Search

Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.

C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria., In C-glycosides the sugar moiety is linked through a carbon-carbon bond to the non-sugar moiety, which can be cleaved by intestinal microbes. Here, the authors use bioinformatics analysis to identify C-glycoside deglycosidase enzymes in intestinal and soil bacteria, biochemically characterise them and determine their structures and probe catalytic important residues in mutagenesis experiments.

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid β aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies., The calcium binding protein S100B is an abundantly expressed protein in the brain and has neuro-protective functions by inhibiting Aβ aggregation and metal ion toxicity. Here, the authors combine cell biology and biochemical experiments with chemical kinetics and NMR measurements and show that S100B protein is an extracellular Tau chaperone and further characterize the interactions between S100B and Tau.

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, βLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant βL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the βHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

Rsm22-family proteins are conserved putative SAM-dependent methyltransferases with important functions in mitochondrial translation. Here, the results of a comparative bioinformatics analysis of Rsm22-type proteins are presented, the expression, biophysical characterization and crystallization of Saccharomyces cerevisiae Rsm22 are reported, a low-resolution SAXS structure of the protein is revealed, and SAM-dependent RNA methyl transferase activity of the protein is demonstrated., The Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2 kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117–Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.

Immunity-related GTPase B10 (IRGB10) belongs to the interferon (IFN)-inducible GTPases, a family of proteins critical to host defense. It is induced by IFNs after pathogen infection, and plays a role in liberating pathogenic ligands for the activation of the inflammasome by directly disrupting the pathogen membrane. Although IRGB10 has been intensively studied owing to its functional importance in the cell-autonomous immune response, the molecular mechanism of IRGB10-mediated microbial membrane disruption is still unclear. In this study, we report the structure of mouse IRGB10. Our structural study showed that IRGB10 bound to GDP forms an inactive head-to-head dimer. Further structural analysis and comparisons indicated that IRGB10 might change its conformation to activate its membrane-binding and disruptive functions. Based on this observation, we propose a model of the working mechanism of IRGB10 during pathogen membrane disruption., Ha et al. present a crystal structure of mouse IRGB10, a mouse interferon-inducible GTPase that mediates bacteriolysis in cell autonomous immunity. With further mutagenesis studies, they show that IRGB10 bound to GDP forms an inactive head-to-head dimer, which changes its conformation to activate its membrane-binding and disruptive functions.

Lichens are symbiotic photosynthesizing organisms with thalli formed by fungi and algae/cyanobacteria that possess high stress tolerance. One of the factors that contributes to the ability of a lichen to tolerate harsh environmental conditions is the presence of unique metabolites, including high-molecular-weight dark pigments termed melanins. The chemical composition and structure of lichen melanins remain poorly studied. We analyzed the elemental composition, the main functional groups, and the physicochemical properties of melanin extracted from Cetraria islandica and Pseudevernia furfuracea lichens. Based on the C/N ratio, this pigment is allomelanin. We also identified functional groups that provide photoprotective and antioxidant properties of melanin. Melanin synthesis might be an essential defense mechanism contributing to the survival of lichens under exposure to UV radiation.

A dual-receptor interaction with a polysialoganglioside and synaptic vesicle glycoprotein 2 (SV2) is required for botulinum neurotoxin A (BoNT) toxicity. Here, we review what is currently known about the BoNT/A-SV2 interaction based on structural studies. Currently, five crystal structures of the receptor-binding domain (Hc) of BoNT subtypes A1 and A2 complexed to the large luminal domain (LD4) of SV2C have been determined. On the basis of the available structures, we will discuss the importance of protein-protein and protein-carbohydrate interactions for BoNT/A toxicity as well as the high plasticity of BoNT/A for receptor recognition by tolerating a variety of side-chain interactions at the interface. A plausible explanation how receptor-binding specificity of BoNT/A may be achieved without an extensive and conserved side chain-side chain interaction network will be provided.

Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 A. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication. Significance Statement Alphaviruses are a group of viruses that comprise several important pathogens that provoke encephalitis or arthritis in humans. Through its RdRp activity, nsP4 of alphaviruses plays a crucial role in viral RNA replication. We determined the crystal structure of nsP4, disclosing a dynamic structure resembling the core catalytic domain of RNA polymerases from pestiviruses, picornaviruses, and noroviruses. These findings expand our knowledge of the molecular basis for alphavirus RNA replication and should accelerate the design of specific antiviral compounds targeting RNA polymerase activity.

