SUMO Suppresses the Activity of the Jasmonic Acid Receptor CORONATINE INSENSITIVE1.

Plants respond rapidly to sudden environmental cues, often responding prior to changes in the hormone levels that coordinate these responses. How this is achieved is not fully understood. The integrative role of the phytohormone jasmonic acid (JA) relies upon the plant's ability to control the levels of JASMONATE ZIM (JAZ) domain-containing repressor proteins. Here, we demonstrate that regardless of intrinsic JA levels, Small Ubiquitin-like Modifier (SUMO)-conjugated JAZ proteins inhibit the JA receptor CORONATINE INSENSITIVE1 (COI1) from mediating non-SUMOylated JAZ degradation. The SUMO-deconjugating proteases OVERLY TOLERANT TO SALT1 (OTS1) and OTS2 regulate JAZ protein SUMOylation and stability. The ots1 ots2 double mutants accumulate SUMOylated and non-SUMOylated JAZ repressor proteins but show no change in endogenous JA levels compared with wild-type plants. SUMO1-conjugated JAZ proteins bind to COI1 independently of the JA mimic coronatine. SUMO inhibits JAZ binding to COI1. We identify the SUMO interacting motif in COI1 and demonstrate that this is vital to SUMO-dependent inhibition of COI1. Necrotroph infection of Arabidopsis thaliana promotes SUMO protease degradation, and this increases JAZ SUMOylation and abundance, which in turn inhibits JA signaling. This study reveals a mechanism for rapidly regulating JA responses, allowing plants to adapt to environmental changes.

Structural Basis of Oligomerization of N-Terminal Domain of Spider Aciniform Silk Protein.

Spider silk is self-assembled from water-soluble silk proteins through changes in the environment, including pH, salt concentrations, and shear force. The N-terminal domains of major and minor ampullate silk proteins have been found to play an important role in the assembly process through salt- and pH-dependent dimerization. Here, we identified the sequences of the N-terminal domains of aciniform silk protein (AcSpN) and major ampullate silk protein (MaSpN) from Nephila antipodiana (NA). Different from MaSpN, our biophysical characterization indicated that AcSpN assembles to form large oligomers, instead of a dimer, upon condition changes from neutral to acidic pH and/or from a high to low salt concentration. Our structural studies, by nuclear magnetic resonance spectroscopy and homology modelling, revealed that AcSpN and MaSpN monomers adopt similar overall structures, but have very different charge distributions contributing to the differential self-association features. The intermolecular interaction interfaces for AcSp oligomers were identified using hydrogen-deuterium exchange mass spectrometry and mutagenesis. On the basis of the monomeric structure and identified interfaces, the oligomeric structures of AcSpN were modelled. The structural information obtained will facilitate an understanding of silk fiber formation mechanisms for aciniform silk protein.

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Channeling of cAMP in PDE-PKA Complexes Promotes Signal Adaptation.

Spatiotemporal control of the cAMP signaling pathway is governed by both hormonal stimulation of cAMP generation by adenylyl cyclases (activation phase) and cAMP hydrolysis by phosphodiesterases (PDEs) (termination phase). The termination phase is initiated by PDEs actively targeting the protein kinase A (PKA) R-subunit through formation of a PDE-PKAR-cyclic adenosine monophosphate (cAMP) complex (the termination complex). Our results using PDE8 as a model PDE, reveal that PDEs mediate active hydrolysis of cAMP bound to its receptor RIα by enhancing the enzymatic activity. This accelerated cAMP turnover occurs via formation of a stable PDE8-RIα complex, where the protein-protein interface forms peripheral contacts and the central ligand cements this ternary interaction. The basis for enhanced catalysis is active translocation of cAMP from its binding site on RIα to the hydrolysis site on PDE8 through direct "channeling." Our results reveal cAMP channeling in the PDE8-RIα complex and a molecular description of how this channel facilitates processive hydrolysis of unbound cAMP. Thus, unbound cAMP maintains the PDE8-RIα complex while being hydrolyzed, revealing an undiscovered mode for amplification of PKA activity by cAMP-mediated sequestration of the R-subunit by PDEs. This novel regulatory mode explains the paradox of cAMP signal amplification by accelerated PDE-mediated cAMP turnover. This highlights how target effector proteins of small-molecule ligands can promote enzyme-mediated ligand hydrolysis by scaffolding effects. Enhanced activity of the PDE8-RIα complex facilitates robust desensitization, allowing the cell to respond to dynamic levels of cAMP rather than steady-state levels. The PDE8-RIα complex represents a new class of PDE-based complexes for specific drug discovery targeting the cAMP signaling pathway.

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Secreted M-ficolin anchors onto monocyte transmembrane G protein-coupled receptor 43 and cross talks with plasma C-reactive protein to mediate immune signaling and regulate host defense.

Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin-GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin-GPCR43 complex. The collaboration among CRP-M-ficolin-GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin-CRP provides a powerful template for future design of potential immunomodulators.

Ligand-induced shifts in conformational ensembles that describe transcriptional activation.

Nuclear receptors function as ligand-regulated transcription factors whose ability to regulate diverse physiological processes is closely linked with conformational changes induced upon ligand binding. Understanding how conformational populations of nuclear receptors are shifted by various ligands could illuminate strategies for the design of synthetic modulators to regulate specific transcriptional programs. Here, we investigate ligand-induced conformational changes using a reconstructed, ancestral nuclear receptor. By making substitutions at a key position, we engineer receptor variants with altered ligand specificities. We combine cellular and biophysical experiments to characterize transcriptional activity, as well as elucidate mechanisms underlying altered transcription in receptor variants. We then use atomistic molecular dynamics (MD) simulations with enhanced sampling to generate ensembles of wildtype and engineered receptors in combination with multiple ligands, followed by conformational analysis and correlation of MD-based predictions with functional ligand profiles. We determine that conformational ensembles accurately describe ligand responses based on observed population shifts. These studies provide a platform which will allow structural characterization of physiologically-relevant conformational ensembles, as well as provide the ability to design and predict transcriptional responses in novel ligands.

Disrupting enzyme fluidity.

A combination of X-ray crystallography, NMR, and mass spectrometry has revealed how diverse small-molecule inhibitors bind Bruton's tyrosine kinase and alter the conformation of this enzyme.

Botanical Medicine and Natural Products Used for Erectile Dysfunction.

INTRODUCTION: Erectile dysfunction is the persistent or recurrent inability to achieve or maintain an erection sufficient for intercourse. Despite various treatment options, not all patients respond adequately and their usefulness is limited by adverse effects and cost. Botanical medicine and natural products have been and continue to be invaluable and untapped sources of new drugs, including potentially those to treat erectile dysfunction. OBJECTIVES: To review the current literature on botanical medicine traditionally used as aphrodisiacs and treatment of erectile dysfunction, in particular, scientific and clinical investigations that have been performed, possible active phytoconstituents, and mechanisms of action and to identify gaps in current knowledge to better guide future research efforts. METHODS: A comprehensive literature search was conducted via PubMed, Scopus, Science Direct, and Web of Science on English publications, using various keywords, for example, "herb", "natural product", combined with "erectile dysfunction", "aphrodisiac", and "sexual performance". RESULTS: 369 relevant articles studying medicinal plants used for erectile dysfunction were analyzed. A total of 718 plants from 145 families and 499 genera were reported to be used traditionally as aphrodisiacs and treatment of erectile dysfunction. Top plants used include Pausinystalia johimbe, Lepidium meyenii, and Panax ginseng. Different plant parts are used, with roots being the most common. Less than half of these plants have been evaluated scientifically, using various research methodologies. Clinical trials conducted were collated. Current scientific investigation shows mixed results about their usefulness in enhancing sexual performance. A limited number of studies have attempted to elucidate the mechanisms of action of these medicinal plants. CONCLUSION: A comprehensive literature review on botanical medicine and natural products used for treatment of erectile dysfunction was successfully conducted. Although medicinal plants serve as a potential source of lead compounds for erectile dysfunction drugs, further studies are warranted to further evaluate their efficacy and safety. SinVJ-E, Anand GS, Koh H-L. Botanical Medicine and Natural Products Used for Erectile Dysfunction. Sex Med Rev 2021;9:568-592.

Uncovering metastability and disassembly hotspots in whole viral particles.

Viruses are metastable macromolecular assemblies that toggle between multiple conformational states through molecular rearrangements that are critical for mediating viral host entry. Viruses respond to different host specific environmental cues to form disassembly intermediates for the eventual release of genomic material required for replication. Although static snapshots of these intermediates have been captured through structural techniques such as X-ray crystallography and cryo-EM, the mechanistic details of these conformational rearrangements underpinning viral metastability have been poorly understood. Amide hydrogen deuterium exchange mass spectrometry (HDXMS) is a powerful tool that measures hydrogen bonding propensities to probe changes in the dynamics of different macromolecular interactions. Chaotropic agents such as urea can be used to disrupt hydrogen bonds between different subunits, thereby ranking regions of the virus that are critical in maintaining viral stability. By controlled urea denaturation with HDXMS, we have identified specific loci in a Turnip Crinkle Virus (TCV) model showing increased deuterium exchange with even minimally disruptive concentrations of urea. These loci represent dynamic disassembly hotspots. These hotspots are predominantly present at the quaternary contacts at the 3-fold and 5-fold axes. This approach can be applied to detect vulnerabilities in virus icosahedral structures to uncover the molecular mechanism of viral disassembly.

