Nucleotide Binding Region Research Articles

NLRP9 involved in antiviral innate immunity via binding VIM in IPEC-J2 cells

BackgroundNucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) was the first nucleotide-binding region receptor (NLR) proposed to be expressed and function only in the reproductive system. Recent evidence suggests that NLRP9 is also capable of playing a role in infectious and inflammatory diseases. Results and conclusionsIn this study, we examined the expression of NLRP9 in various tissues of piglets and IPEC-J2 cells. The results showed that high expression of NLRP9 mRNA and protein were detected in both intestine of piglets and IPEC-J2 cells. Both LPS and poly I:C significantly up-regulated NLRP9 protein levels in the IPEC-J2 cells. Besides, poly I:C upregulated the level of transcriptional elements NF-κB, IRF3, IRF7, ISG15, ISG56, OAS1, and IFNB1. Furthermore, interference with the NLRP9 gene in the presence of poly I:C strongly downregulated the expression of all the above genes. Moreover, we demonstrated for the first time that NLRP9 acts in combination with VIM (Vimentin). These results suggested that NLRP9 may participate in the antiviral innate immune by binding to VIM in the porcine intestine. The findings provide preliminary insights into the molecular mechanisms involved in the regulation of mucosal immunity in the porcine intestine by NLRP9.

Effector-mediated partial and nonhost disease resistance in wheat.

Effector-mediated partial and nonhost disease resistance in wheat.

Barley Ror1 encodes a class XI myosin required for mlo-based broad-spectrum resistance to the fungal powdery mildew pathogen.

Loss-of-function alleles of plant MLO genes confer broad-spectrum resistance to powdery mildews in many eudicot and monocot species. Although barley (Hordeum vulgare) mlo mutants have been used in agriculture for more than 40 years, understanding of the molecular principles underlying this type of disease resistance remains fragmentary. Forward genetic screens in barley have revealed mutations in two Required for mlo resistance (Ror) genes that partially impair immunity conferred by mlo mutants. While Ror2 encodes a soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE), the identity of Ror1, located at the pericentromeric region of barley chromosome 1H, remained elusive. We report the identification of Ror1 based on combined barley genomic sequence information and transcriptomic data from ror1 mutant plants. Ror1 encodes the barley class XI myosin Myo11A (HORVU.MOREX.r3.1HG0046420). Single amino acid substitutions of this myosin, deduced from non-functional ror1 mutant alleles, map to the nucleotide-binding region and the interface between the relay-helix and the converter domain of the motor protein. Ror1 myosin accumulates transiently in the course of powdery mildew infection. Functional fluorophore-labeled Ror1 variants associate with mobile intracellular compartments that partially colocalize with peroxisomes. Single-cell expression of the Ror1 tail region causes a dominant-negative effect that phenocopies ror1 loss-of-function mutants. We define a myosin motor for the establishment of mlo-mediated resistance, suggesting that motor protein-driven intracellular transport processes are critical for extracellular immunity, possibly through the targeted transfer of antifungal and/or cell wall cargoes to pathogen contact sites.

The to and fro of Rho

The functions of Ras members are largely governed by the structural dynamics of the nucleotide-binding switch regions. In this issue of Structure, Lin etal. (2021) take a close look at the dynamic equilibrium of RhoA, the founding member of the Rho family of Ras GTPases.

Thalidomide may be an effective medicine for Blau Syndrome

nucleotide-binding domain region of the NOD2/CARD15 gene. We reported the case of a patient with a typical triad of rash, arthritis, and uveitis. Sanger sequencing indicated a NOD2 missense variant (c.1759 C >T, p.R587C) in the patient that was inherited from the mother. After treatment with methotrexate, a TNF-α inhibitor, and corticosteroids, the patient’s clinical symptoms and inflammatory indicators remained uncontrolled. In the meantime, the patient experienced multiple side effects, such as hypertension and growth retardation attributed to prolonged use of corticosteroids. After treatment with thalidomide, the condition was controlled without recurrence or side effects, and corticosteroids were stopped as soon as possible. This report suggests that thalidomide may be an effective drug for BS treatment.