Zinc homeostasis in mammals is constantly and precisely maintained by sophisticated regulatory proteins. Among them, the Zrt/Irt-like protein (ZIP) regulates the influx of zinc into the cytoplasm. In the current work, we have employed all-atom molecular dynamics simulations to investigate the Zn(2+) transport mechanism in prokaryotic ZIP obtained from Bordetella bronchiseptica (BbZIP) in a membrane bilayer. Additionally, the structural and dynamical transformations of BbZIP during this process have been analyzed. The present study allowed us to develop a hypothesis for the zinc influx mechanism and metal-binding site formation. We have created a model for the outward facing form of BbZIP (experimentally only the inward facing form has been characterized) which has allowed us, for the first time, to observe the Zn(2+) ion entering the channel and binding to the negatively charged M2 site. It is thought that the M2 site is less favored than the M1 site, which then leads to metal ion egress; however, we have not observed the M1 site being occupied in our simulations. Furthermore, removing both the Zn(2+) ions from this complex resulted in the collapse of the metal-binding site illustrating the “structural role” of metal ions in maintaining the binding site and holding the proteins together. Finally, due to the long Cd(2+)-residue bond distances observed in the X-ray structures, we have proposed the existence of an H(3)O(+) ion at the M2 site which plays an important role in the protein stability in the absence of the metal ion. Understanding the molecular-level Zn(2+) transport process sheds light on the development of small molecules that can affect intracellular transition metal ion concentrations to treat diseases like diabetes and cancer.

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins., New virions of Ff bacteriophages are extruded from the host cell via the channel built from phage protein pIV, homologous to bacterial secretins. Here, the authors report the structure of this channel from the f1 filamentous bacteriophage and propose its use as an adjuvant to increase the uptake and efficacy of antibiotics.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Background: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. Results: The solution structure of isolated Nb23 nanobody was determined. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure-function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:297 issue:2 ispartof: location:United States status: published

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen–deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

BACKGROUND AND PURPOSE: The heteromeric α4β2 nicotinic acetylcholine receptor (nAChR) is abundant in the human brain and is associated with a range of CNS disorders. This nAChR subtype has been recently crystallised in a conformation that was proposed to represent a desensitised state. Here, we investigated the conformational transition mechanism of this nAChR from a desensitised to a closed/resting state. EXPERIMENTAL APPROACH: The competitive antagonist dihydro‐β‐erythroidine (DHβE) was modelled by replacement of the agonist nicotine in the α4β2 nAChR experimental structure. DHβE is used both in vitro and in vivo for its ability to block α4β2 nAChRs. This system was studied by three molecular dynamics simulations with a combined simulation time of 2.6 μs. Electrophysiological studies of mutated receptors were performed to validate the simulation results. KEY RESULTS: The relative positions of the extracellular and transmembrane domains in the models are distinct from those of the desensitised state structure and are compatible with experimental structures of Cys‐loop receptors captured in a closed/resting state. CONCLUSIONS AND IMPLICATIONS: Our model suggests that the side chains of α4 L257 (9′) and α4 L264 (16′) are the main constrictions in the transmembrane pore. The involvement of position 9′ in channel gating is well established, but position 16′ was only previously identified as a gate for the bacterial channels, ELIC and GLIC. L257 but not L264 was found to influence the slow component of desensitisation. The structure of the antagonist‐bound state proposed here should be valuable for the development of therapeutic or insecticide compounds.

Immune response stimulation and inactivation of chondroitinase ABC I in physiological condition have been limited its use in various clinical conditions as a bacterial enzyme drug. In the present study, we have investigated some structural and functional features of N∆89, C∆274 and N∆89C∆274; three designed truncated cABC I, in order to clarify the unclear role of two terminal parts of cABC I i.e., the 1-89 and 747-1021 amino acids sequences of the full length enzyme through truncation. As a result, the numbers of potential epitopes, the susceptibility to trypsin digestion, ANS fluorescence spectra, and fluorescence quenching using KI and acrylamide were diminished for N∆89 and C∆274 in comparison to the wild type. Secondary and tertiary structure investigation for N∆89 and C∆274 revealed that the intrinsic fluorescence was increased and Far-UV CD spectra were changed accordingly. Relative to the wild type enzyme, 0.164, 0.195 remaining activity and lack of activity was shown with the zymographic assay for N∆89, C∆274 and N∆89C∆274 variants, respectively. The diminished enzyme activity and structural changes suggested a reorientation of microenvironments interactions including cation-π interactions around structural elements toward lowering regional mobility. Constructing applicable truncated cABC I with improved features could be regarded as a strategy to regain new possible functional advantages over the full length enzyme.

