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Noncanonical amino acids have proved extremely useful for modifying the properties of proteins. Among them, extensively fluorinated (fluorous) amino acids seem particularly effective in increasing protein stability; however, in the absence of structural data, the basis of this stabilizing effect remains poorly understood. To address this problem, we solved X-ray structures for three small proteins with hydrophobic cores that are packed with either fluorocarbon or hydrocarbon side chains and compared their stabilities. Although larger, the fluorinated residues are accommodated within the protein with minimal structural perturbation, because they closely match the shape of the hydrocarbon side chains that they replace. Thus, stability increases seem to be better explained by increases in buried hydrophobic surface area that accompany fluorination than by specific fluorous interactions between fluorinated side chains. This finding is illustrated by the design of a highly fluorinated protein that, by compensating for the larger volume and surface area of the fluorinated side chains, exhibits similar stability to its nonfluorinated counterpart. These structure-based observations should inform efforts to rationally modulate protein function using noncanonical amino acids. [ABSTRACT FROM AUTHOR]

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions-a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals. [ABSTRACT FROM AUTHOR]

Aims Chloroquine, an anti-malarial quinoline, is structurally similar to quinidine. Both drugs have been shown to block ion channels. We tested the hypothesis that chloroquine's mode of interaction with the vestibule of the cytoplasmic domain of the inward rectifier potassium channel Kir2.1 makes it a more effective IK1 blocker and anti-fibrillatory agent than quinidine. Methods and results We used comparative molecular modelling and ligand docking of the three-dimensional structures of quinidine and chloroquine in the intracellular domain of Kir2.1. Simulations predicted that chloroquine effectively blocks potassium flow by binding at the centre of the ion permeation vestibule of Kir2.1. In contrast, quinidine binds the vestibular side, only partially blocking ion movement. We tested the modelling predictions in Kir2.1-expressing human embryonic kidney (HEK)-293 cells. The half-maximal inhibitory concentration for chloroquine block of IK1 was 1.2 µM, while that of quinidine was 57 µM. Finally, we used optical mapping of Langendorff-perfused mouse hearts with cardiac-specific Kir2.1 up-regulation to compare the anti-fibrillatory effects of the drugs. In five of six hearts, 10 μM quinidine slowed the frequency but did not terminate the tachyarrhythmia. In five of five hearts, 10 μM chloroquine terminated the arrhythmia, restoring sinus rhythm. Conclusion Quinidine only partially blocks IK1. Chloroquine binds at the centre of the ion permeation vestibule of Kir2.1, which makes it a more effective IK1 blocker and anti-fibrillatory agent than quinidine. Integrating the structural biology of drug-ion channel interactions with cellular electrophysiology and optical mapping is an excellent approach to understand the molecular mechanisms of anti-arrhythmic drug action and for drug discovery. [ABSTRACT FROM AUTHOR]

Kinesin motors drive the intracellular transport of multiple cargoes along microtubule tracks; yet, how kinesins discriminate among their many potential cargoes is unknown. We tested whether Kinesin-1 cargoes compete, co-operate or are transported independently of each other. We focused on Kinesin-1 cargoes that bind directly to the kinesin light chain (KLC) subunit, namely the c-Jun NH2-terminal kinase-interacting proteins (JIPs) 1 and 3, Kidins220/ARMS and PAT1. Overexpression of individual cargo proteins in differentiated CAD cells resulted in mislocalization of the endogenous protein but had no effect on localization of other cargo proteins to neurite tips. Thus, while transport of distinct cargoes is saturable, they do not compete with each other. Interestingly, we found that low expression of JIP1 or JIP3 enhanced the transport of the other JIP to neurite tips. Moreover, JIP1 and JIP3 require each other for transport. Co-operative transport is due to an interaction between JIP1 and JIP3 as well as distinct binding sites on the KLC tetratricopeptide repeat (TPR) bundle: the TPR groove binds to C-terminal residues of JIP1, whereas the TPR surface binds to internal residues in JIP3. Formation of a JIP1/JIP3/KLC complex is necessary for efficient JIP1 or JIP3 transport in neuronal cells. Thus, JIP scaffolding proteins are transported in a co-operative manner, despite the independent transport of other Kinesin-1 cargoes. [ABSTRACT FROM AUTHOR]

