Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in non-structural protein 14 (nsp14), which excises nucleotides including antiviral drugs mis-incorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here we determined a 1.6-Å resolution crystal structure of SARS-CoV-2 ExoN in complex with its essential co-factor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. Molecular dynamics simulations further show remarkable flexibility of multi-domain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA-binding to support its exoribonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anti-coronaviral drugs or strategies to attenuate the viral virulence.

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q.

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.

Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands.

Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format. By examining chemical libraries of bioactive lipids, we provided evidence for the selective interaction of CRBP2 with a subset of nonretinoid ligands with the highest affinity for sn-1 and sn-2 MAGs that contain polyunsaturated C18-C20 acyl chains. We also elucidated the structure-affinity relationship for nonretinoid ligands of this protein. We further dissect the molecular basis for this ligand's specificity by analyzing high-resolution crystal structures of CRBP2 in complex with selected derivatives of MAGs. Finally, we identify T51 and V62 as key amino acids that enable the broadening of ligand selectivity to MAGs in CRBP2 as compared with retinoid-specific CRBP1. Thus, our study provides the molecular framework for understanding the lipid selectivity and diverse functions of CRBPs in controlling lipid homeostasis.

Crystal structure of the human PRPK-TPRKB complex.

Mutations of the p53-related protein kinase (PRPK) and TP53RK-binding protein (TPRKB) cause Galloway-Mowat syndrome (GAMOS) and are found in various human cancers. We have previously shown that small compounds targeting PRPK showed anti-cancer activity against colon and skin cancer. Here we present the 2.53 Å crystal structure of the human PRPK-TPRKB-AMPPNP (adenylyl-imidodiphosphate) complex. The structure reveals details in PRPK-AMPPNP coordination and PRPK-TPRKB interaction. PRPK appears in an active conformation, albeit lacking the conventional kinase activation loop. We constructed a structural model of the human EKC/KEOPS complex, composed of PRPK, TPRKB, OSGEP, LAGE3, and GON7. Disease mutations in PRPK and TPRKB are mapped into the structure, and we show that one mutation, PRPK K238Nfs*2, lost the binding to OSGEP. Our structure also makes the virtual screening possible and paves the way for more rational drug design.

Structural basis for multifunctional roles of human Ints3 C-terminal domain.

Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1), and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.

Structural basis of superinfection exclusion by bacteriophage T4 Spackle.

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages - a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

Harmaline Analogs as Substrate-Selective Cyclooxygenase-2 Inhibitors.

We report the design, synthesis, and evaluation of a series of harmaline analogs as selective inhibitors of 2-arachidonylglycerol (2-AG) oxygenation over arachidonic acid (AA) oxygenation by purified cyclooxygenase-2 (COX-2). A fused tricyclic harmaline analog containing a CH3O substituent at C-6 and a CH3 group at the C-1 position of 4,9-dihydro-3H-pyrido[3,4-b]indole (compound 3) was the best substrate-selective COX-2 inhibitor of those evaluated, exhibiting a 2AG-selective COX-2 inhibitory IC50 of 0.022 µM as compared to >1 µM for AA. The 2.66 Å resolution crystal complex of COX-2 with compound 3 revealed that this series of tricyclic indoles binds in the cyclooxygenase channel by flipping the side chain of L531 toward the dimer interface. This novel tricyclic indole series provides the foundation for the development of promising substrate-selective molecules capable of increasing endocannabinoid (EC) levels in the brain to offer new treatments for a variety of diseases, from pain and inflammation to stress and anxiety disorders.

Chlamydia trachomatis glyceraldehyde 3-phosphate dehydrogenase: Enzyme kinetics, high-resolution crystal structure, and plasminogen binding.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an evolutionarily conserved essential enzyme in the glycolytic pathway. GAPDH is also involved in a wide spectrum of non-catalytic cellular 'moonlighting' functions. Bacterial surface-associated GAPDHs engage in many host interactions that aid in colonization, pathogenesis, and virulence. We have structurally and functionally characterized the recombinant GAPDH of the obligate intracellular bacteria Chlamydia trachomatis, the leading cause of sexually transmitted bacterial and ocular infections. Contrary to earlier speculations, recent data confirm the presence of glucose-catabolizing enzymes including GAPDH in both stages of the biphasic life cycle of the bacterium. The high-resolution crystal structure described here provides a close-up view of the enzyme's active site and surface topology and reveals two chemically modified cysteine residues. Moreover, we show for the first time that purified C. trachomatis GAPDH binds to human plasminogen and plasmin. Based on the versatility of GAPDH's functions, data presented here emphasize the need for investigating the Chlamydiae GAPDH's involvement in biological functions beyond energy metabolism.

Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing.

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Šresolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

Erratum: Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly. Corrigendum.

[This corrects the article DOI: 10.1107/S2052252518012459.].

Structural and Functional Characterization of Phosphatidylinositol-Phosphate Biosynthesis in Mycobacteria.

In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B.

The single-stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3-family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high-resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2-pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

Structural insights into the promiscuous DNA binding and broad substrate selectivity of fowlpox virus resolvase.