Protoporphyrinogen IX oxidase (PPO) is the last common enzyme in chlorophyll and heme biosynthesis pathways. In human, point mutations on PPO are responsible for the dominantly inherited disorder disease, Variegate Porphyria (VP). Of the VP-causing mutation site, the Arg59 is by far the most prevalent VP mutation residue identified. Multiple sequences alignment of PPOs shows that the Arg59 of human PPO (hPPO) is not conserved, and experiments have shown that the equivalent residues in PPO from various species are essential for enzymatic activity. In this work, it was proposed that the Arg59 performs its function by forming a hydrogen-bonding (HB) network around it in hPPO, and we investigated the role of the HB network via site-directed mutagenesis, enzymatic kinetics and computational studies. We found the integrity of the HB network around Arg59 is important for enzyme activity. The HB network maintains the substrate binding chamber by holding the side chain of Arg59, while it stabilizes the micro-environment of the isoalloxazine ring of FAD, which is favorable for the substrate-FAD interaction. Our result provides a new insight to understanding the relationship between the structure and function for hPPO that non-conserved residues can form a conserved element to maintain the function of protein.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 A. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Histone lysine demethylase 4D (KDM4D), also known as JMJD2D, plays an important role in cell proliferation and survival and has been associated with several tumor types. KDM4D has emerged as a potential target for the treatment of human cancer. Here, we reported crystal complex structures for two KDM4D inhibitors, OWS [2-(1H-pyrazol-3-yl)isonicotinic acid] and 10r (5-hydroxy-2-methylpyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile), which were both determined to 2.0 A. OWS is a newly discovered KDM4D inhibitor (IC50 = 4.28 μM) and the critical pharmacophores of this compound are confirmed by the complex structure. Compound 10r is a KDM4D inhibitor reported by us previously. To clarify the binding mode in more detail, the crystal structure was determined and the comparison analysis revealed unique interactions that had never been observed before. Overall, our data provide new structural insights for rational design and offer an opportunity for optimization of KDM4D inhibitors.

Biochemical and biophysical research communications 539, 42 - 47 (2021). doi:10.1016/j.bbrc.2020.12.069, In this report, we describe a truncated Deinococcus radiodurans 1-deoxy-D-xylulose-5-phosphate synthase (DXS) protein that retains enzymatic activity, while slowing protein degradation and showing improved crystallization properties. With modern drug-design approaches relying heavily on the elucidation of atomic interactions of potential new drugs with their targets, the need for co-crystal structures with the compounds of interest is high. DXS itself is a promising drug target, as it catalyzes the first reaction in the 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway for the biosynthesis of the universal precursors of terpenes, which are essential secondary metabolites. In contrast to many bacteria and pathogens, which employ the MEP pathway, mammals use the distinct mevalonate-pathway for the biosynthesis of these precursors, which makes all enzymes of the MEP-pathway potential new targets for the development of anti-infectives. However, crystallization of DXS has proven to be challenging: while the first X-ray structures from Escherichia coli and D. radiodurans were solved in 2004, since then only two additions have been made in 2019 that were obtained under anoxic conditions. The presented site of truncation can potentially also be transferred to other homologues, opening up the possibility for the determination of crystal structures from pathogenic species, which until now could not be crystallized. This manuscript also provides a further example that truncation of a variable region of a protein can lead to improved structural data., Published by Academic Press, Orlando, Fla.

The recognition of ACE2 by the receptor-binding domain (RBD) of spike protein mediates host cell entry. The objective of the work is to identify SARS-CoV2 spike variants that emerged during the pandemic and evaluate their binding affinity with ACE2. Evolutionary analysis of 2178 SARS-CoV2 genomes identifies RBD variants that are under selection bias. The binding efficacy of these RBD variants to the ACE2 has been analyzed by using protein-protein docking and binding free energy calculations. Pan-proteomic analysis reveals 113 mutations among them 33 are parsimonious. Evolutionary analysis reveals five RBD variants A348T, V367F, G476S, V483A, and S494P are under strong positive selection bias. Variations at these sites alter the ACE2 binding affinity. A348T, G476S, and V483A variants display reduced affinity to ACE2 in comparison to the Wuhan SARS-CoV2 spike protein. While the V367F and S494P population variants display a higher binding affinity towards human ACE2. Reorientation of several crucial residues at the RBD-ACE2 interface facilitates additional hydrogen bond formation for the V367F variant which enhances the binding energy during ACE2 recognition. On the other hand, the enhanced binding affinity of S494P is attributed to strong interfacial complementarity between the RBD and ACE2., Highlights • The S gene in few SARS-CoV2 strains is emerging with distinct pattern disparity. • RBD variants A348T, V367F, G476S, V483A, and S494P under positive selection bias. • V367F and S494P population variants bind ACE2 with higher affinity. • V367 spike variant forms enhanced hydrogen-bonding interactions ACE2. • S494P alteration leads to strong surface complementarity between the RBD and ACE2.

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational opening of the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, the relevance of this for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant enhanced activity. These data are consistent with CD81 cholesterol sensing resulting in a switch between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81’s function as a molecular scaffold; these insights are relevant to CD81’s varied roles in both health and disease.

