X-ray Crystal Structure Research Articles

Structure-Based Design of Covalent SARS-CoV-2 Papain-like Protease Inhibitors.

The COVID-19 pandemic is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Like other RNA viruses, SARS-CoV-2 continues to evolve with or without drug selection pressure, and many variants have emerged since the beginning of the pandemic. The papain-like protease, PLpro, is a cysteine protease that cleaves viral polyproteins as well as ubiquitin and ISG15 modifications from host proteins. Leveraging our recently discovered Val70Ub binding site in PLpro, we designed covalent PLpro inhibitors by connecting cysteine reactive warheads to the biarylphenyl PLpro inhibitors via flexible linkers. Several leads displayed potent enzymatic inhibition (IC50 = 0.1-0.3 μM) and antiviral activity (EC50 = 0.09-0.96 μM). Fumaramide inhibitors Jun13567 (15), Jun13728 (16), and Jun13714 (18) showed favorable in vivo pharmacokinetic properties with intraperitoneal injection. The X-ray crystal structure of PLpro with Jun13567 (15) validated our design strategy, revealing covalent conjugation between the catalytic Cys111 and the fumaramide warhead. The results suggest these covalent PLpro inhibitors are promising SARS-CoV-2 antiviral drug candidates.

Exploring C2 and N6 Substituent Effects on Truncated 4'-Thioadenosine Derivatives as Dual A2A and A3 Adenosine Receptor Ligands.

Based on high binding affinity of truncated 2-hexynyl-4'-thioadenosine (3a) at both A2A adenosine receptor (AR) and A3 AR, we explored structure-activity relationship (SAR) of the C2-substitution by altering chain length of the 2-hexynyl moiety, thereby evaluating the hydrophobic pocket size. A series of truncated N6-substituted 4'-thioadenosine derivatives with C2-alkynyl substitution were successfully synthesized from D-mannose, using a palladium-catalyzed Sonogashira coupling reaction as the key step, whose structures were confirmed by the X-ray crystal structure of 4h. As the size of the alkynyl group at the C2-position increased, the binding affinity improved; however, when the substituted group was larger than hexynyl, the binding affinity decreased. The introduction of a bulky hydrophobic group such as 3-halobenzyl group at the free N6-amino group decreased the binding affinity at hA2AAR. These results confirm our previous findings that a free amino group at N6-position and longer hydrophobic chain at C2-position are essential for hA2A AR binding affinity. The introduction of a bulky hydrophobic group at free N6-amino group maintained the binding affinity at hA3 AR. The binding mode of truncated 2-substituted-4'-thioadenosine derivatives to hA2A and hA3 AR were predicted by a molecular docking study.

Synthesis of (Z)-Enediynes via Stereoinvertive Nucleophilic Substitution of (E)-Sulfonylethenes with Arylethynide, and Their Aggregation-Induced Optical Properties.

(Z)-Enediynes were successfully synthesized from a trio of terminal ethynes through consecutive three-step reactions: iodosulfonylation of ethyne with I2/PhSO2Na, followed by ethynylations of iodo and sulfonyl moieties of the resulting iodosulfonylethene via Sonogashira-Hagihara coupling and nucleophilic addition-elimination, respectively. The molecular structure of the obtained (Z)-enediyne was fully characterized by X-ray crystal structure analysis, revealing that the nucleophilic substitution of (E)-sulfonylethene with arylethynide underwent a selective stereoinversion. The (Z)-enediynes exhibited photoluminescence in both the solution and solid states (crystals and powders). Ph2N-substituted derivatives showed remarkable solvatofluorochromism, and upon replacing the solvent from toluene to acetonitrile, the emission color changed from blue to yellow.

Stoichiometry of ligand binding and role of C-terminal lysines in Mycobacterium tuberculosis and human GAPDH multifunctionality.

Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; EC1.2.1.12) has several functions in Mycobacterium tuberculosis (Mtb) and the human host. Apart from its role in glycolysis, it serves both as a cell surface and a secreted receptor for plasmin(ogen) (Plg/Plm), transferrin (Tf), and lactoferrin (Lf). Plg sequestration by Mtb GAPDH facilitates bacterial adhesion and tissue invasion, while an equivalent interaction with host GAPDH regulates immune cell migration. In both, host and microbe, internalization of Tf/Lf-GAPDH complexes serves as a route for iron acquisition. To date, the structure of Mtb GAPDH or the residues involved in these moonlighting interactions have not been identified. This study provides the first known X-ray crystal structure of Mtb GAPDH. Through further mutagenesis and functional assays, we found that the C-terminal lysines of Mtb and human GAPDH affect enzyme activity and ligand binding. We also establish the stoichiometry of Plg, Tf and Lf interactions with the GAPDH tetramer. Lastly, molecular simulation studies reveal the interactions of the C-terminal lysine residues.

Deciphering the Conformations of Glutathione Oxidized Peptide: A Comparative NMR Study in Solution and Solid-State Environments.

Glutathione (GSH) and its oxidized dimer (GSSG) play an important role in living systems as an antioxidant, balancing the presence of reactive oxygen species (ROS). The central thiol (-S-S-) bond in GSSG can undergo free rotation, providing multiple conformations with respect to the S-S bridge. The six titratable sites of GSSG, which are influenced by pH variations, affect these conformations in solution, whereas in solids, additionally crystal packing effects come into play. In view of differing reports about the structure of GSSG in literature, we have here conducted an extensive reexamination of its conformations using NMR, and contrasting results have been obtained for solution and solid state. In solution, the existence of more than one antiparallel orientation of the monomer unit with different hydrogen bonding schemes has been indicated by NOE and amide temperature coefficient results. On the other hand, in the solid-state, a 1H-1H double-quantum (DQ) to 13C single-quantum (SQ) correlation study has confirmed a parallel orientation, consistent with the reported X-ray crystal structure. Experimentally assigned solid-state NMR resonances have been validated using GIPAW calculations incorporated in the Quantum ESPRESSO package.

NeIle, a Genetically Encoded Indicator for Branched-Chain Amino Acids Based on mNeonGreen Fluorescent Protein and LIVBP Protein.

Branched-chain amino acids (BCAAs) play an important role in the functioning of mammalian cells and the central nervous system. However, available genetically encoded indicators for BCAAs are based on Förster resonance energy transfer and have a limited dynamic range. We developed a single fluorescent protein-based sensor for BCAAs, called NeIle, which is composed of circularly permutated mNeonGreen protein inserted into the leucine-isoleucine-valine binding protein (LIVBP) from Escherichia coli bacteria. In solution, the NeIle indicator displayed a positive fluorescence response to adding isoleucine, leucine, and valin amino acids with high ΔF/F dynamic ranges of 27-, 19-, and 11-fold and the corresponding affinity values of 5.0, 2.9, and 75 mM, respectively. The spectral and biochemical properties of the NeIle indicator were characterized in solution. We characterized the brightness of the NeIle indicator in living mammalian cells, including cultured neurons. Using the NeIle indicator, we successfully visualized the dynamics of isoleucine transients in different organelles of mammalian cells. We obtained and analyzed the X-ray crystal structure of the NeIle indicator in an isoleucine-bound state. Structure-guided directed mutagenesis of the NeIle indicator revealed the basis of its fluorescence response and selectivity to isoleucine.

Crystal Structure of Caryolan-1-ol Synthase, a Sesquiterpene Synthase Catalyzing an Initial Anti-Markovnikov Cyclization Reaction.

In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus, with and without an inactive analog of the farnesyl diphosphate (FPP) substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP structure was solved to 2.65 Å resolution and showed the ligand in an elongated orientation, incapable of undergoing the initial cyclization event to form a C1-C11 bond. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case, well fit by a curled-up tetraethylene glycol molecule recruited, presumably, from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotation of some active-site residues in comparison to the 2FFPP-structure. Most notably, W56 and F183 undergo 90° rotations between the 2FFPP complex and apoenzyme structures, suggesting that these residues provide interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L and F183A variants were observed to be severely restricted in their ability to catalyze C1-C11 cyclization of the FPP substrate and instead produced predominantly acyclic terpene products dominated by farnesol, β-farnesene, and nerolidol.