Modulation of post-powerstroke dynamics in myosin II by 2′-deoxy-ADP

Muscle myosins are molecular motors that hydrolyze ATP and generate force through coordinated interactions with actin filaments, known as cross-bridge cycling. During the cross-bridge cycle, functional sites in myosin ‘sense’ changes in interactions with actin filaments and the nucleotide binding region, resulting in allosteric transmission of information throughout the structure. We investigated whether the dynamics of the post-powerstroke state of the cross-bridge cycle are modulated in a nucleotide-dependent fashion. We compared molecular dynamics simulations of the myosin II motor domain (M) from Dictyostelium discoideum in the presence of ADP (M.ADP) versus 2′-deoxy-ADP bound myosin (M.dADP). We found that dADP was more flexible than ADP and the two nucleotides interacted with myosin in different ways. Replacement of ADP with dADP in the post-powerstroke state also altered the conformation of the actin binding region in myosin heads. Our results provide atomic level insights into allosteric communication networks in myosin that provide insight into the nucleotide-dependent dynamics of the cross-bridge cycle.

FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms

SUMMARYMany chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the Gα C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal α5 helix before allowing GTP-gated release of Gα from Ric-8A.

Dynamic View of Allosteric Regulation in the Hsp70 Chaperones by J-Domain Cochaperone and Post-Translational Modifications: Computational Analysis of Hsp70 Mechanisms by Exploring Conformational Landscapes and Residue Interaction Networks

Structural and biochemical studies of Hsp70 chaperones have provided a molecular view of the chaperone biochemical cycle by revealing a complex interplay between allosteric conformational states that controls the feedback loop between stimulation of the adenosinetriphosphatase (ATPase) activity and the substrate release. Allosteric regulation in the Hsp70 chaperones and efficient substrate targeting are mediated by J-domain cochaperones through a dynamic interaction network controlled by the regulatory hotspots. In the current work, we have simulated conformational landscapes and residue interaction networks in the open, closed, and cochaperone-bound DnaK structures. The results of this work have shown that J-domain can selectively enhance direction-specific signal propagation from the substrate-binding domain to the catalytic center and promote the structural environment required for ATP hydrolysis. By employing different network-based approaches, we examined the role and contribution of post-translational modification sites in allosteric regulation of human Hsp70. The central finding of this analysis indicated that conserved phosphorylation sites localized preferentially in the nucleotide-binding domain regions are often aligned with the allosteric control points and serve as effector centers in Hsp70. We have found that cooperation of post-translational modifications sites is based on the governing role of phosphorylation sites in dictating regulatory switching functions, whereas the bulk of acetylation sites can be involved in sensing the long-range signals and executing allosteric changes during the ATPase cycle. The results of this study highlight the important role of phosphorylation sites in exerting control over allosteric changes in Hsp70. The network-centric framework for the analysis of conformational dynamics and chaperone landscapes can explain a range of structural and functional experiments, providing a robust dynamic model of Hsp70 regulation by cochaperones and sites of post-translational modifications.

Stronger When Together: Clustering of Plant NLR Disease resistance Genes.

Gene clustering is rare in eukaryotes. However, nucleotide-binding leucine-rich repeat (NLR)-encoding disease resistance (R) genes show consistent clustering in plant genomes. These arrangements are likely to provide coregulatory benefits, as suggested by growing evidence that the gene products of both paired and larger clusters of NLRs act together in triggering immunity. Head-to-head gene pairs where one of the encoded NLRs includes an integrated decoy domain appear to behave differently than clusters evolved from closely related typical NLRs. These patterns may help to explain the broad resistance that most plants have despite their finite number of R genes. By taking into consideration the relationship between genomic arrangement and function, we can improve our understanding of and ability to predict plant immune detection.

Characterization of gonadotropin releasing hormone receptor gene in Sokoto and Kalahari Red goats

The objective of this study was to characterize GnRHR gene in Sokoto (n = 70) and Kalahari Red (n = 70) goats. Three SNPs, (g.-29T > G, g.48 G > A and g.209 T > G), were detected in Sokoto Red (SR) and one (g.48 G > A) in Kalahari Red (KR) goats. All the mutations occurred within the 5′UTR and Exon one of the gene and the g.209 T > G was non-synonymous and, therefore, resulted in an amino acid change from methionine to arginine at Position 70 of the GnRHR polypeptide. The homozygous mutant genotypes at the three SNP loci were not detected in both breeds but minor allele frequencies were ≥ 0.1 for the three SNP loci in SR goats. Frequency of the T allele, however, was 0.93 at the only SNP locus detected in KR goats. There was a strong linkage disequilibrium (LD; r2>0.98) among the detected mutations in SR goats resulting in two haplotypes (T-G-T and G-A-G) with a frequency of 86% and 13%, respectively. There was no significant association between genotypes at the polymorphic loci and litter size (P > 0.05) in the two breeds. The non-synonymous mutation (g.209T>G) appears to have changed the nucleotide binding region and area spanning exposed/buried regions on the predicted secondary structure of the two variants of the receptor. This change led to variation in the tertiary structure between the two variants of the receptor and can influence the function of the transmembrane receptor. Comparison of the GnRH receptor domains for goats, sheep, cattle and swine confirmed that the seven transmembrane domains of the receptor are conserved in all the farm animals considered.