Protein–carbohydrate interactions play a major role in several cellular and biological processes. Elucidating the factors influencing the binding affinity of protein–carbohydrate complexes and predicting their free energy of binding provide deep insights for understanding the recognition mechanism. In this work, we have collected the experimental binding affinity data for a set of 389 protein–carbohydrate complexes and derived several structure-based features such as contact potentials, interaction energy, number of binding residues and contacts between different types of atoms. Our analysis on the relationship between binding affinity and structural features revealed that the important factors depend on the type of the complex based on number of carbohydrate and protein chains. Specifically, binding site residues, accessible surface area, interactions between various atoms and energy contributions are important to understand the binding affinity. Further, we have developed multiple regression equations for predicting the binding affinity of protein–carbohydrate complexes belonging to six categories of protein–carbohydrate complexes. Our method showed an average correlation and mean absolute error of 0.731 and 1.149 kcal/mol, respectively, between experimental and predicted binding affinities on a jackknife test. We have developed a web server PCA-Pred, Protein–Carbohydrate Affinity Predictor, for predicting the binding affinity of protein–carbohydrate complexes. The web server is freely accessible at https://web.iitm.ac.in/bioinfo2/pcapred/. The web server is implemented using HTML and Python and supports recent versions of major browsers such as Chrome, Firefox, IE10 and Opera.

Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen–antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.

Alzheimer disease (AD) is the most common neurodegenerative disease worldwide. AD is pathologically characterized by the deposition of senile plaques in the brain, which are composed of an amyloid-β peptide (Aβ) that is produced through the multistep cleavage of amyloid precursor protein (APP) by γ-secretase. γ-Secretase is a membrane protein complex, which includes its catalytic subunit presenilin 1 (PS1). However, much about the structural dynamics of this enzyme remain unclear. We have previously demonstrated that movements of the transmembrane domain (TMD) 1 and TMD3 of PS1 are strongly associated with decreased production of the Aβ peptide ending at the 42nd residue (i.e. Aβ42), which is the aggregation-prone, toxic species. However, the association between these movements as well as the sequence of these TMDs remains unclear. In this study, we raised the possibility that the vertical movement of TMD1 is a prerequisite for expansion of the catalytic cavity around TMD3 of PS1, resulting in reduced Aβ42 production. Our results shed light on the association between the conformational changes of TMDs and the regulation of γ-secretase activity.

This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26 Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65 Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug.

Endonuclease III (EndoIII) is nearly ubiquitous in all three domains of life. EndoIII family proteins exhibit a bifunctional (glycosylase/lyase) activity on oxidative/saturated pyrimidine bases, such as thymine glycol. Previous studies on EndoIII homologs have reported the presence of important residues involved in substrate binding and catalytic activity. However, a biochemical clarification of the roles of these residues as well as details of their evolutionary conservation is still lacking. This is particularly true for archaeal orthologs. The current study demonstrated the roles of the evolutionarily conserved residues of euryarchaeon Thermococcus kodakarensis EndoIII (TkoEndoIII). We utilized amino acid sequence analysis and homology modeling to identify highly conserved regions with potential key residues in the EndoIII proteins. Using Ala-substituted TkoEndoIII mutant proteins, residues of interest were quantitatively examined via DNA binding, glycosylase/AP lyase/bifunctional activity, and DNA trapping assays. The obtained results allowed us to determine the roles, as well as the significance of these roles in Schiff base formation (Lys140 as a nucleophile and Asp158), Tg recognition (His160), substrate binding (Arg59, Leu101, Trp102, and Gly136), β-elimination activities (Ser57 and Asp62), and [4Fe-4S] cluster formation (Cys208 and Cys215). Interestingly, a critical role played by the highly conserved Lys105 (predicted as being away from the catalytic site) in substrate binding, accompanied by a significant indirect effect on catalytic activity, were detected. Our results suggest that these particular residues play conserved roles among EndoIII orthologs across the domains. In addition to identifying the critical role of the highly conserved Lys105, the study provides a comprehensive understanding of the functions attributable to the evolutionarily conserved residues found in the EndoIII family, from Escherichia coli to humans.

Rosmarinic acid (RA) is a natural compound that is gaining wide popularity owing to its broad-spectrum biological activities. RA is known for its wide range of medicinal properties and therapeutic applications in a vast range of neurodegenerative disorders thus making it a vital natural compound. Human transferrin (hTf) is a clinically significant protein that plays a pivotal role in maintaining iron homeostasis. The importance of studies pertaining to hTf is attributable to the pivotal role of iron deposition in CNS in neurodegenerative disorders. The study was intended to have an insight into the interaction between RA and hTf employing multispectroscopic approach, molecular docking, and molecular dynamic simulation studies. Fluorescence quenching studies revealed that RA shows an excellent binding affinity to hTf with a binding constant ( K ) of 107 M-1 and is guided by static mode of quenching. Isothermal titration calorimetry (ITC) further validated the spontaneous nature of binding. The estimation of enthalpy change (∆H) and entropy change (∆S) suggested that the RA-hTf complex formation is driven by hydrogen bonding, thereby making this process seemingly specific. Further, Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that RA induces conformational and structural changes in hTf. Additionally, molecular dynamics (MD) studies were carried out to investigate the stability of the hTf and hTf–RA system and suggested that binding of RA induces structural alteration in hTf with free hTf being more stable. This study provides a rationale to use RA in drug development against neurodegenerative disorders by designing novel functional foods containing RA.

