Non-conservative Substitutions Research Articles

The Use of Direct Genetic Markers K-Casein and DGAT1, Involved in Milk Production Characteristics

Application of molecular markers for improved production relies on the ability to genotype individuals for specific genetic loci and to find the significant association between DNA polymorphism and important economic traits. Milk yield, milk composition, fat %, protein %, total solids and solids-non-fat are the most important characteristics in diary industry. Effects of K-casein and DGAT1 genes have been shown to be involved in the genetic determinism of these characteristics in cattle. The present study seeks a non-conservative substitution K232A (lysine by alanine) in the DGAT1 gene and for a point mutation, in a 350 bp fragment of kCN gene (exon IV), which distinguishes between the A from B alleles, the latter being desirable for cheese manufacturing. The genetic variation on these two loci was estimated in two cattle populations with different origins, in view of future studies to determine the effect of single gene and interaction effect of genotypes on milk yield and milk composition.

Genetic variants affecting human TRPA1 or TRPM8 structure can be classified in vitro as 'well expressed', 'poorly expressed' or 'salvageable'.

Multiple mis-sense variants of TRPA1 (transient receptor potential A1) and TRPM8 (transient receptor potential M8) are recorded in the human genome single nt polymorphism (SNP) database, but their potential impact on channel signalling in patho-physiology is not fully explored. Variants, mostly quite rare in the general human population, alter sites in different structural domains of these homo-tetrameric ion channel proteins. The effects of individual SNPs affecting the large cytoplasmic N-terminal domain have not been completely documented for TRPM8 or TRPA1. We examined the Ca(2+) signalling properties of a short-list of eight variants affecting the N-terminal domain by individual expression in human embryonic kidney HEK293 or neuroblastoma (SH-SY5Y) cell lines (four SNP variants for TRPM8: G150R, K423N, R475C, R485W and four for TRPA1: Y69C, A366D, E477K, D573A). These were compared with TRPA1 SNP variants affecting intracellular loops located beyond the N-terminal domain and associated with gain of function (such as increased sensitivity to agonists: TRPA1 R797T and N855S). A substitution in TRPA1 (Y69C) exhibited high expression/sensitivity to agonists (high iCa(2+)max (maximum level of intracellular calcium ion), similar to R797T, but less sensitive than N855S), whereas each of the other non-conservative substitutions exhibited poor signalling response (low iCa(2+)max). Responses from these poorly expressed variants could be salvaged, to different extents, by pre-treating cells with the Src (Src protein) family inhibitor protein kinase inhibitor PP2 (PP2: 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), or with micromolar Zn(2+). The TRPA1 variants and several experimental mutants (TRPA1 Y97F, Y226F and YY654-655FF) expressed poorly in SH-SY5Y compared with HEK293 cells. More in-depth studies are needed to identify SNP variants eliciting gain of function in these TRP (transient receptor potential) channels and to assess their roles in medical conditions.

The HOXB13 G84E Mutation Is Associated with an Increased Risk for Prostate Cancer and Other Malignancies.

A rare nonconservative substitution (G84E) in the HOXB13 gene has been shown to be associated with risk of prostate cancer. DNA samples from male patients included in the Mayo Clinic Biobank (MCB) were genotyped to determine the frequency of the G84E mutation and its association with various cancers. Subjects were genotyped using a custom TaqMan (Applied Biosystems) assay for G84E (rs138213197). In addition to donating a blood specimen, all MCB participants completed a baseline questionnaire to collect information on medical history and family history of cancer. Forty-nine of 9,012 male patients were carriers of G84E (0.5%). Thirty-one percent (n = 2,595) of participants had been diagnosed with cancer, including 51.1% of G84E carriers compared with just 30.6% of noncarriers (P = 0.004). G84E was most frequently observed among men with prostate cancer compared with men without cancer (P < 0.0001). However, the mutation was also more commonly observed in men with bladder cancer (P = 0.06) and leukemia (P = 0.01). G84E carriers were more likely to have a positive family history of prostate cancer in a first-degree relative compared to noncarriers (36.2% vs. 16.0%, P = 0.0003). Our study confirms the association between the HOXB13 G84E variant and prostate cancer and suggests a novel association between G84E and leukemia and a suggestive association with bladder cancer. Future investigation is warranted to confirm these associations in order to improve our understanding of the role of germline HOXB13 mutations in human cancer. The associations between HOXB13 and prostate, leukemia, and bladder suggest that this gene is important in carcinogenesis.