A novel ABCC6 variant causative of pseudoxanthoma elasticum

Pseudoxanthoma elasticum is an autosomal recessive heritable disorder caused by mutations in ABCC6. We describe two siblings showing typical skin lesions and a clinical diagnosis of pseudoxanthoma elasticum. Genetic analysis of ABCC6 revealed a novel homozygous c.4041G > A variant located in the last position of exon 28 that compromises the splicing donor site, resulting in a shorter messenger RNA. The deletion impairs the nucleotide-binding fold region, which is crucial for ABCC6 function.

Hepatic protein Carbonylation profiles induced by lipid accumulation and oxidative stress for investigating cellular response to non-alcoholic fatty liver disease in vitro

BackgroundNon-alcoholic fatty liver disease (NAFLD) is caused by excessive accumulation of fat within the liver, leading to further severe conditions such as non-alcoholic steatohepatitis (NASH). Progression of healthy liver to steatosis and NASH is not yet fully understood in terms of process and response. Hepatic oxidative stress is believed to be one of the factors driving steatosis to NASH. Oxidative protein modification is the major cause of protein functional impairment in which alteration of key hepatic enzymes is likely to be a crucial factor for NAFLD biology. In the present study, we aimed to discover carbonylated protein profiles involving in NAFLD biology in vitro.MethodsHepatocyte cell line was used to induce steatosis with fatty acids (FA) in the presence and absence of menadione (oxidative stress inducer). Two-dimensional gel electrophoresis-based proteomics and dinitrophenyl hydrazine derivatization technique were used to identify carbonylated proteins. Sequentially, in order to view changes in protein carbonylation pathway, enrichment using Funrich algorithm was performed. The selected carbonylated proteins were validated with western blot and carbonylated sites were further identified by high-resolution LC-MS/MS.ResultsProteomic results and pathway analysis revealed that carbonylated proteins are involved in NASH pathogenesis pathways in which most of them play important roles in energy metabolisms. Particularly, carbonylation level of ATP synthase subunit α (ATP5A), a key protein in cellular respiration, was reduced after FA and FA with oxidative stress treatment, whereas its expression was not altered. Carbonylated sites on this protein were identified and it was revealed that these sites are located in nucleotide binding region. Modification of these sites may, therefore, disturb ATP5A activity. As a consequence, the lower carbonylation level on ATP5A after FA treatment solely or with oxidative stress can increase ATP production.ConclusionsThe reduction in carbonylated level of ATP5A might occur to generate more energy in response to pathological conditions, in our case, fat accumulation and oxidative stress in hepatocytes. This would imply the association between protein carbonylation and molecular response to development of steatosis and NASH.

A lipid gating mechanism for the channel-forming O antigen ABC transporter

Extracellular glycan biosynthesis is a widespread microbial protection mechanism. In Gram-negative bacteria, the O antigen polysaccharide represents the variable region of outer membrane lipopolysaccharides. Fully assembled lipid-linked O antigens are translocated across the inner membrane by the WzmWzt ABC transporter for ligation to the lipopolysaccharide core, with the transporter forming a continuous transmembrane channel in a nucleotide-free state. Here, we report its structure in an ATP-bound conformation. Large structural changes within the nucleotide-binding and transmembrane regions push conserved hydrophobic residues at the substrate entry site towards the periplasm and provide a model for polysaccharide translocation. With ATP bound, the transporter forms a large transmembrane channel with openings toward the membrane and periplasm. The channel’s periplasmic exit is sealed by detergent molecules that block solvent permeation. Molecular dynamics simulation data suggest that, in a biological membrane, lipid molecules occupy this periplasmic exit and prevent water flux in the transporter’s resting state.