Fowlpox virus resolvase (Fpr) is an endonuclease that cleaves a broad range of branched DNA structures, including the Holliday junction (HJ), with little sequence-specificity. To better understand the mechanisms underlying its relaxed substrate specificity, we determined the crystal structures of Fpr and that in a novel complex with HJ at 3.1-Å resolution. In the Fpr-HJ complex, two Fpr dimers use several distinct regions to interact with different DNA structural motifs, showing versatility in DNA-binding. Biochemical and solution NMR data support the existence of non-canonical modes of HJ interaction in solution. The binding of Fpr to various DNA motifs are mediated by its flat DNA-binding surface, which is centered on a short loop spanning K61 to I72 and flanked by longer α-helices at the outer edges, and basic side grooves near the dimer interface. Replacing the Fpr loop K61~I72 with a longer loop from Thermus thermophilus RuvC (E71~A87) endows Fpr with an enhanced selectivity toward HJ cleavage but with a target sequence preference distinct from that of RuvC, highlighting a unique role of this loop region in Fpr-HJ interaction. Our work helps explain the broad substrate selectivity of Fpr and suggests a possible mode of its association with poxvirus hairpin telomeres.

An overview of structure, function, and regulation of pyruvate kinases.

In the last step of glycolysis Pyruvate kinase catalyzes the irreversible conversion of ADP and phosphoenolpyruvate to ATP and pyruvic acid, both crucial for cellular metabolism. Thus pyruvate kinase plays a key role in controlling the metabolic flux and ATP production. The hallmark of the activity of different pyruvate kinases is their tight modulation by a variety of mechanisms including the use of a large number of physiological allosteric effectors in addition to their homotropic regulation by phosphoenolpyruvate. Binding of effectors signals precise and orchestrated movements in selected areas of the protein structure that alter the catalytic action of these evolutionarily conserved enzymes with remarkably conserved architecture and sequences. While the diverse nature of the allosteric effectors has been discussed in the literature, the structural basis of their regulatory effects is still not well understood because of the lack of data representing conformations in various activation states. Results of recent studies on pyruvate kinases of different families suggest that members of evolutionarily related families follow somewhat conserved allosteric strategies but evolutionarily distant members adopt different strategies. Here we review the structure and allosteric properties of pyruvate kinases of different families for which structural data are available.

Fluorescent indomethacin-dansyl conjugates utilize the membrane-binding domain of cyclooxygenase-2 to block the opening to the active site.

Many indomethacin amides and esters are cyclooxygenase-2 (COX-2)-selective inhibitors, providing a framework for the design of COX-2-targeted imaging and cancer chemotherapeutic agents. Although previous studies have suggested that the amide or ester moiety of these inhibitors binds in the lobby region, a spacious alcove within the enzyme's membrane-binding domain, structural details have been lacking. Here, we present observations on the crystal complexes of COX-2 with two indomethacin-dansyl conjugates (compounds 1 and 2) at 2.22-Å resolution. Both compounds are COX-2-selective inhibitors with IC50 values of 0.76 and 0.17 µm, respectively. Our results confirmed that the dansyl moiety is localized in and establishes hydrophobic interactions and several hydrogen bonds with the lobby of the membrane-binding domain. We noted that in both crystal structures, the linker tethering indomethacin to the dansyl moiety passes through the constriction at the mouth of the COX-2 active site, resulting in displacement and disorder of Arg-120, located at the opening to the active site. Both compounds exhibited higher inhibitory potency against a COX-2 R120A variant than against the WT enzyme. Inhibition kinetics of compound 2 were similar to those of the indomethacin parent compound against WT COX-2, and the R120A substitution reduced the time dependence of COX inhibition. These results provide a structural basis for the further design and optimization of conjugated COX reagents for imaging of malignant or inflammatory tissues containing high COX-2 levels.

Abnormal Cannabidiol Modulates Vitamin A Metabolism by Acting as a Competitive Inhibitor of CRBP1.

Cellular retinol-binding proteins (CRBPs) facilitate the uptake and intracellular transport of vitamin A. They integrate retinoid metabolism, playing an important role in regulating the synthesis of bioactive vitamin A metabolites. Thus, CRBPs constitute potential pharmacological targets to modulate cellular retinoid status that in turn may have applications in the treatment of certain immunological, metabolic, and ocular disorders. Here we identify abnormal cannabidiol (abn-CBD) as a nonretinoid inhibitor of cellular retinol-binding protein 1 (CRBP1). X-ray crystal structures of CRBP1 in complex with abn-CBD and its derivatives revealed a distinctive mode of protein-ligand interaction and provided a molecular basis for the high affinity and selectivity of this compound. We demonstrated that abn-CBD modulates the flux of retinoids via the retinoid cycle in vivo. Furthermore, the biological activity of abn-CBD was evidenced by its ability to protect against light-induced retinal damage in Balb/cJ mice. Altogether, our findings indicate that targeting selected CRBPs with a small-molecule inhibitor can potentially lead to the development of new therapeutic agents to counteract diseases with etiologies involving imbalance in retinoid metabolism or signaling.

Crystal Structure of the Double Homeodomain of DUX4 in Complex with DNA.

Double homeobox (DUX) transcription factors are unique to eutherian mammals. DUX4 regulates expression of repetitive elements during early embryogenesis, but misexpression of DUX4 causes facioscapulohumeral muscular dystrophy (FSHD) and translocations overexpressing the DUX4 double homeodomain cause B cell leukemia. Here, we report the crystal structure of the tandem homeodomains of DUX4 bound to DNA. The homeodomains bind DNA in a head-to-head fashion, with the linker making anchoring DNA minor-groove interactions and unique protein contacts. Remarkably, despite being tandem duplicates, the DUX4 homeodomains recognize different core sequences. This results from an arginine-to-glutamate mutation, unique to primates, causing alternative positioning of a key arginine side chain in the recognition helix. Mutational studies demonstrate that this primate-specific change is responsible for the divergence in sequence recognition that likely drove coevolution of embryonically regulated repeats in primates. Our work provides a framework for understanding the endogenous function of DUX4 and its role in FSHD and cancer.