Structural characterisation of the acyl CoA: diacylglycerol acyltransferase 1 (DGAT1) gene and association studies with milk traits in Assaf sheep breed

Because DGAT1 plays a fundamental role in triacylglycerol synthesis, existing SNPs in DGAT1 gene might provide important information in partially explaining, the variation of milk fat content in dairy sheep. Therefore, the main objective of this study was to sequence the complete ovine acyl CoA:diacylglycerol acyltransferase 1 (DGAT1)gene in order to identify polymorphisms and to look for its possible association with milk traits in Assaf sheep breed. Polymorphisms identification in the DGAT1gene was carried out in 50 individuals belonging to five sheep breeds reared in Spain: Rasa Aragonesa (n=10), Manchega (n=10), Churra Tensina (n=10), Latxa (n=10) and Spanish Assaf (n=10). The association studies between polymorphisms, and milk traits were carried out using animals belonging to three flocks of Assaf breed (n=402). Four SNPs were detected, one in exon 1 (EU178818: g.358C>A), two in exon 17 (g.8522C>T and g.8539C>T), and one in intron 10 (g.7457C>A). The SNP in exon 1, g.358C>A,generates a non-conservative substitution at position p.Asp53Glu (GenBank ABW24130). The first SNP in exon 17 (g.8522C>T)causes an amino acid change at position p.Arg482Cys.The genotype frequencies were studied in a panel of 9 breed reared in Spain: Ansotana (n=50), Latxa (n=36), Romanov (n=33), Rasa aragonesa (n=55), Churra (n=52), Churra tensina (n=57), Churra lebrijana (n=50), Manchega (n=48) and Assaf (n=402). All breeds were in Hardy–Weinberg equilibrium for all SNPs, except for the SNPs g.8522C>T and g.8539C>T SNPs which showed a deficit of heterozygous animals in Ansotana breed. This gene show low variability in a panel of 9 breeds reared in Spain. The only polymorphism not fixed in Assaf breed was the SNP g.8539C>T, and was used to test possible association with milk traits. The allelic frequency of the 8539C allele was 0.96, being 373 animals homozygous for the CC genotype and 29 heterozygous. The association studies showed that lactose, fatty acids C4:0, C16:1 c9, and the ratio n-6:n-3 were affected by the SNP g.8539C>T. Animals carrying the CC genotype had greater lactose, C4:0 and C16:1 c9 contents and lower ratio of n-6:n-3 compare to the CT ones. Probably the SNP g.8539C>T, is not causative of the variation observed in the lactose content but might be in linkage disequilibrium with the causal mutation located in the same or other closer gene.

Structural basis for the antipolymer activity of Hb [formula omitted] trapped in a tense conformation

The phenotypical severity of sickle cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α2β2s) to contain embryonic ζ globin in place of adult α-globin subunits (Hb ζ2β2s). Crystallographical analyses of liganded Hb ζζ2β2s, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of α → ζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the βs subunits of T-state deoxyHb α2β2s and T-state CO-liganded Hb ζ2β2s reveal significant displacements of both mutant βsVal6 and conserved β-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ2βs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological βsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2β2s suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ2β2s appear to arise from both repositioning and replacement of specific α- and βs-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ2β2s, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.

Key residues at the membrane-distal surface of KACL, but not glycosylation, determine the functional interaction of the keratinocyte-specific C-type lectin-like receptor KACL with its high-affinity receptor NKp65.

Keratinocyte-associated C-type lectin (KACL) is a peculiar C-type lectin-like receptor (CTLR) due to its selective expression by human keratinocytes and cognate interaction with the genetically coupled CTLR NKp65. KACL and NKp65 are members of the CLEC2 and NKRP1 subfamilies of natural killer gene complex (NKC)-encoded CTLR, respectively. Most NKRP1 molecules are expressed on NK cells and T cells and act as receptors of CLEC2 glycoproteins with their genes being intermingled in a certain sub-region of the mammalian NKC. The reasons for the tight genetic linkage of these dedicated receptor/ligand pairs are unknown, as is the physiological expression of NKp65. Recently, we reported that the CTLR NKp65 and KACL interact with high affinity, resulting in activation of NKp65-expressing NK-92MI cells. Here, we address the molecular basis of this high-affinity interaction by analysing KACL mutants with KACL-specific monoclonal antibodies (mAb), soluble NKp65 (sNKp65) and NK-92MI-NKp65 cells. We find that none of the three N-linked carbohydrates of KACL glycoproteins significantly contributes to KACL surface expression and NKp65 interaction. However, KACL mutants with non-conservative amino acid substitutions of arginine 158 or isoleucine 161 abrogated binding of both KACL-specific mAb OMA1 and sNKp65, well in line with the blockade of NKp65-KACL interaction by OMA1. Accordingly, functional recognition of these KACL mutants by NK-92M-NKp65 cells was completely abolished. Arginine 158 and isoleucine 161 located at the membrane-distal surface of KACL were defined as residues, decisively determining functional KACL-NKp65 interaction that is independent of KACL glycosylation.

Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Segment-2 sequencing and cross-neutralization studies confirm existence of a neutralization resistant VP2 phenotypic variant of bluetongue virus serotype 1 in India

Segment-2 (seg-2) of a bluetongue virus seropype-1 (BTV-1) isolate WGV104/08/Ind of Indian origin was sequenced and its neutralization behavior was studied to understand the antigenic similarity and relationship with other BTV-1 isolates. Multiple alignments of the coding region of seg-2 of WGV104/08/Ind revealed 97.6–99.0% and 97.2–98.4% similarity with other Indian BTV-1 isolates at nucleotide and deduced amino acid sequence level respectively. Several conservative and non-conservative substitutions were observed on the deduced VP2 amino acid sequence of WGV104/08/Ind. Non-conservative substitution of Lys119Glu on the B-cell epitope and Arg330Gly on the neutralizing epitope of VP2 of this isolate was observed. Using isolate-specific heterologous hyperimmune serum (HIS) the phenotypic antigenic relationship (r) was determined between WGV104/08/Ind and other Indian BTV-1 isolates which ranged from 0.092 to 0.208. The relationship score ranged from 0.203 to 0.295 when neutralization behavior of other Indian BTV-1 isolates was studied with the HIS of WGV104/08/Ind. Antigenic similarity (R) between WGV104/08/Ind and other Indian BTV-1 isolates was estimated from a reciprocal cross-neutralization study and ranged from 14.70% to 24.80% indicating existence of major subtype antigenic divergence and neutralization resistant behavior of WGV104/08/Ind.

Functional role of a polymorphism in the Pannexin1 gene in collagen-induced platelet aggregation.

Pannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific (10)Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1-400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1-400A allele. Conversely, the frequency of Panx1-400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1-400C channels compared with Panx1-400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1-400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1-400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

Mitochondrial ND3 G10398A mutation: a biomarker for breast cancer.

Mitochondrial DNA mutations have been found to play important roles in carcinogenesis. The most common G10398A mutation, a non-conservative amino acid substitution from Thr to Ala, seems to be involved in the tumorigenesis of breast cancer. Results from studies concerning this mutation remain inconclusive. In the current study, we first took clinical and molecular datasets from case-control studies to determine the association between the G10398A mutation and breast cancer. We further used the Phylotree to determine the haplogroups of this mutation. The frequencies of this mutation in 500 unrelated healthy controls were also screened. We found that this mutation is very common in the human population, and may be a polymorph.

Biochemical characterization of unusual meso-2,3-butanediol dehydrogenase from a strain of Bacillus subtilis

meso-2,3-Butanediol dehydrogenase (BDH) catalyzes the redox reaction between (R)-acetoin and meso-2,3-butanediol (meso-BD). The BDHs isolated from bacteria are generally characterized as homo-tetramers. In the present work, an isolated and identified high acetoin (AC)-yielding (41.63g/L) bacteria strain belonging to Bacillus subtilis but without 2,3-butanediol (BD) byproduct during its fermentation was selected. To understand the characteristics of BDH activity from this high AC-yielding strain, we cloned, purified and compared the BDH from Enterobacter aerogenes (CICC10293) (E. a-BDH) with the BDH from this high AC-yielding strain B. subtilis. Sequence alignments indicate a non-conservative amino acid substitution from Asp to Gly at site 194 on the B. s-BDH compared to that of E. a-BDH. Enzymatic analysis of E. a-BDH and D194G B. s-BDH shows D194G B. s-BDH has almost lost its entire enzymatic activity. Moreover, Isothermal titration calorimetry (ITC) measurements detected the substrate binding for the D194G B. s-BDH but no reaction was detected. Circular dicroism (CD) spectroscopy characterization revealed an identical secondary structure of E. a-BDH and D194G B. s-BDH. Remarkably, D194G B. s-BDH is highly susceptible to protease digestion, suggesting that the aspartic acid to glycine substitution might cause the proteolytic susceptibility of D194G B. s-BDH. Furthermore, by homology modeling with meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae (K. p-BDH) as a template, Gly194 seems to lose the hydrogen bond interactions with the surrounding residues (Gly206, Gly207 and Thr209), resulting in a putative conformational changes of D194G B. s-BDH which might be responsible for the loss of activity.