Biophysical characterization of anticoagulant hemextin AB complex from the venom of snake Hemachatus haemachatus.

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of beta-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.

Ligand-mediated changes in conformational dynamics of NpmA: implications for ribosomal interactions.

Aminoglycosides are broad-spectrum antibiotics that bind to the 30S ribosomal subunit (30S) of bacteria and disrupt protein translation. NpmA, a structurally well-characterized methyltransferase identified in an E. coli clinical isolate, catalyzes methylation of 30S at A1408 of the 16S rRNA and confers aminoglycoside resistance. Using sucrose cushion centrifugation and isothermal titration calorimetry, we first confirmed the binding between NpmA and 30S. Next, we performed amide Hydrogen/Deuterium Exchange Mass Spectrometry (HDXMS) of apo NpmA and in the presence and absence of SAM/SAH. We observed that ligand binding resulted in time-dependent differences in deuterium exchange not only at the ligand-binding pocket (D25-D55 and A86-E112) but also in distal regions (F62-F82 and Y113-S144) of NpmA. These results provide insights into methylation group donor cofactor-mediated allostery in NpmA in the ligand-bound states, which could not be observed in the static endpoint crystal structures. We predict that the two distal sites in NpmA form part of the allosteric sites that importantly are part of the main 16S rRNA binding interface. Thus HDXMS helped uncover allosteric communication relays that couple SAM/SAH binding sites with the ribosome-binding site. This highlights how HDXMS together with X-ray crystallography can provide important allosteric insights in protein-ligand complexes.

ß-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase.

The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, ß and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the ß-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the ßCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.

Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2.

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

Heart extracellular matrix supports cardiomyocyte differentiation of mouse embryonic stem cells.

We have evaluated the effect of heart extracellular matrix (ECM) on the cardiomyocyte differentiation of mouse embryonic stem cells (ES cells) using de-cellularized heart tissue. Several lines of evidence indicate that ECM plays significant roles in cell proliferation, cell death and differentiation, but role of ECM possessing a 3D structure in differentiation has not been studied in detail. We found that there are substantial differences in the quantitative protein profiles of ECM in SDS-treated heart tissue compared to that of liver tissue, as assessed by iTRAQ™ quantitative proteomics analysis. When mouse ES cells were cultured on thin (60 µm) sections of de-cellularized tissue, the expression of cardiac myosin heavy chain (cMHC) and cardiac troponin I (cTnI) was high in ES cells cultured on heart ECM compared with those cultured on liver ECM. In addition, the protein expression of cMHC and cTnI was detected in cells on heart ECM after 2 weeks, which was not detectable in cells on liver ECM. These results indicate that heart ECM plays a critical role in the cardiomyocyte differentiation of ES cells. We propose that tissue-specific ECM induced cell lineage specification through mechano-transduction mediated by the structure, elasticity and components of ECM.

Interaction of Mycobacterium tuberculosis RshA and SigH is mediated by salt bridges.

The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1-25, 58-69, 90-111, 115-132 and 157-196 and residues 35-57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.

Pathophysiological condition changes the conformation of a flexible FBG-related protein, switching it from pathogen-recognition to host-interaction.

Although homeostatic disturbance of the blood pH and calcium in the vicinity of tissue injury/malignancy/local infection seems subtle, it can cause substantial pathophysiological consequences, a phenomenon which has remained largely unexplored. The fibrinogen-related proteins (FREPs) containing fibrinogen-like domain (FBG) represent a conserved protein family with a common calcium-binding region, implying the presence of elements responsive to physiological perturbation. Here, we studied the molecular interaction between a representative FREP, the M-ficolin, and an acute phase blood protein, the C-reactive protein (CRP), both of which are known to trigger and control seminal pathways in infection and injury. Using hydrogen-deuterium exchange mass spectrometry, we showed that the C-terminal region of M-ficolin FBG underwent dramatic conformational change upon pH and calcium perturbations. Biochemical and biophysical assays showed that under defined pathophysiological condition (pH 6.5, 2.0 mM calcium), the FBG:CRP interaction occurred more strongly compared to that under physiological condition (pH 7.4, 2.5 mM calcium). We identified the binding interface between CRP and FBG, locating it to the pH- and calcium-sensitive C-terminal region of FBG. By site-directed mutagenesis, we determined H284 in the N-acetylglucosamine (GlcNAc)-binding pocket of the FBG, to be the critical CRP-binding residue. This conformational switch involving H284, explains how the pathophysiologically-driven FBG:CRP interaction diverts the M-ficolin away from GlcNAc/pathogen-recognition to host protein-protein interaction, thus enabling the host to regain homeostatic control. Our elucidation of the binding interface at the flexible FBG domain provides insights into the bioactive centre of the M-ficolin, and possibly other FREPs, which might aid future development of immunomodulators.