Work on the electronic structures of metal–oxo complexes began in Copenhagen over 50 years ago. This work led to the prediction that tetragonal multiply bonded transition metal–oxos would not be stable beyond the iron–ruthenium–osmium oxo wall in the periodic table and that triply bonded metal–oxos could not be protonated, even in the strongest Brønsted acids. In this theory, only double bonded metal–oxos could attract protons, with basicities being a function of the electron donating ability of ancillary ligands. Such correlations of electronic structure with reactivity have gained importance in recent years, most notably owing to the widespread recognition that high-valent iron–oxos are intermediates in biological reactions critical to life on Earth. In this Account, we focus attention on the oxygenations of inert organic substrates by cytochromes P450, as these reactions involve multiply bonded iron–oxos. We emphasize that P450 iron–oxos are strong oxidants, so strong that they would destroy nearby amino acids if substrates are not oxygenated rapidly; it is our view that these high-valent iron–oxos are such dangerous reactive oxygen species that Nature surely found ways to disable them. Looking more deeply into this matter, mainly by examining many thousands of structures in the Protein Data Bank, we have found that P450s and other enzymes that require oxygen for function have chains of tyrosines and tryptophans that extend from active-site regions to protein surfaces. Tyrosines are near the heme active sites in bacterial P450s, whereas tryptophan is closest in most human enzymes. High-valent iron–oxo survival times taken from hole hopping maps range from a few nanoseconds to milliseconds, depending on the distance of the closest Trp or Tyr residue to the heme. In our proposed mechanism, multistep hole tunneling (hopping) through Tyr/Trp chains guides the damaging oxidizing hole to the protein surface, where it can be quenched by soluble protein or small molecule reductants. As the Earth’s oxygenic atmosphere is believed to have developed about 2.5 billion years ago, the increase in occurrence frequency of tyrosine and tryptophan since the last universal evolutionary ancestor may be in part a consequence of enzyme protective functions that developed to cope with the environmental toxin, O₂.

The selective removal of B800 bacteriochlorophyll (BChl) a from light-harvesting complex 2 (LH2) in purple photosynthetic bacteria is a clue about elucidation of the mechanism for the transfer of energy from these pigments to B850 BChl a and their roles in the LH2 protein structure. We demonstrated that the kinetics of the removal of B800 BChl a from two representative LH2 proteins derived from Phaeospirillum molischianum and Rhodoblastus acidophilus differed significantly, in contrast to the calculated binding enthalpy. These results may be interpreted as changes in the local structure near B800 BChl a with respect to the geometries of the original crystal structures upon removal of B800 BChl a. Despite the difficulty of removing B800 BChl a from molischianum-LH2, we prepared the molischianum-LH2 protein lacking B800 BChl a by combination of two detergents, n-dodecyl β-d-maltoside and n-octyl β-d-glucoside, under acidic conditions. Spectral and atomic force microscopy analyses indicated that the absence of B800 BChl a had little effect on the local structure in the vicinity of B850 BChl a and the circular arrangement in this protein. These results suggest that the hydrophobic domain near B850 BChl a is rigid and plays a major role in the structural formation of molischianum-LH2.

γ-Resorcylate decarboxylase (γ-RSD) has evolved to catalyze the reversible decarboxylation of 2,6-dihydroxybenzoate to resorcinol in a nonoxidative fashion. This enzyme is of significant interest because of its potential for the production of γ-resorcylate and other benzoic acid derivatives under environmentally sustainable conditions. Kinetic constants for the decarboxylation of 2,6-dihydroxybenzoate catalyzed by γ-RSD from Polaromonas sp. JS666 are reported and the enzyme is shown to be active with 2,3-dihydroxybenzoate, 2,4,6-trihydroxybenzoate and 2,6-dihydroxy-4 methylbenzoate. The three-dimensional structure of γ-RSD with the inhibitor 2-nitroresorcinol (2-NR) bound in the active site is reported. 2-NR is directly ligated to a Mn(2+) bound in the active site and the nitro-substituent of the inhibitor is tilted significantly from the plane of the phenyl ring. The inhibitor exhibits a different binding mode compared to the substrate bound in the previously determined structure of γ-RSD from Rhizobium sp. MTP-10005. On the basis of the crystal structure of the enzyme from Polaromonas sp. JS666, complementary density functional calculations were performed to investigate the reaction mechanism. In the proposed reaction mechanism, γ-RSD binds 2,6-dihydroxybenzoate by direct coordination of the active site manganese ion to the carboxylate anion of the substrate and one of the adjacent phenolic oxygens. The enzyme subsequently catalyzes the transfer of a proton to C1 of γ-resorcylate prior to the actual decarboxylation step. The reaction mechanism proposed previously, based on the structure of γ-RSD from Rhizobium sp. JS666, is shown to be associated with high energies and thus less likely to be correct.