A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis

Defects in the completion of cell division by cytokinesis have long been proposed to foster carcinogenesis by engendering chromosome instability, but few tumor suppressor mechanisms controlling this process have so far been identified. Here, we identify a carboxyl (C)-terminal region of the high-mobility group protein HMG20b that is essential for cytokinesis, and report that it is inactivated by a cancer-associated mutation. We find that a C-terminal region of HMG20b spanning residues 173–317 is necessary and sufficient not only for its localization to cytokinetic structures, but also for its interaction with the tumor suppressor BRCA2, implicated in the abscission step of cytokinesis. Indeed, expression of this C-terminal HMG20b region suffices to restore cytokinesis in HMG20b-depleted cells. The non-conservative substitution of HMG20b residue Ala247 with Pro, reported in human lung cancer, disrupts these activities of HMG20b, impairing cytokinesis in a trans-dominant manner. Our findings provide fresh insight into the mechanism by which the HMG20b-BRCA2 complex controls mitotic cell division, and implicate heterozygous HMG20b mutations affecting cytokinesis regulation in the genesis of human cancers.

A novel variant in GABRB2 associated with intellectual disability and epilepsy.

The γ-aminobutyric acid type A (GABAA ) receptor is one of the three main classes of receptors activated by GABA, the principal inhibitory neurotransmitter in the central nervous system. Mutations in genes encoding various subunits of this receptor (GABRA1, GABRA2, GABRA4, GABRA5, GABRA6, GABRB1, GABRB3, GABRG1, GABRG2, GABRG3, and GABRD) are implicated in a number of neurological and developmental disorders, including epilepsy and autism. To date, no human genetics studies have implicated mutations in GABRB2, encoding the β2 subunit of the GABAA receptor, with neurodevelopmental disorders. Here we present a 12-year-old girl with intellectual disability and epilepsy, who was discovered by whole exome sequencing to have a de novo heterozygous missense variant in exon 4 of GABRB2 (c.236T>C; p.M79T). This variant is likely pathogenic, based on in silico analyses, as well as the fact that it results in the non-conservative substitution of a non-polar amino acid with a polar amino acid at a position that is evolutionarily conserved across multiple species. Our findings underscore the need for further investigation into the mechanisms by which mutations in GABRB2 contribute to neurological and developmental dysfunction.

Interplay between chaperones and protein disorder promotes the evolution of protein networks.

Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular context and integrated approaches for understanding proteome evolution. We feel that the development of λ may be a valuable addition to the toolbox applied to understand the molecular basis of evolution.

Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance.

Pgp (P-glycoprotein) is a prototype ABC (ATP-binding-cassette) transporter involved in multidrug resistance of cancer. We used directed evolution to replace six cytoplasmic Cys (cysteine) residues in Pgp with all 20 standard amino acids and selected for active mutants. From a pool of 75000 transformants for each block of three Cys, we identified multiple mutants that preserved drug resistance and yeast mating activity. The most frequent substitutions were glycine and serine for Cys427 (24 and 20%, respectively) and Cys1070 (37 and 25%) of the Walker A motifs in the NBDs (nucleotide-binding domains), Cys1223 in NBD2 (25 and 8%) and Cys638 in the linker region (24 and 16%), whereas close-by Cys669 tolerated glycine (16%) and alanine (14%), but not serine (absent). Cys1121 in NBD2 showed a clear preference for positively charged arginine (38%) suggesting a salt bridge with Glu269 in the ICL2 (intracellular loop 2) may stabilize domain interactions. In contrast, three Cys residues in transmembrane α-helices could be successfully replaced by alanine. The resulting CL (Cys-less) Pgp was fully active in yeast cells, and purified proteins displayed drug-stimulated ATPase activities indistinguishable from WT (wild-type) Pgp. Overall, directed evolution identified site-specific, non-conservative Cys substitutions that allowed building of a robust CL Pgp, an invaluable new tool for future functional and structural studies, and that may guide the construction of other CL proteins where alanine and serine have proven unsuccessful.

Association of the HOXB13 G84E mutation with increased risk for prostate cancer and other malignancies.

1558 Background: A rare non-conservative substitution (G84E) in the highly conserved MEIS-interacting domain of the homeobox transcription factor gene HOXB13 has been shown to be associated with an...

