ResearchPad - solid-state-physics https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Crystal structure of <i>Thermus thermophilus</i> methylenetetrahydrofolate dehydrogenase and determinants of thermostability]]> https://www.researchpad.co/article/elastic_article_13865 The elucidation of mechanisms behind the thermostability of proteins is extremely important both from the theoretical and applied perspective. Here we report the crystal structure of methylenetetrahydrofolate dehydrogenase (MTHFD) from Thermus thermophilus HB8, a thermophilic model organism. Molecular dynamics trajectory analysis of this protein at different temperatures (303 K, 333 K and 363 K) was compared with homologous proteins from the less temperature resistant organism Thermoplasma acidophilum and the mesophilic organism Acinetobacter baumannii using several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis. These methods enabled the determination of important residues for the thermostability of this enzyme. The description of rotamer distributions by Gini coefficients and Kullback–Leibler (KL) divergence both revealed significant correlations with temperature. The emerging view seems to indicate that a static salt bridge/charged residue network plays a fundamental role in the temperature resistance of Thermus thermophilus MTHFD by enhancing both electrostatic interactions and entropic energy dispersion. Furthermore, this analysis uncovered a relationship between residue mutations and evolutionary pressure acting on thermophilic organisms and thus could be of use for the design of future thermostable enzymes.

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<![CDATA[ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient <i>hsdRMS</i> restriction system broadening conjugation host range]]> https://www.researchpad.co/article/elastic_article_7739 Horizontal gene transfer is the main mechanism by which bacteria acquire and disseminate new traits, such as antibiotic resistance genes, that allow adaptation and evolution. Here we identified a gene, ardC, that enables a plasmid to increase its conjugative host range, and thus positively contributes to plasmid fitness. The crystal structure of the antirestriction protein ArdC revealed a fold different from other antirestriction proteins. Our results have wide implications for understanding how a gene enlarges the environments a plasmid can colonize and point to new targets to harness the bacterial DNA uptake control.

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<![CDATA[Crystal structures of Triosephosphate Isomerases from Taenia solium and Schistosoma mansoni provide insights for vaccine rationale and drug design against helminth parasites]]> https://www.researchpad.co/article/N340e3046-cb91-4c84-8d1b-fb2a65cf4cdb

Triosephosphate isomerases (TPIs) from Taenia solium (TsTPI) and Schistosoma mansoni (SmTPI) are potential vaccine and drug targets against cysticercosis and schistosomiasis, respectively. This is due to the dependence of parasitic helminths on glycolysis and because those proteins elicit an immune response, presumably due to their surface localization. Here we report the crystal structures of TsTPI and SmTPI in complex with 2-phosphoglyceric acid (2-PGA). Both TPIs fold into a dimeric (β-α)8 barrel in which the dimer interface consists of α-helices 2, 3, and 4, and swapping of loop 3. TPIs from parasitic helminths harbor a region of three amino acids knows as the SXD/E insert (S155 to E157 and S157 to D159 in TsTPI and SmTPI, respectively). This insert is located between α5 and β6 and is proposed to be the main TPI epitope. This region is part of a solvent-exposed 310–helix that folds into a hook-like structure. The crystal structures of TsTPI and SmTPI predicted conformational epitopes that could be used for vaccine design. Surprisingly, the epitopes corresponding to the SXD/E inserts are not the ones with the greatest immunological potential. SmTPI, but not TsTPI, habors a sole solvent exposed cysteine (SmTPI-S230) and alterations in this residue decrease catalysis. The latter suggests that thiol-conjugating agents could be used to target SmTPI. In sum, the crystal structures of SmTPI and TsTPI are a blueprint for targeted schistosomiasis and cysticercosis drug and vaccine development.

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<![CDATA[Molecular features of steroid-binding antidins and their use for assaying serum progesterone]]> https://www.researchpad.co/article/5c76fe5fd5eed0c484e5b998

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10−15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.

