed available transcriptome data to identify potential insecticide resistance-associated CPs. RESULTS Genome analysis revealed 238 putative CPs belonging to 11 different families; 136 proteins possessing a Rebers and Riddiford motif (CPR), 12 CPs analogous to peritrophins with a single chitin-binding domain (CPAP1), eight CPs analogous to peritrophins with three chitin-binding domains (CPAP3), four proteins with a 44 amino acid motif (CPF), five CPF-like (CPFL) proteins, nine Tweedle proteins, three proteins of low-complexity with alanine residues (CPLCA), 26 proteins of low-complexity with conserved glycine residues (CPLCG), seven proteins of low-complexity with invariant W residues (CPLCW), 27 proteins of low-complexity with proline residues (CPLCP), and one protein with two or three copies of a C-X(5)-C motif (CPCFC). Two CPs belonging to CPR and CPAP3 families were over-expressed in a deltamethrin-resistant strain. CONCLUSION These results establish an information framework for An. sinensis CPs that provides a basis for further molecular research.

The aggregation of α-synuclein (α-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and innocuous α-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of α-syn has hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of this transition. The recent 3D structure of a pathogenic α-syn fibril provides a template for this kind of studies. The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of α-syn in different environments. The new covalent link biases the native structural ensemble of α-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early α-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD. Keywords: α-synuclein, Disulfide bond, Amyloid, Protein aggregation, Parkinson's disease

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

Norovirus (NV) RNA-dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer-template duplex was investigated using X-ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back-tracked state of the enzyme, in which the (Formula presented.) -end of the primer occupies the position expected for the post-incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435–1445. © 2017 Wiley Periodicals, Inc.

Emerging evidence supports the role for the intracellular domains of amyloid precursor protein (APP) in the physiology and function of APP. In this short report, I discuss the hypothesis that mutation of Tyr682 on the Y682 ENPTY687 C-terminal motif of APP may be directly or indirectly associated with alterations in APP functioning and activity, leading to neuronal defects and deficits. Mutation of Tyr682 induces an early and progressive age-dependent cognitive and locomotor decline that is associated with a loss of synaptic connections, a decrease in cholinergic tone, and defects in NGF signaling. These findings support a model in which APP-C-terminal domain exerts a pathogenic function in neuronal development and decline, and suggest that Tyr682 potentially could modulate the properties of APP metabolites in humans.

Expression of the MHC class I chain related molecules A and B (MICA/B) on tumor cell surface can signal the immune receptor NKG2D for tumor immune destruction. However, MIC was found to be shed by tumors in cancer patients, which negatively regulates host immunity and promotes tumor immune evasion and progression. The mechanisms by which tumors shed MIC are not well understood although diverse groups of enzymes are suggested to be involved. The functional complexity of these enzymes makes them unfeasible therapeutic targets for inhibiting MIC shedding. Here we identified an six-amino acid (6-aa) motif in the alpha3 domain of MIC that is critical for the interaction of MIC with ERp5 to enable shedding. Mutations in this motif prevented MIC shedding but did not interfere with NKG2D-mediated recognition of MIC. Our study suggests that the 6-aa motif is a feasible target to inhibit MIC shedding for cancer therapy.

5′-Deoxy-5′-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of Δ H cal (850 kcal/mol) and T max (39 °C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200–Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138–Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.

Background The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. Scope of review p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. Major conclusions The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. General significance The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets.

When the export of E. coli SecM is blocked, a 17 amino acid motif near the C terminus of the protein induces a translation arrest from within the ribosome tunnel. Here we used a recently described application of fluorescence resonance energy transfer (FRET) to gain insight into the mechanism of translation arrest. We found that the SecM C terminus adopted a compact conformation upon synthesis of the arrest motif. This conformational change did not occur spontaneously, but rather was induced by the ribosome. Translation arrest required both compaction of the SecM C terminus and the presence of key residues in the arrest motif. Further analysis showed that the arrested peptidyl-tRNA was resistant to puromycin treatment and revealed additional changes in the ribosome-nascent SecM complex. Based on these observations, we propose that translation arrest results from a series of reciprocal interactions between the ribosome and the C terminus of the nascent SecM polypeptide.

This review deals with recent data on the structure and biochemical properties of dehydrins, proteins that are normally synthesized in maturating seeds during their desiccation, and also in vegetative tissues of plants treated with abscisic acid or exposed to environmental stress factors that result in cellular dehydration. The dehydrins are considered as stress proteins involved in formation of plant protective reactions against dehydration. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y-, S-, and K-segments. The K-segment representing a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) forming amphiphilic α-helix has been found in all dehydrins. The pathways of regulation of dehydrin gene expression, putative functions of dehydrins, and molecular mechanisms of their actions are discussed.

Processing of Abeta-precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the (682) YENPTY(687) motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Y(682) to Gly (APP(YG/YG) mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP-like protein 2 KO background. This evidence prompted us to characterize further the APP(YG/YG) mice. Here, we show that APP(YG/YG) mice develop aging-dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP(YG/YG) mice. The profound deficits caused by the Y(682) mutation underscore the biological importance of APP and indicate that APP(YG/YG) are a valuable mouse model to study APP functions in physiological and pathological processes.