Cysteine-Persulfide Sulfane Sulfur-Ligated Zn Complex of Sulfur-Carrying SufU in the SufCDSUB System for Fe-S Cluster Biosynthesis.

SufU, a component of the SufCDSUB Fe-S cluster biosynthetic system, serves as a Zn-dependent sulfur-carrying protein that delivers inorganic sulfur in the form of cysteine persulfide from SufS to SufBCD. To understand this sulfur delivery mechanism, we studied the X-ray crystal structure of SufU and its sulfur-carrying state (persulfurated SufU) and performed functional analysis of the conserved amino acid residues around the Zn sites. Interestingly, sulfur-carrying SufU with Cys41-persulfide (Cys41-Sγ-Sδ-) exhibited a unique Zn coordination structure, in which electrophilic Sγ is ligated to Zn and nucleophilic/anionic Sδ is bound to distally conserved Arg125. This structure is distinct from those of other Cys-persulfide-Sδ-ligated metals of metalloproteins, such as hybrid cluster proteins and SoxAX. Functional analysis of SufU variants with Zn-ligand and Arg125 substitutions revealed that both Zn and Arg125 are critical for the function of SufU with SufS. The Zn-persulfide structure of SufU provides insight into the sulfur-transfer process, suggesting that persulfide-Sδ- is stabilized via bridging by Zn and Arg125 of SufU.

Structural insights into phosphatidylethanolamine N-methyltransferase PmtA mediating bacterial phosphatidylcholine synthesis.

Phosphatidylethanolamine N-methyltransferase (PmtA) catalyzes the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE). Although PC is one of the major phospholipids constituting bilayer membranes in eukaryotes, certain bacterial species encode PmtA, a membrane-associated methyltransferase, to produce PC, which is correlated with cellular stress responses, adaptability to environmental changes, and symbiosis or virulence with eukaryotic hosts. Depending on the organism, multiple PmtAs may be required for producing monomethyl- and dimethyl-PE derivatives along with PC, whereas in organisms such as Rubellimicrobium thermophilum, a single enzyme is sufficient to direct all three methylation steps. In this study, we present the x-ray crystal structures of PmtA from R. thermophilum in complex with dimethyl-PE and S-adenosyl-l-homocysteine, as well as in its lipid-free form. Moreover, we demonstrate that the enzyme associates with the cellular membrane via electrostatic interactions facilitated by a group of critical basic residues and can successively methylate PE and its methylated derivatives, culminating in the production of PC.

Isolation, X-ray crystal structure and DFT calculations for 9β,13β-epoxy-7-abietene from the essential oil of Tetradenia riparia

Isolation, X-ray crystal structure and DFT calculations for 9β,13β-epoxy-7-abietene from the essential oil of Tetradenia riparia

Identification of the high-pressure phases of α-SnWO4 combining x-ray diffraction and crystal structure prediction

We have characterized the high-pressure behavior of -SnWO4. The compound has been studied up to 30 GPa using a diamond-anvil cell and synchrotron powder X-ray diffraction. We report evidence of two structural phase transitions in the pressure range covered in our study, and we propose a crystal structure for the two high-pressure phases. The first one, observed around 12.9 GPa, has been obtained combining indexation using DICVOL and density-functional theory calculations. The second high-pressure phase, observed around 17.5 GPa, has been determined by using the CALYPSO code, the prediction of which was supported by a Le Bail fit to the experimental X-ray diffraction patterns. The proposed structural sequence involves two successive collapses of the unit-cell volume and an increase in the coordination number of Sn and W atoms. The room-temperature equations of state, the principal axes of compression and their compressibility, the elastic constants, and the elastic moduli are reported for -SnWO4 and for the two high-pressure phases.