The field of protein structure prediction has recently been revolutionized through the introduction of deep learning. The current state-of-the-art tool AlphaFold2 can predict highly accurate structures; however, it has a prohibitively long inference time for applications that require the folding of hundreds of sequences. The prediction of protein structure annotations, such as amino acid distances, can be achieved at a higher speed with existing tools, such as the ProSPr network. Here, we report on important updates to the ProSPr network, its performance in the recent Critical Assessment of Techniques for Protein Structure Prediction (CASP14) competition, and an evaluation of its accuracy dependency on sequence length and multiple sequence alignment depth. We also provide a detailed description of the architecture and the training process, accompanied by reusable code. This work is anticipated to provide a solid foundation for the further development of protein distance prediction tools.

β-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αβ domains conjugated by an interdomain loop and can effectively bind and inactivate class A β-lactamases, which are responsible for resistance of bacteria to β-lactam antibiotics. The varied ability of BLIP to bind different β-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A β-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135–145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135–145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135–145, could be desirable determinants for enhancing inhibition of BLIP to class A β-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein–protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein–protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.

We acknowledge Funda??o Amaz?nia de Amparo a Estudos e Pesquisas do Par? (FAPESPA) for financially supporting (ICAAF 099/2014) our project. Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) also supported individual authors through Grant 311686/2015-0 (ECG). Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Tecnologia Biomolecular. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Tecnologia Biomolecular. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Exatas e Naturais. Laborat?rios de Investiga??o Sistem?tica em Biotecnologia e Biodiversidade Molecular. Bel?m, PA, Brazil. Universidade Federal do Par?. Instituto de Ci?ncias Exatas e Naturais. Laborat?rios de Investiga??o Sistem?tica em Biotecnologia e Biodiversidade Molecular. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Tecnologia Biomolecular. Bel?m, PA, Brazil. Scytovirin is a lectin isolated from the cyanobacterium Scytonema varium that has shown activity against HIV, SARS coronavirus and Zaire Ebola virus. Its 95 amino acids are divided into two structural domains (SD), the first spanning amino acids 1?48 (SD1) and the second 49?95 (SD2). Interestingly, the domains are nearly identical but differ in their affinities for carbohydrates. With the aim of enhancing understanding of the binding properties of scytovirin, we performed molecular dynamics (MD) simulations of scytovirin complexed with Man4. We set up three systems: (i) Man4 bound to both domains (SD1+SD2) using the full-length protein; (ii) Man4 bound to an incomplete protein, containing only SD1 and (iii) Man4 bound to an incomplete protein containing only SD2. Contrary to other reports, binding free energy results suggest that Man4 can bind simultaneously to SD1 and SD2 binding regions, but SD1 individually has the best values of energy and the best affinity for Man4. Decomposition of the binding free energy showed that the residues that interact with Man4 were different in the three systems, suggesting that the binding mechanism of Man4 varies between full-length protein, SD1 and SD2. The results presented here may help to formulate strategies to use scytovirin and promote mutagenesis studies to improve the antiviral activity of scytovirin.

Vertebrate hypoxia tolerance can emerge from modifications to the oxygen (O2) transport cascade, but whether there is adaptive variation to O2 binding at the terminus of this cascade, mitochondrial cytochrome c oxidase (COX), is not known. In order to address the hypothesis that hypoxia tolerance is associated with enhanced O2 binding by mitochondria we undertook a comparative analysis of COX O2 kinetics across species of intertidal sculpins (Cottidae, Actinopterygii) that vary in hypoxia tolerance. Our analysis revealed a significant relationship between hypoxia tolerance (critical O2 tension of O2 consumption rate; Pcrit), mitochondrial O2 binding affinity (O2 tension at which mitochondrial respiration was half maximal; P50), and COX O2-binding affinity (apparent Michaelis-Menten constant for O2 binding to COX; Km,app O2). The more hypoxia tolerant species had both a lower mitochondrial P50 and lower COX Km,app O2, facilitating the maintenance of mitochondrial function to a lower O2 tension than in hypoxia intolerant species. Additionally, hypoxia tolerant species had a lower overall COX Vmax but higher mitochondrial COX respiration rate when expressed relative to maximal electron transport system respiration rate. In silico analyses of the COX3 subunit postulated as the entry point for O2 into the COX protein catalytic core, points to variation in COX3 protein stability (estimated as free energy of unfolding) contributing to the variation in COX Km,app O2. We propose that interactions between COX3 and cardiolipin at four amino acid positions along the same alpha-helix forming the COX3 v-cleft represent likely determinants of interspecific differences in COX Km,app O2.