The bacterial pathogen Listeria monocytogenes replicates within the cytosol of mammalian cells. Mechanisms by which the bacterium exploits the host cytosolic environment for essential nutrients are poorly defined. L. monocytogenes is a lipoate auxotroph and must scavenge this critical cofactor, using lipoate ligases to facilitate attachment of the lipoyl moiety to metabolic enzyme complexes. Although the L. monocytogenes genome encodes two putative lipoate ligases, LplA1 and LplA2, intracellular replication and virulence require only LplA1. Here we show that LplA1 enables utilization of host-derived lipoyl peptides by L. monocytogenes. LplA1 is dispensable for growth in the presence of free lipoate, but necessary for growth on low concentrations of mammalian lipoyl peptides. Furthermore, we demonstrate that the intracellular growth defect of the Δ lplA1 mutant is rescued by addition of exogenous lipoic acid to host cells, suggesting that L. monocytogenes dependence on LplA1 is dictated by limiting concentrations of available host lipoyl substrates. Thus, the ability of L. monocytogenes and other intracellular pathogens to efficiently use host lipoyl peptides as a source of lipoate may be a requisite adaptation for life within the mammalian cell. [ABSTRACT FROM AUTHOR]

Arsenic, a contaminant of water supplies worldwide, is one of the most toxic inorganic ions. Despite arsenic's health impact, there is relatively little structural detail known about its interactions with proteins. Bacteria such as Escherichia co/i have evolved arsenic resistance using the Ars operon that is regulated by ArsR, a repressor protein that dissociates from DNA when As(III) binds. This protein undergoes a critical conformational change upon binding As(III) with three cysteine residues. Unfortunately, structures of ArsR with or without As(III) have not been reported. Alternatively, de novo designed peptides can bind As(III) in an endo configuration within a thiolate-rich environment consistent with that proposed for both ArsR and ArsD. We report the structure of the As(III) complex of Coil Ser L9C to a 1.8-Å resolution, providing x-ray characterization of As(III) in a Tris thiolate protein environment and allowing a structural basis by which to understand arsenated ArsR. [ABSTRACT FROM AUTHOR]

It is often difficult to determine which of the sequence and structural differences between divergent members of multigene families are functionally important. Here we use a laboratory evolution approach to determine functionally important structural differences between two distantly related disulfide isomerases, DsbC and DsbG from Escherichia coil. Surprisingly, we found single amino acid substitutions in DsbG that were able to complement dsbC in vivo and have more DsbC-like isomerase activity in vitro. Crystal structures of the three strongest point mutants, DsbG K113E, DsbG V216M, and DsbG T200M, reveal changes in highly surface-exposed regions that cause DsbG to more closely resemble the distantly related DsbC. In this case, laboratory evolution appears to have taken a direct route to allow one protein family member to complement another, with single substitutions apparently bypassing much of the need for multiple changes that took place over ≈0.5 billion years of evolution. Our findings suggest that, for these two proteins at least, regions important in determining functional differences may represent only a tiny fraction of the overall protein structure. [ABSTRACT FROM AUTHOR]

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacteriumYersinia. Yersiniadelivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site (‘site 2’) within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain (‘site 1’) to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein–protein interactions for determining protein tyrosine phosphatase specificityin vivo, and highlights the sophisticated nature of microbial pathogenicity factors. [ABSTRACT FROM AUTHOR]

The first crystal structure of a phospholipase D (PLD) family member has been determined at 2.0 Å resolution. The PLD superfamily is defined by a common sequence motif, HxK(x)4D(x)6GSxN, and includes enzymes involved in signal transduction, lipid biosynthesis, endonucleases and open reading frames in pathogenic viruses and bacteria. The crystal structure suggests that residues from two sequence motifs form a single active site. A histidine residue from one motif acts as a nucleophile in the catalytic mechanism, forming a phosphoenzyme intermediate, whereas a histidine residue from the other motif appears to function as a general acid in the cleavage of the phosphodiester bond. The structure suggests that the conserved lysine residues are involved in phosphate binding. Large-scale genomic sequencing revealed that there are many PLD family members. Our results suggest that all of these proteins may possess a common structure and catalytic mechanism. [ABSTRACT FROM AUTHOR]

Presents the structures of the unliganded and tungstate-bound crystal forms of protein tyrosine phosphatases (PTPase). Ribbon diagram of the unliganded Yersinia PTPase structure; Yersinia PTPase P-loop and hydrogen-bonding arrays.