Motivation: We have previously demonstrated that proteins may be aligned not only by sequence or structural homology, but also using their dynamical properties. Dynamics-based alignments are sensitive and powerful tools to compare even structurally dissimilar protein families. Here, we propose to use this method to predict protein regions involved in the binding of nucleic acids. We have used the OB-fold, a motif known to promote protein–nucleic acid interactions, to validate our approach. Results: We have tested the method using this well-characterized nucleic acid binding family. Protein regions consensually involved in statistically significant dynamics-based alignments were found to correlate with nucleic acid binding regions. The validated scheme was next used as a tool to predict which regions of the AXH-domain representatives (a sub-family of the OB-fold for which no DNA/RNA complex is yet available) are putatively involved in binding nucleic acids. The method, therefore, is a promising general approach for predicting functional regions in protein families on the basis of comparative large-scale dynamics. Availability: The software is available upon request from the authors, free of charge for academic users. Contact: michelet@sissa.it Supplementary information: Supplementary data are available at Bioinformatics online.

We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.

We review and further develop an analytical model that describes how thermodynamic constraints on the stability of the native state influence protein evolution in a site-specific manner. To this end, we represent both protein sequences and protein structures as vectors: Structures are represented by the principal eigenvector (PE) of the protein contact matrix, a quantity that resembles closely the effective connectivity of each site; Sequences are represented through the ``interactivity'' of each amino acid type, using novel parameters that are correlated with hydropathy scales. These interactivity parameters are more strongly correlated than the other hydropathy scales that we examine with: (1) The change upon mutations of the unfolding free energy of proteins with two-states thermodynamics; (2) Genomic properties as the genome-size and the genome-wide GC content; (3) The main eigenvectors of the substitution matrices. The evolutionary average of the interactivity vector correlates very strongly with the PE of a protein structure. Using this result, we derive an analytic expression for site-specific distributions of amino acids across protein families in the form of Boltzmann distributions whose ``inverse temperature'' is a function of the PE component. We show that our predictions are in agreement with site-specific amino acid distributions obtained from the Protein Data Bank, and we determine the mutational model that best fits the observed site-specific amino acid distributions. Interestingly, the optimal model almost minimizes the rate at which deleterious mutations are eliminated by natural selection., 11 pages, 5 figures

The presence of highly conserved amino acid stretches in G protein-coupled receptors (GPCRs) usually predicts an important role in receptor function. Considerable attention has therefore been focused on the involvement of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif at the cytoplasmic end of transmembrane domain 3 in the regulation of GPCR conformational states and/or the mediation of G protein activation. In the present study, we investigated the role of Glu129 and Arg130 in the ERY of thromboxane A2 receptor alpha (TPalpha) in transfected human embryonic kidney 293 cells. We show that no conservative or nonconservative substitutions of Glu129 and Arg130 generated a constitutively active TPalpha mutant, but a nonconservative mutation of Arg130 (R130V) yielded a mutant receptor with significantly impaired 9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619)-induced accumulation of inositol phosphates (IPs). This loss-of-function phenotype seems to be caused by the uncoupling of the TPalpha receptor from Gq, as demonstrated by the loss of high-affinity agonist binding, and not by receptor internalization, as shown by localization studies with the R130V-green fluorescent protein fusion protein. It is interesting to note that U46619-induced activation of the nonconservative E129V mutant stimulated the production of IPs with a approximately 10-fold lower EC50 and a approximately 2-fold higher Emax than in the wild-type receptor. Collectively, these data demonstrate that, unlike other GPCRs, mutations of Glu129 do not induce constitutive activity, whereas Arg130 is involved in G protein coupling or recognition, and they suggest the existence within class A GPCRs of at least two different subclasses that make different uses of the highly conserved E/DRY motif.

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A main challenge for structural biologists is to understand the mechanisms that discriminate between molecular interactions and determine function. Here, we show how partner recognition of the AXH domain of the transcriptional co-regulator ataxin-1 is fine-tuned by a subtle balance between self- and hetero-associations. Ataxin-1 is the protein responsible for the hereditary spinocerebellar ataxia type 1, a disease linked to protein aggregation and transcriptional dysregulation. Expansion of a polyglutamine tract is essential for ataxin-1 aggregation, but the sequence-wise distant AXH domain plays an important aggravating role in the process. The AXH domain is also a key element for non-aberrant function as it intervenes in interactions with multiple protein partners. Previous data have shown that AXH is dimeric in solution and forms a dimer of dimers when crystallized. By solving the structure of a complex of AXH with a peptide from the interacting transcriptional repressor CIC, we show that the dimer interface of AXH is displaced by the new interaction and that, when blocked by the CIC peptide AXH aggregation and misfolding are impaired. This is a unique example in which palindromic self- and hetero-interactions within a sequence with chameleon properties discriminate the partner. We propose a drug design strategy for the treatment of SCA1 that is based on the information gained from the AXH/CIC complex.

Pancreatic reg proteins are exocrine proteins which are mitogenic to pancreatic ductal and ∃-cells (1,2). Using multiple alignment sequence comparison of the reg proteins with known mitogenic activity, we identified an amino acid sequence (RegP) highly conserved between rat, mouse and human reg’s (3), and postulated that it is the bioactive fragment. This sequence contained an ‘WIGL’ amino acid motif, considered important in calcium-dependant lectins. We also postulated that the action of reg would be through activation of mitogen-activated protein kinases (MAPK).

A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [–(hydrophobic)–k–(hydrophobic)–x–x–(hydrophobic)– (polar)–(hydrophobic)–(polar)–(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.