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<![CDATA[Temporal evolution and pathway models of poly(ethylene-terephthalate) degradation under multi-factor accelerated weathering exposures]]> https://www.researchpad.co/article/5c70673ad5eed0c4847c6c71

Photolytic and hydrolytic degradation of poly(ethylene-terephthalate) (PET) polymers with different stabilizers were performed under multiple accelerated weathering exposures and changes in the polymers were monitored by various evaluation techniques. Yellowing was caused by photolytic degradation and haze formation was induced by combined effects of photolytic and hydrolytic degradation. The formation of light absorbing chromophores and bleaching of the UV stabilizer additive were recorded through optical spectroscopy. Chain scission and crystallization were found to be common mechanisms under both photolytic and hydrolytic conditions, based on the infrared absorption of the carbonyl (C = O) band and the trans ethylene glycol unit, respectively. The degradation mechanisms determined from these evaluations were then used to construct a set of degradation pathway network models using the network structural equation modeling (netSEM) approach. This method captured the temporal evolution of degradation by assessing statistically significant relationships between applied stressors, mechanistic variables, and performance level responses. Quantitative pathway equations provided the contributions from mechanistic variables to the response changes.

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<![CDATA[Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor-binding head domains]]> https://www.researchpad.co/article/5c61e8e4d5eed0c48496f33e

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The “breathing” of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.

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<![CDATA[Biophysical and structural characterization of a zinc-responsive repressor of the MarR superfamily]]> https://www.researchpad.co/article/5c6c75e4d5eed0c4843d0401

The uptake of zinc, which is vital in trace amounts, is tightly controlled in bacteria. For this control, bacteria of the Streptococcaceae group use a Zn(II)-binding repressor named ZitR in lactococci and AdcR in streptococci, while other bacteria use a Zur protein of the Ferric uptake regulator (Fur) superfamily. ZitR and AdcR proteins, characterized by a winged helix-turn-helix DNA-binding domain, belong to the multiple antibiotic resistance (MarR) superfamily, where they form a specific group of metallo-regulators. Here, one such Zn(II)-responsive repressor, ZitR of Lactococcus lactis subspecies cremoris strain MG1363, is characterized. Size Exclusion Chromatography-coupled to Multi Angle Light Scattering, Circular Dichroism and Isothermal Titration Calorimetry show that purified ZitR is a stable dimer complexed to Zn(II), which is able to bind its two palindromic operator sites on DNA fragments. The crystal structure of ZitR holo-form (Zn(II)4-ZitR2), has been determined at 2.8 Å resolution. ZitR is the fourth member of the MarR metallo-regulator subgroup whose structure has been determined. The folding of ZitR/AdcR metallo-proteins is highly conserved between both subspecies (cremoris or lactis) in the Lactococcus lactis species and between species (Lactococcus lactis and Streptococcus pneumoniae or pyogenes) in the Streptococcaceae group. It is also similar to the folding of other MarR members, especially in the DNA-binding domain. Our study contributes to better understand the biochemical and structural properties of metallo-regulators in the MarR superfamily.

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<![CDATA[Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM]]> https://www.researchpad.co/article/5c50c481d5eed0c4845e8843

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

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<![CDATA[Inherent versus induced protein flexibility: Comparisons within and between apo and holo structures]]> https://www.researchpad.co/article/5c5b52c9d5eed0c4842bd003

Understanding how ligand binding influences protein flexibility is important, especially in rational drug design. Protein flexibility upon ligand binding is analyzed herein using 305 proteins with 2369 crystal structures with ligands (holo) and 1679 without (apo). Each protein has at least two apo and two holo structures for analysis. The inherent variation in structures with and without ligands is first established as a baseline. This baseline is then compared to the change in conformation in going from the apo to holo states to probe induced flexibility. The inherent backbone flexibility across the apo structures is roughly the same as the variation across holo structures. The induced backbone flexibility across apo-holo pairs is larger than that of the apo or holo states, but the increase in RMSD is less than 0.5 Å. Analysis of χ1 angles revealed a distinctly different pattern with significant influences seen for ligand binding on side-chain conformations in the binding site. Within the apo and holo states themselves, the variation of the χ1 angles is the same. However, the data combining both apo and holo states show significant displacements. Upon ligand binding, χ1 angles are frequently pushed to new orientations outside the range seen in the apo states. Influences on binding-site variation could not be easily attributed to features such as ligand size or x-ray structure resolution. By combining these findings, we find that most binding site flexibility is compatible with the common practice in flexible docking, where backbones are kept rigid and side chains are allowed some degree of flexibility.