To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N2-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N2-dG. To investigate structural differences between Y-Family-classes, Dpo4 (a canonical η/V-class-member) is modified to make it more κ/IV-like, as evaluated via primer-extension studies with a BP-N2-dG-containing template. Three protein structural elements are identified that promote fidelity. (1) Watson-Crick-like [dCTP:BP-N2-dG] pairing requires the BP-moiety to be in the minor groove. Thus, as expected, dCTP insertion is facilitated by having large openings in the protein surface that can accommodate BP-bulk in the minor groove. The BP-moiety is also in the minor groove during dATP and dTTP misinsertion, though evidence suggests that each of these three minor groove BP-conformations differ. (2) Plugging an opening on the major groove side of the protein suppresses dGTP misinsertion, implying BP-N2-dG bulk is in the major groove during Hoogsteensyn-adduct-dG:dGTP pairing. (3) Y-Family DNAPs have non-covalent bridges (NCBs) holding their little finger-domain in contact with their catalytic core-domain; dATP/dGTP/dTTP misinsertions are suppressed by the quantity and quality of NCBs, including one near and another distal to the active site on the minor groove side. In conclusion, three protein structural elements enhance dCTP and/or suppress dATP/dGTP/dTTP insertion; four different BP-adduct conformations are responsible for the four different dNTP insertional pathways opposite BP-N2-dG; generalizations about Y-Family structure are also considered.

21 p.-4 fig.-1 tab., Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat cancer patients. Pironetin is a natural product that displays promising anticancer properties by binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin–pironetin complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are essential for establishing longitudinal tubulin contacts in microtubules, this result explains how pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin variants with improved activity profiles. They further extend our knowledge on strategies evolved by natural products to target and perturb the microtubule cytoskeleton., This work was supported by grants from the Ministerio de Economía y Competitividad and Conselleria d’Educaciò and Investigaciò, Cultura i Sport de la Generalitat Valenciana (CTQ2014-52949-P and PROMETEO 2013/027, respectively; to J.M.), from the Ministerio de Economia y Competitividad and Comunidad Autónoma de Madrid (BIO2013-42984-R and S2010/BMD-2457 BIPEDD2, respectively; to J.F.D), and from the Swiss National Science Foundation (310030B_138659 and 31003A_166608; to M.O.S.).

FSH-FSHR interaction is critical for folliculogenesis, spermatogenesis and progression of several cancers. Therefore, FSHR is an attractive target for fertility regulation and cancer therapeutics. Based on homology and structural analysis of hFSH-FSHR(ECD) complex, a minimal continuous stretch within FSHβ seat-belt loop (FSHβ (89-97)) was identified to be crucial for FSHR interaction. The ability of FSHβ (89-97) peptide to neutralize FSHR activity was evaluated by a panel of in vitro and in vivo experiments. The synthetic peptide significantly inhibited binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. In immature rats, FSHβ (89-97) peptide administration reduced FSH-mediated increase in ovarian weight. The peptide inhibited transition of follicles from pre-antral to antral stage and hindered the cell cycle progression of granulosa cells beyond G0/G1 phase. In adult rats, administration of the peptide inhibited estradiol synthesis and significantly perturbed folliculogenesis.

Tuberculosis (TB) is caused by bacterial pathogen Mycobacterium tuberculosis (Mtb) and remains a major health problem worldwide. The increasing prevalence of drug-resistant Mtb strains and the extended duration of anti-TB regimens have created an urgent need for new anti-tuberculosis antibiotics with novel targets or inhibitory strategies. Anthracenedione compound bostrycin has been shown to inhibit the growth of Mtb in vitro and inhibit the activity of the effector protein tyrosine phosphatase (MptpB) secreted by Mtb. In this study, we characterized the proteomic profile of the Mtb strain H37Ra exposed to 1 mg/L and 25 mg/L of bostrycin for 24 h. Bioinformatic analysis of the differential abundant proteins indicated that bostrycin treatment may induce oxidative stress and interfere with essential processes such as synthesis of NAD(+) and the tricarboxylic acid cycle in mycobacteria. Then, the molecular docking of bostrycin and 15 candidates of targeted proteins showed that Rv3684 and Rv1908c got higher scores compared to MptpB, suggesting the direct interaction of bostrycin and these two proteins. Further docking of potential targeted proteins with the functional group-removal derivatives of bostrycin revealed possible key functional groups of bostrycin and provides direction for the modification of bostrycin in future. Biological significance It is a challenging work to determine the potential target(s) of an antibiotic accurately and quickly. In this study, we conducted a proteomic analysis of Mtb responding to the treatment of bostrycin, and provided insight into the inhibiting mechanism of this anti-Mtb compound. The proper interaction of bostrycin and targeted proteins, as well as the interacting residues of targets, and functional groups of bostrycin were also identified within the docking surface, providing a direction for further modification of bostrycin. Our study also suggests a reference for the interaction analysis between mycobacteria and antibiotics, and provides potential targets information for other active anthraquinones.