Focuses on the structure and function of the protein phosphatases. Structure of serine/threonine protein phosphatases; Structure of protein tyrosine phosphatases; Molecular reaction mechanism.

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2's efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye. [ABSTRACT FROM AUTHOR]

An abstract of the article "Conserved Residues in the HAMP Domain Define a New Family of Proposed Bipartite Energy Taxis Receptors," by Kathryn T. Elliott, Igor B. Zhulin, Jeanne A. Stuckey and Victor J. DiRita is presented.

Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn2+ or Fe2+ in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd2+. The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe2+, but not for Zn2+. However, maximal activity is again achieved by substitution of the endogenous metal with Cd2+. We have determined the X-ray structures of the Cd2+ H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd2+ bound, suggesting that coordination by His185 is not essential to retain Cd2+ in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd2+ with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A. aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion. [Copyright &y& Elsevier]

The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens. A nanoparticle vaccine platform that preserves conformational epitopes of protein antigens elicits robust humoral immune responses against HIV-1 and SARS-CoV-2. Unlabelled Image • Vaccine nanoparticles effectively deliver antigens to dendritic cells in draining lymph nodes. • Vaccine nanoparticles allow conformational display of viral antigens from HIV-1 or SARS-CoV-2. • Vaccine nanoparticles elicit strong neutralizing antibody responses against HIV-1 in rabbits. • Vaccine nanoparticles induce robust and long-lasting antibody responses against SARS-CoV-2 in mice. [ABSTRACT FROM AUTHOR]

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi–pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi–pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi–pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi–pi stacking without disrupting the overall lectin structure. These findings show that pi–pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Infection of animal cells by numerous viruses is detected and countered by a variety of means, including recognition of nonself nucleic acids. The zinc finger antiviral protein (ZAP) depletes cytoplasmic RNA that is recognized as foreign in mammalian cells by virtue of its elevated CG dinucleotide content compared with endogenous mRNAs. Here, we determined a crystal structure of a protein-RNA complex containing the N-terminal, 4-zinc finger human (h) ZAP RNA-binding domain (RBD) and a CG dinucleotide-containing RNA target. The structure reveals in molecular detail how hZAP is able to bind selectively to CG-rich RNA. Specifically, the 4 zinc fingers create a basic patch on the hZAP RBD surface. The highly basic second zinc finger contains a pocket that selectively accommodates CG dinucleotide bases. Structure guided mutagenesis, cross-linking immunoprecipitation sequencing assays, and RNA affinity assays show that the structurally defined CG-binding pocket is not required for RNA binding per se in human cells. However, the pocket is a crucial determinant of high-affinity, specific binding to CG dinucleotide-containing RNA. Moreover, variations in RNA-binding specificity among a panel of CG-binding pocket mutants quantitatively predict their selective antiviral activity against a CGenriched HIV-1 strain. Overall, the hZAP RBD RNA structure provides an atomic-level explanation for how ZAP selectively targets foreign, CG-rich RNA. [ABSTRACT FROM AUTHOR]

Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1's function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis. [ABSTRACT FROM AUTHOR]