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<![CDATA[New interfaces on MiD51 for Drp1 recruitment and regulation]]> https://www.researchpad.co/article/5c5ca2b5d5eed0c48441e93f

Mitochondrial fission is facilitated by dynamin-related protein Drp1 and a variety of its receptors. However, the molecular mechanism of how Drp1 is recruited to the mitochondrial surface by receptors MiD49 and MiD51 remains elusive. Here, we showed that the interaction between Drp1 and MiD51 is regulated by GTP binding and depends on the polymerization of Drp1. We identified two regions on MiD51 that directly bind to Drp1, and found that dimerization of MiD51, relevant to residue C452, is required for mitochondrial dynamics regulation. Our Results have suggested a multi-faceted regulatory mechanism for the interaction between Drp1 and MiD51 that illustrates the potentially complicated and tight regulation of mitochondrial fission.

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<![CDATA[Expression, purification and characterization of the dimeric protruding domain of Macrobrachium rosenbergii nodavirus capsid protein expressed in Escherichia coli]]> https://www.researchpad.co/article/5c5df342d5eed0c484581038

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1–252 and 253–371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.

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<![CDATA[R pyocin tail fiber structure reveals a receptor-binding domain with a lectin fold]]> https://www.researchpad.co/article/5c633948d5eed0c484ae63f5

R pyocins are ɸCTX-like myophage tailocins of Pseudomonas sp. Adsorption of R pyocins to target strains occurs by the interaction of tail fiber proteins with core lipopolysaccharide (LPS). Here, we demonstrate that N-terminally truncated R pyocin tail fibers corresponding to a region of variation between R-subtypes are sufficient to bind target strains according to R-subtype. We also report the crystal structures of these tail fiber proteins and show that they form an elongated helical trimer composed of three domains arranged linearly from N- to C-terminus: a baseplate proximal head, medial shaft, and distal foot. The head and shaft domains contain novel structural motifs. The foot domain, however, is composed of a conserved jellyroll fold and shares high structural similarity to the tail fiber of myophage AP22, podophage tailspike C-terminal domains (LKA-1 and ɸ297), and several eukaryotic adhesins (discoidin I/II, agglutinin, and octocoral lectin). Many of these proteins bind polysaccharides by means of their distal loop network, a series of highly variable loops at one end of the conserved jellyroll fold backbone. Our structures reveal that the majority of R-subtype specific polymorphisms cluster in patches covering a cleft formed at the oligomeric interface of the head domain and in a large patch covering much of the foot domain, including the distal loop network. Based on the structural variation in distal loops within the foot region, we propose that the foot is the primary sugar-binding domain of R pyocins and R-subtype specific structural differences in the foot domain distal loop network are responsible for binding target strains in an R-subtype dependent manner.

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<![CDATA[Structure of the DP1–DP2 PolD complex bound with DNA and its implications for the evolutionary history of DNA and RNA polymerases]]> https://www.researchpad.co/article/5c4b7f56d5eed0c48484117d

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo–electron microscopy (cryo-EM) structure of the heterodimeric DP1–DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1–DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome.

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<![CDATA[Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF]]> https://www.researchpad.co/article/5c61e8f4d5eed0c48496f4de

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure–function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF–RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand–receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor–synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

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<![CDATA[Structural basis of human ORP1-Rab7 interaction for the late-endosome and lysosome targeting]]> https://www.researchpad.co/article/5c63397ed5eed0c484ae68d3

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of lipid transfer proteins conserved in eukaryotes. ORP1 transports cholesterol at the interface between the late endosomes/lysosomes (LELs) and the endoplasmic reticulum (ER). ORP1 is targeted to the endosomal membranes by forming a tripartite complex with the LE GTPase Rab7 and its effector RILP (Rab7-interacting lysosomal protein). Here, we determined the crystal structure of human ORP1 ANK domain in complex with the GTP-bound form of Rab7. ORP1 ANK binds to the helix α3 of Rab7 located away from the switching regions, which makes the interaction independent of the nucleotide-binding state of Rab7. Thus, the effector-interacting switch regions of Rab7 are accessible for RILP binding, allowing formation of the ORP1-Rab7-RILP complex. ORP1 ANK binds to Rab7 and the Rab7-RILP complex with similar micro-molar affinities, which is consistent with the independence binding of ORP1 and RILP to Rab7. The structural model of the ORP1-Rab7-RILP complex correlates with the recruitment of ORP1 at the LEL-ER interface and the role in lipid transport and regulation.