To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-β sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recently, cryoEM analyses have produced close-to-complete models of all 45 subunits in the bovine, ovine and porcine complexes, and identifed two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically-relevant model system, in the ‘active’ state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside-kinase homolog, and define mechanistically-critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the ‘deactive’ state and with known bacterial structures we identify differences in helical geometry in the membrane domain that occur upon activation, or that alter the positions of catalytically-important charged residues. Our results demonstrate the capability of cryoEM analyses to challenge and develop mechanistic models for mammalian complex I.

Protein Direct Coupling Analysis (DCA), which predicts residue-residue contacts based on covarying positions within a multiple sequence alignment, has been remarkably effective. This suggests that there is more to learn from sequence correlations than is generally assumed, and calls for deeper investigations into DCA and perhaps into other types of correlations. Here we describe an approach that enables such investigations by measuring, as an estimated p-value, the statistical significance of the association between residue-residue covariance and structural interactions, either internal or homodimeric. Its application to thirty protein superfamilies confirms that direct coupling (DC) scores correlate with 3D pairwise contacts with very high significance. This method also permits quantitative assessment of the relative performance of alternative DCA methods, and of the degree to which they detect direct versus indirect couplings. We illustrate its use to assess, for a given protein, the biological relevance of alternative conformational states, to investigate the possible mechanistic implications of differences between these states, and to characterize subtle aspects of direct couplings. Our analysis indicates that direct pairwise correlations may be largely distinct from correlated patterns associated with functional specialization, and that the joint analysis of both types of correlations can yield greater power. Data, programs, and source code are freely available at http://evaldca.igs.umaryland.edu., Author summary The success of Direct Coupling Analysis (DCA) for protein structure prediction suggests that multiple sequence alignments implicitly contain more structural information than had previously been realized, and prompts deeper investigations of the sequence correlations uncovered by either DCA or other approaches. To aid such investigations and thereby broaden the utility of and improve DCA, we describe an approach that measures the statistical significance of the association between DCA and either 3D structure or correlated patterns associated with functional specialization. This approach can be used to evaluate the relative performance of DCA methods, their ability to distinguish direct from indirect couplings, and the potential biological relevance and mechanistic implications of alternative conformations and homodimeric interactions.

Polyamines such as putrescine, spermidine, and spermine are small aliphatic cations that serve myriad biological functions in all forms of life. While polyamine biosynthesis and cellular trafficking pathways are generally well defined, it is only recently that the molecular basis of reversible polyamine acetylation has been established. In particular, enzymes that catalyze polyamine deacetylation reactions have been identified and structurally characterized: histone deacetylase 10 (HDAC10) from Homo sapiens and Danio rerio (zebrafish) is a highly specific N(8)-acetylspermidine deacetylase, and its prokaryotic counterpart, acetylpolyamine amidohydrolase (APAH) from Mycoplana ramosa, is a broad-specificity polyamine deacetylase. Similar to the greater family of HDACs, which mainly serve as lysine deacetylases, both enzymes adopt the characteristic arginase-deacetylase fold and employ a Zn(2+)-activated water molecule for catalysis. In contrast with HDACs, however, the active sites of HDAC10 and APAH are sterically constricted to enforce specificity for long, slender polyamine substrates and exclude bulky peptides and proteins containing acetyl-L-lysine. Crystal structures of APAH and D. rerio HDAC10 reveal that quaternary structure, i.e., dimer assembly, provides the steric constriction that directs the polyamine substrate specificity of APAH, whereas tertiary structure – a unique 3(10) helix defined by the P(E,A)CE motif – provides the steric constriction that directs the polyamine substrate specificity of HDAC10. Given the recent identification of HDAC10 and spermidine as mediators of autophagy, HDAC10 is rapidly emerging as a biomarker and target for the design of isozyme-selective inhibitors that will suppress autophagic responses to cancer chemotherapy, thereby rendering cancer cells more susceptible to cytotoxic drugs.

Although not being classified as the most fundamental protein structural elements like α-helices and β-strands, the loop segment may play considerable roles for protein stability, flexibility, and dynamic activity. Meanwhile, the protein loop is also quite elusive; i.e. its interactions with the other parts of protein as well as its own shape-maintaining forces have still remained as a puzzle or at least not quite clear yet. Here, we report a molecular force, the so-called polar hydrogen-π interaction (Hp-π), which may play an important role in supporting the backbones of protein loops. By conducting the potential energy surface scanning calculations on the quasi π-plane of peptide bond unit, we have observed the following intriguing phenomena: (1) when the polar hydrogen atom of a peptide unit is perpendicularly pointing to the π-plane of other peptide bond units, a remarkable Hp-π interaction occurs; (2) the interaction is distance and orientation dependent, acting in a broad space, and belonging to the 'point-to-plane' one. The molecular force reported here may provide useful interaction concepts and insights into better understanding the loop's unique stability and flexibility feature, as well as the driving force of the protein global folding.

Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH−membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.