MMP13 mutations are the cause of recessive metaphyseal dysplasia, Spahr type

Metaphyseal dysplasia, Spahr type (MDST; OMIM 250400) was described in 1961 based on the observation of four children in one family who had rickets-like metaphyseal changes but normal blood chemistry and moderate short stature. Its molecular basis and nosologic status remained unknown. We followed up on those individuals and diagnosed the disorder in an additional member of the family. We used exome sequencing to ascertain the underlying mutation and explored its consequences on three-dimensional models of the affected protein. The MDST phenotype is associated with moderate short stature and knee pain in adults, while extra-skeletal complications are not observed. The sequencing showed that MDST segregated with a c.619T>G single nucleotide transversion in MMP13. The predicted non-conservative amino acid substitution, p.Trp207Gly, disrupts a crucial hydrogen bond in the calcium-binding region of the catalytic domain of the matrix metalloproteinase, MMP13. The MDST phenotype is associated with recessive MMP13 mutations, confirming the importance of this metalloproteinase in the metaphyseal growth plate. Dominant MMP13 mutations have been associated with metaphyseal anadysplasia (OMIM 602111), while a single child homozygous for a MMP13 mutation had been previously diagnosed as "recessive metaphyseal anadysplasia," that we conclude is the same nosologic entity as MDST. Molecular confirmation of MDST allows distinction of it from dominant conditions (e.g., metaphyseal dysplasia, Schmid type; OMIM # 156500) and from more severe multi-system conditions (such as cartilage-hair hypoplasia; OMIM # 250250) and to give precise recurrence risks and prognosis.

The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells

Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.

Comparative Structural and Functional Analysis of Orthomyxovirus Polymerase Cap-Snatching Domains

Orthomyxovirus Influenza A virus (IAV) heterotrimeric polymerase performs transcription of viral mRNAs by cap-snatching, which involves generation of capped primers by host pre-mRNA binding via the PB2 subunit cap-binding site and cleavage 10–13 nucleotides from the 5′ cap by the PA subunit endonuclease. Thogotoviruses, tick-borne orthomyxoviruses that includes Thogoto (THOV), Dhori (DHOV) and Jos (JOSV) viruses, are thought to perform cap-snatching by cleaving directly after the cap and thus have no heterogeneous, host-derived sequences at the 5′ extremity of their mRNAs. Based on recent work identifying the cap-binding and endonuclease domains in IAV polymerase, we determined the crystal structures of two THOV PB2 domains, the putative cap-binding and the so-called ‘627-domain’, and the structures of the putative endonuclease domains (PA-Nter) of THOV and DHOV. Despite low sequence similarity, corresponding domains have the same fold confirming the overall architectural similarity of orthomyxovirus polymerases. However the putative Thogotovirus cap-snatching domains in PA and PB2 have non-conservative substitutions of key active site residues. Biochemical analysis confirms that, unlike the IAV domains, the THOV and DHOV PA-Nter domains do not bind divalent cations and have no endonuclease activity and the THOV central PB2 domain does not bind cap analogues. On the other hand, sequence analysis suggests that other, non-influenza, orthomyxoviruses, such as salmon anemia virus (isavirus) and Quaranfil virus likely conserve active cap-snatching domains correlating with the reported occurrence of heterogeneous, host-derived sequences at the 5′ end of the mRNAs of these viruses. These results highlight the unusual nature of transcription initiation by Thogotoviruses.

Kinesin-5 Motility is Regulated by the Residue Chemistry of Loop-5

Kinesin-5 drives separation of microtubules and organizes the mitotic spindle via its motor domains. More than one component within Kinesin-5 has been implicated in its motile ability; these are Loop-5, the necklinker and the cover neck. In all studies, measured changes in structure, or their implied dynamic motion, were deemed key to the role of these components. Unaddressed is whether these protein components chemically direct mechanical output. Herein, we show that specific side-chain chemistry of Loop-5 must be conserved for productive Kinesin-5 motility. Six substitutions of Loop-5 residues were created in the Eg5 dimer by mutagenesis, and their microtubule-gliding velocities were measured. Mutant Eg5 gliding velocities ranged from nearly equivalent to wildtype to a complete loss of motility. There were key differences between the mutations in the N- versus C-terminus of Loop-5. Substitutions near the N-terminus retained the ability to glide microtubules; alteration of gliding velocity paralleled changes in microtubule-stimulated catalytic rates. In contrast, nonconservative substitutions near the C-terminus of Loop-5 bound microtubules in rigor despite having robust ATPase activity. Collectively, these results suggest the integrity of the active site remains intact and communication between the active site and microtubule site is not compromised. Mechanical output is challenged, however. Therefore, we conclude that side-chain interactions of these C-terminal residues with the surrounding protein matrix are required for the terminal step in Eg5 mechanotransduction. Given our kinetic data, we speculate that aberrant interactions may result in changes in force and/or coordination of the two motor domains in the dimer, rendering Eg5 incapable of motility. This work is funded by the support of the National Institutes of Health (R01 GM097350 S.K.;P20GM103424 and 5G12RR026260 T.H.) and the LSU School of Graduate Studies (R.B.).