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<![CDATA[Kinetic and thermodynamic insights into sodium ion translocation through the μ-opioid receptor from molecular dynamics and machine learning analysis]]> https://www.researchpad.co/article/5c536a92d5eed0c484a47639

The differential modulation of agonist and antagonist binding to opioid receptors (ORs) by sodium (Na+) has been known for decades. To shed light on the molecular determinants, thermodynamics, and kinetics of Na+ translocation through the μ-OR (MOR), we used a multi-ensemble Markov model framework combining equilibrium and non-equilibrium atomistic molecular dynamics simulations of Na+ binding to MOR active or inactive crystal structures embedded in an explicit lipid bilayer. We identify an energetically favorable, continuous ion pathway through the MOR active conformation only, and provide, for the first time: i) estimates of the energy differences and required timescales of Na+ translocation in inactive and active MORs, ii) estimates of Na+-induced changes to agonist binding validated by radioligand measurements, and iii) testable hypotheses of molecular determinants and correlated motions involved in this translocation, which are likely to play a key role in MOR signaling.

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<![CDATA[Potential off-target effects of beta-blockers on gut hormone receptors: In silico study including GUT-DOCK—A web service for small-molecule docking]]> https://www.researchpad.co/article/5c57e6e2d5eed0c484ef412a

The prolonged use of many currently available drugs results in the severe side effect of the disruption of glucose metabolism leading to type 2 diabetes mellitus (T2DM. Gut hormone receptors including glucagon receptor (GCGR) and the incretin hormone receptors: glucagon-like peptide 1 receptor (GLP1R) and gastric inhibitory polypeptide receptor (GIPR) are important drug targets for the treatment of T2DM, as they play roles in the regulation of glucose and insulin levels and of food intake. In this study, we hypothesized that we could compensate for the negative influences of specific drugs on glucose metabolism by the positive incretin effect enhanced by the off-target interactions with incretin GPCR receptors. As a test case, we chose to examine beta-blockers because beta-adrenergic receptors and incretin receptors are expressed in a similar location, making off-target interactions possible. The binding affinity of drugs for incretin receptors was approximated by using two docking scoring functions of Autodock VINA (GUT-DOCK) and Glide (Schrodinger) and juxtaposing these values with the medical information on drug-induced T2DM. We observed that beta-blockers with the highest theoretical binding affinities for gut hormone receptors were reported as the least harmful to glucose homeostasis in clinical trials. Notably, a recently discovered beta-blocker compound 15 ([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide was among the top-scoring drugs, potentially supporting its use in the treatment of hypertension in diabetic patients. Our recently developed web service GUT-DOCK (gut-dock.miningmembrane.com) allows for the execution of similar studies for any drug-like molecule. Specifically, users can compute the binding affinities for various class B GPCRs, gut hormone receptors, VIPR1 and PAC1R.

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<![CDATA[Crystal structure of a Thermus aquaticus diversity-generating retroelement variable protein]]> https://www.researchpad.co/article/5c40f7b0d5eed0c484386634

Diversity-generating retroelements (DGRs) are widely distributed in bacteria, archaea, and microbial viruses, and bring about unparalleled levels of sequence variation in target proteins. While DGR variable proteins share low sequence identity, the structures of several such proteins have revealed the C-type lectin (CLec)-fold as a conserved scaffold for accommodating massive sequence variation. This conservation has led to the suggestion that the CLec-fold may be useful in molecular surface display applications. Thermostability is an attractive feature in such applications, and thus we studied the variable protein of a DGR encoded by a prophage of the thermophile Thermus aquaticus. We report here the 2.8 Å resolution crystal structure of the variable protein from the T. aquaticus DGR, called TaqVP, and confirm that it has a CLec-fold. Remarkably, its variable region is nearly identical in structure to those of several other CLec-fold DGR variable proteins despite low sequence identity among these. TaqVP was found to be thermostable, which appears to be a property shared by several CLec-fold DGR variable proteins. These results provide impetus for the pursuit of the DGR variable protein CLec-fold in molecular display applications.

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<![CDATA[Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity]]> https://www.researchpad.co/article/5c40f818d5eed0c484387080

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.

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<![CDATA[Potent, multi-target serine protease inhibition achieved by a simplified β-sheet motif]]> https://www.researchpad.co/article/5c50c48fd5eed0c4845e88ff

Engagement of an extended β-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like β-sheet to enzyme inhibition. Here we report the crystal structure of an simplified β-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by β–sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

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