Impact of Human Beta-Cardiac Myosin Mutation Implicated in Both Hypertrophic and Dilated Cardiomyopathy

Hypertrophic (HCM) and dilated cardiomyopathy (DCM) are autosomal dominant diseases in which affected individuals are heterozygous for gene mutations in sarcomeric proteins, including beta cardiac myosin. HCM is characterized by left ventricular (LV) hypertrophy, myocyte disarray, and LV hypercontraction. DCM is indicated by LV dilation with impaired contraction of the left or both ventricles. There is evidence to suggest that mutations which increase myosin contractility cause HCM, and mutations that reduce myosin contractility cause DCM. We examined a missense mutation (R243H) in the switch 1 nucleotide-sensing region of the human beta-myosin heavy chain (β-MYHC, MYH7) implicated in both HCM and DCM. R243 is highly conserved in myosins and participates in a salt-bridge between the switch I and switch II nucleotide binding regions. We observed very minimal actin-activated ATPase activity for the mutant compared to WT (2-fold compared to 100-fold activation, respectively), while the basal ATPase was unchanged. R243H bound to actin in an ATP-dependent manner based on actin-cosedimentation assays. We also observed no actin-sliding by R243H in the in vitro motility assay. In the mixed motility assay, low ratios (≤50%) of R243H did not change or only slightly decreased sliding velocities. At higher ratios of R243H (≥50%) the velocity decreased dramatically. We speculate that at low ratios of R243H/WT the mutant myosin may cooperatively activate the actin thin filaments to allow more opportunity for WT myosin heads to bind actin without slowing shortening velocity, which would correlate with HCM. At higher ratios of R243/WT, the inhibitory action of R243H may dominate and slow muscle shortening, which would correlate with DCM.

An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1)

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.

Mutations in the Converter Domain of Myosin V Demonstrate Coupling Between Lever Arm Swing and Phosphate Release

Myosin V is an actin based molecular motor that utilizes ATP to convert chemical energy into mechanical work. The converter domain plays a crucial role in the allosteric communication between the lever arm and the actin- and nucleotide-binding regions. Interestingly, mutations in the converter domain of human beta cardiac myosin are associated with hypertrophic and dilated cardiac myopathies. Thus, we have made the corresponding mutations, R712G and F750L, in the converter domain of myosin V to investigate the effect of these mutations on the myosin kinetics, structural dynamics, and motor properties. The R712G and F750L mutations did not change the actin concentration at which the ATPase activity is one-half maximal (KATPase) and the maximum ATPase rate was increased 15% in the F750L mutant but unchanged in the R712G mutant. In vitro motility assays revealed sliding velocities similar to WT for both R712G and F750L. Actin-activated phosphate release was increased (43%) in the R712G mutant, while the actin-dependence of phosphate release was disrupted in the F750L mutant. We followed the motion of the lever-arm during the ATP binding (recovery stroke) and actin-activated product release (power stroke) steps using stopped-flow FRET. The R712G mutation reduced the rate of the recovery stroke by 30% while the power stroke was increased by 60%, whereas the F750L mutation had the opposite effect on the recovery and power strokes. Mechanosensitivity studies demonstrated that both mutants are more sensitive to the addition of load in the in vitro motility assay. Our results provide direct evidence that the converter domain is crucial for lever arm rotation and we provide further evidence that the lever arm gates phosphate release.

Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange.

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to a lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-RasG12C. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent binder that modifies the inactive, GDP-bound state of K-RasG12C. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these new compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.

Molecular cloning and tissue distribution of a novel marmoset ABC transporter.

Common marmosets (Callithrix jacchus) have been recognized as a useful small non-human primate model in preclinical testing for drug development. In this study, a cDNA of novel ATP-dependent efflux transporter ABCB1 was cloned from marmoset liver tissue. Marmoset ABCB1 cDNA encodes a protein of 1279 amino acid residues (MW=141.4kDa) containing characteristic regions of an ATP-binding cassette (ABC) protein, two hydrophobic transmembrane regions and two cytoplasmic nucleotide-binding regions, similar to human ABCB1. The deduced amino acid sequences were more highly identical (95%) to those of human ABCB1 compared with non-primate species such as dogs, pigs and rodents (79-90%). A close evolutionary relationship of ABCB1 among marmosets, cynomolgus and rhesus monkeys and humans was evident from a phylogenetic analysis using ABCB1 amino acid sequences from primates, dogs, pigs and rodents. Tissue distribution analyses by quantitative reverse transcription-polymerase chain reaction indicated that marmoset ABCB1 mRNA was most abundant in kidneys, followed by small intestines and livers, similar to human ABCB1, and marmoset ABCB1 proteins in these tissues were also detected by immunoblotting. These results indicated that the primary structure and tissue distribution of ABCB1 in marmosets were similar to those of humans, suggesting similar molecular characteristics of ABCB1 between marmosets and humans.

Lessons in Effector and NLR Biology of Plant-Microbe Systems.

A diversity of plant-associated organisms secrete effectors-proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat region (NLR)-containing proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.