Abstract One obstacle in de novo protein design is the vast sequence space that needs to be searched through to obtain functional proteins. We developed a new method using structural profiles created from evolutionarily related proteins to constrain the simulation search process, with functions specified by atomic-level ligand–protein binding interactions. The approach was applied to redesigning the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP), whose primary function is to suppress the cell death by inhibiting caspase-9 activity; however, the function of the wild-type XIAP can be eliminated by the binding of Smac peptides. Isothermal calorimetry and luminescence assay reveal that the designed XIAP domains can bind strongly with the Smac peptides but do not significantly inhibit the caspase-9 proteolytic activity in vitro compared with the wild-type XIAP protein. Detailed mutation assay experiments suggest that the binding specificity in the designs is essentially determined by the interplay of structural profile and physical interactions, which demonstrates the potential to modify apoptosis pathways through computational design. Graphical abstract Unlabelled Image Highlights • Potential to modify apoptosis pathways through computational protein design • New protein design algorithm built on evolutionary profiles • Design functional XIAP BIR3 domain using evolutionary profiles • Design binding specificity by combining profile and physical interactions • Testify protein design by hybrid structure prediction and biochemistry experiments [ABSTRACT FROM AUTHOR]

The transcription factor BCL11A has recently been reported to be a driving force in triple-negative breast cancer (TNBC), contributing to the maintenance of a chemoresistant breast cancer stem cell (BCSC) population. Although BCL11A was shown to suppress γ-globin and p21 and to induce MDM2 expression in the hematopoietic system, its downstream targets in TNBC are still unclear. For its role in transcriptional repression, BCL11A was found to interact with several corepressor complexes; however, the mechanisms underlying these interactions remain unknown. Here, we reveal that BCL11A interacts with histone methyltransferase (PRC2) and histone deacetylase (NuRD and SIN3A) complexes through their common subunit, RBBP4/7. In fluorescence polarization assays, we show that BCL11A competes with histone H3 for binding to the negatively charged top face of RBBP4. To define that interaction, we solved the crystal structure of RBBP4 in complex with an N-terminal peptide of BCL11A (residues 2-16, BCL11A(2-16)). The crystal structure identifies novel interactions between BCL11A and the side of the β-propeller of RBBP4 that are not seen with histone H3. We next show that BCL11A(2-16) pulls down RBBP4, RBBP7, and other components of PRC2, NuRD, and SIN3A from the cell lysate of theTNBCcell line SUM149. Furthermore, we demonstrate the therapeutic potential of targeting the RBBP4-BCL11A binding by showing that a BCL11A peptide can decrease aldehyde dehydrogenase-positive BCSCs and mammosphere formation capacity in SUM149. Together, our findings have uncovered a previously unidentified mechanism that BCL11A may use to recruit epigenetic complexes to regulate transcription and promote tumorigenesis. [ABSTRACT FROM AUTHOR]

Tarin, the Colocasia esculenta lectin from the superfamily of α-D-mannose-specific plant bulb lectins, is a tetramer of 47 kDa composed of two heterodimers. Each heterodimer possesses homologous monomers of ~11.9 (A chain) and ~12.7 (B chain) kDa. The structures of apo and carbohydrate-bound tarin were solved to 1.7 Å and 1.91 Å, respectively. Each tarin monomer forms a canonical β-prism II fold, common to all members of Galanthus nivalis agglutinin (GNA) family, which is partially stabilized by a disulfide bond and a conserved hydrophobic core. The heterodimer is formed through domain swapping involving the C-terminal β-strand and the β-sheet on face I of the prism. The tetramer is assembled through the dimerization of the B chains from heterodimers involving face II of each prism. The 1.91 Å crystal structure of tarin bound to Manα(1,3)Manα(1,6)Man reveals an expanded carbohydrate-binding sequence (QxDxNxVxYx4/6WX) on face III of the β-prism. Both monomers possess a similar fold, except for the length of the loop, which begins after the conserved tyrosine and creates the binding pocket for the α(1,6)-terminal mannose. This loop differs in size and amino-acid composition from 10 other β-prism II domain proteins, and may confer carbohydrate-binding specificity among members of the GNA-related lectin family. [ABSTRACT FROM AUTHOR]

The Drug Design Data Resource (D3R) ran Grand Challenge 2015 between September 2015 and February 2016. Two targets served as the framework to test community docking and scoring methods: (1) HSP90, donated by AbbVie and the Community Structure Activity Resource (CSAR), and (2) MAP4K4, donated by Genentech. The challenges for both target datasets were conducted in two stages, with the first stage testing pose predictions and the capacity to rank compounds by affinity with minimal structural data; and the second stage testing methods for ranking compounds with knowledge of at least a subset of the ligand-protein poses. An additional sub-challenge provided small groups of chemically similar HSP90 compounds amenable to alchemical calculations of relative binding free energy. Unlike previous blinded Challenges, we did not provide cognate receptors or receptors prepared with hydrogens and likewise did not require a specified crystal structure to be used for pose or affinity prediction in Stage 1. Given the freedom to select from over 200 crystal structures of HSP90 in the PDB, participants employed workflows that tested not only core docking and scoring technologies, but also methods for addressing water-mediated ligand-protein interactions, binding pocket flexibility, and the optimal selection of protein structures for use in docking calculations. Nearly 40 participating groups submitted over 350 prediction sets for Grand Challenge 2015. This overview describes the datasets and the organization of the challenge components, summarizes the results across all submitted predictions, and considers broad conclusions that may be drawn from this collaborative community endeavor. [ABSTRACT FROM AUTHOR]

The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein is a multifunctional RNA binding protein implicated in a wide range of biological functions. Mechanisms and putative hnRNP A1–RNA interactions have been inferred primarily from the crystal structure of its UP1 domain bound to ssDNA. RNA stem loops represent an important class of known hnRNP A1 targets, yet little is known about the structural basis of hnRNP A1–RNA recognition. Here, we report the first high-resolution structure (1.92 Å) of UP1 bound to a 5′-AGU-3′ trinucleotide that resembles sequence elements of several native hnRNP A1–RNA stem loop targets. UP1 interacts specifically with the AG dinucleotide sequence via a “nucleobase pocket” formed by the β-sheet surface of RRM1 and the inter-RRM linker; RRM2 does not contact the RNA. The inter-RRM linker forms the lid of the nucleobase pocket and we show using structure-guided mutagenesis that the conserved salt-bridge interactions (R75:D155 and R88:D157) on the α-helical side of the RNA binding surface stabilize the linker in a geometry poised to bind RNA. We further investigated the structural basis of UP1 binding HIV i SL3 ESS3 by determining a structural model of the complex scored by small-angle X-ray scattering. UP1 docks on the apical loop of SL3 ESS3 using its RRM1 domain and inter-RRM linker only. The biophysical implications of the structural model were tested by measuring kinetic binding parameters, where mutations introduced within the apical loop reduce binding affinities by slowing down the rate of complex formation. Collectively, the data presented here provide the first insights into hnRNP A1–RNA interactions. [ABSTRACT FROM AUTHOR]

[Display omitted] An elevated plasma level of soluble ST2 (sST2) is a risk biomarker for graft-versus-host disease (GVHD) and death in patients receiving hematopoietic cell transplantation (HCT). sST2 functions as a trap for IL-33 and amplifies the pro-inflammatory type 1 and 17 response while suppressing the tolerogenic type 2 and regulatory T cells activation during GVHD development. We previously identified small-molecule ST2 inhibitors particularly iST2-1 that reduces plasma sST2 levels and improved survival in two animal models. Here, we reported the structure–activity relationship of the furanylmethylpyrrolidine-based ST2 inhibitors based on iST2-1. Based on the biochemical AlphaLISA assay, we improved the activity of iST2-1 by 6-fold (∼6 μM in IC 50 values) in the inhibition of ST2/IL-33 and confirmed the activities of the compounds in a cellular reporter assay. To determine the inhibition of the alloreactivity in vitro, we used the mixed lymphocyte reaction assay to demonstrate that our ST2 inhibitors decreased CD4+ and CD8+ T cells proliferation and increased Treg population. The data presented in this work are critical to the development of ST2 inhibitors in future. [ABSTRACT FROM AUTHOR]

The lectins, a class of proteins that occur widely in animals, plants, fungi, lichens and microorganisms, are known for their ability to specifically bind to carbohydrates. Plant lectins can be classified into 12 families including the Galanthus nivalis agglutinin (GNA)-related lectin superfamily, which is widespread among monocotyledonous plants and binds specifically to mannose, a behavior that confers remarkable anti-tumor, anti-viral and insecticidal properties on these proteins. The present study characterized a mitogenic lectin from this family, called tarin, which was purified from the crude extract from taro ( Colocasia esculenta ). The results showed that tarin is a glycoprotein with 2–3% carbohydrate content, composed of least 10 isoforms with pIs ranging from 5.5 to 9.5. The intact protein is a heterotetramer of 47 kDa composed of two non-identical and non-covalently associated polypeptides, with small subunits of 11.9 kDa and large subunits of 12.6 kDa. The tarin structure is stable and recovers or maintains its functional structure following treatments at different temperatures and pH. Tarin showed a complex carbohydrate specificity, binding with high affinity to high-mannose and complex N-glycans. Many of these ligands can be found in viruses, tumor cells and insects, as well as in hematopoietic progenitor cells. Chemical modifications confirmed that both conserved and non-conserved amino acids participate in this interaction. This study determined the structural and ligand binding characteristics of a GNA-related lectin that can be exploited for several different purposes, particularly as a proliferative therapeutic molecule that is able to enhance the immunological response. [ABSTRACT FROM AUTHOR]

Blocking the oncoprotein murine double minute 2 (MDM2)-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small-molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301), that has been advanced into phase I clinical trials. SAR405838 binds to MDM2 with Ki = 0.88 nmol/L and has high specificity over other proteins. A cocrystal structure of the SAR405838:MDM2 complex shows that, in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional upregulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53. [ABSTRACT FROM AUTHOR]

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-β-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD. [ABSTRACT FROM AUTHOR]

The phosphatase KdsC cleaves 3-deoxy-D-manno-octulosonate 8-phosphate to generate a molecule of inorganic phosphate and Kdo. Kdo is an essential component of the lipopolysaccharide envelope in Gram-negative bacteria. Because lipopolysaccharide is an important determinant of bacterial resistance and toxicity, KdsC is a potential target for novel antibacterial agents. KdsC belongs to the broad haloacid dehalogenase superfamily. In haloacid dehalogenase superfamily enzymes, substrate specificity and catalytic efficiency are generally dictated by a fold feature called the cap domain. It is therefore not clear why KdsC, which lacks a cap domain, is catalytically efficient and highly specific to 3-deoxy-D-manno-octulosonate 8-phosphate. Here, we present a set of seven structures of tetrameric Escherichia coli KdsC (ranging from 1.4 to 3.06 A in resolution) that model different intermediate states in its catalytic mechanism. A crystal structure of product-bound E. coli KdsC shows how the interface between adjacent mono- mers defines the active site pocket. Kdo is engaged in a network of polar and nonpolar interactions with residues at this interface, which explains substrate specificity. Furthermore, this structural and kinetic analysis strongly suggests that the binding of the flexible C-terminal region (tail) to the active site makes KdsC catalytically efficient by facilitating product release. [ABSTRACT FROM AUTHOR]

Abstract: XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics. [Copyright &y& Elsevier]

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH2) showed the highest affinity (Kd = 17.92 nM) and a large dynamic range (ΔmP=231±0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z′ factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng–Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain. [Copyright &y& Elsevier]

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in ∼180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease. [Copyright &y& Elsevier]

In the SWI/SNF chromatin-remodeling complex, the mutually exclusive catalytic ATPase subunits SMARCA2 and SMARCA4 proteins have a synthetic-lethal relationship. Selectively targeting SMARCA2 for degradation is a promising and new therapeutic strategy for human cancers harboring inactivated mutated SMARCA4. In this study, we report the design, synthesis, and biological evaluation of novel SMARCA2/4 ligands and our subsequent design of PROTAC degraders using high-affinity SMARCA ligands and VHL-1 ligands. Our efforts led to the discovery of high-affinity SMARCA2/4 bromodomain ligands and the development of a potent and selective SMARCA2 degrader and a highly potent SMARCA2/4 and PBRM1 degrader.