Ligand-induced CaMKIIα hub Trp403 flip, hub domain stacking, and modulation of kinase activity.

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.

Defining unique structural features in the MAFA and MAFB transcription factors that control Insulin gene activity

MAFA and MAFB are related basic-leucine-zipper domain-containing transcription factors which have important overlapping and distinct regulatory roles in a variety of cellular contexts, including hormone production in pancreatic islet cells. Here we first examined how mutating conserved MAF protein-DNA contact sites obtained from X-ray crystal structure analysis impacted their DNA-binding and Insulin enhancer-driven activity. While most of these interactions were essential and their disruption severely compromised activity, we identified that regions outside of these contact sites also contributed to transcriptional activity. AlphaFold 2, an artificial intelligence-based structural prediction program, was used to determine if there were also differences in the three-dimensional organization of the non-DNA binding/dimerization sequences of MAFA and MAFB. This analysis was conducted on the wildtype (WT) proteins as well as the pathogenic MAFASer64Phe and MAFBSer70Alatrans-activation domain mutants, with differences revealed between MAFAWT and MAFBWT as well as between MAFASer64Phe and MAFAWT, but not between MAFBSer70Ala and MAFBWT. Moreover, dissimilarities between these proteins were also observed in their ability to cooperatively stimulate Insulin enhancer-driven activity in the presence of other islet-enriched transcription factors. Analysis of MAFA and MAFB chimeras disclosed that these properties were influenced by their unique C-terminal region structural differences predicted by AlphaFold 2. Our findings have revealed key structural features of these closely related proteins that impact their ability to regulate gene expression.

The infrared spectrum of the protic ionic liquid 1-methylimidazolium nitrate: An ab initio molecular dynamics simulation study

Complex features observed in the infrared (IR) spectrum of the protic ionic liquid 1-methylimidazolium nitrate, [C1im][NO3], are assigned using ab initio molecular dynamics (AIMD) simulation. AIMD is used at the same level of theory to simulate the known X-ray crystal structure of [C1im][NO3] and a cluster of ionic pairs representing the liquid phase. The calculation of several single particle and collective time correlation functions of velocity forms the basis for the AIMD simulation assignment of the infrared spectra. The AIMD simulation demonstrates that the lift of the degeneracy of the anion asymmetric stretching mode, νas(NO3), due to symmetry breaking and coupling to external modes result in multiple band splitting in the crystal spectrum in the region of the νas(NO3) mode. Owing of the intrinsic anharmonic characteristic of the AIMD simulation, the calculation accounts for the broad band in the high-frequency range of the IR spectrum caused by the cation ν(NH) stretching mode in strong cation–anion hydrogen bond. The AIMD simulation of the liquid captures spectral changes upon melting of [C1im][NO3] due to loosening of the NH···O hydrogen bond and changes in the [NO3]- interaction with other hydrogen atoms of the imidazolium ring. The AIMD simulation assigns the intermolecular stretching mode of the hydrogen bond, ν(NH…O), as the band at 183 cm−1 in the far infrared spectrum of the [C1im][NO3] crystal.

Tuning the Reactivity of Copper(II)-Nitrite Core Towards Nitric Oxide Generation.

Insights into the molecular mechanism and factors affecting nitrite-to-NO transformation at transition metal sites are essential for developing sustainable technologies relevant to NO-based therapeutics, waste water treatment, and agriculture. A set of copper(II)-nitrite complexes1-4have been isolated employing tridentate pincer-type ligands (quL,pyL,ClArOL-,PhOL-) featuring systematically varied donors. Although the X-ray crystal structures of the copper(II)-nitrite cores in1-4are comparable, electrochemical studies on complexes1-4reveal that redox properties of these complexes differ due to the changes in thes-donor abilities of thephenolate / N-heterocycle based donor sites. Reactivity of these nitrite complexes with oxygen-atom-transfer (OAT) reagent (e.g. triphenyl phosphine Ph3P) and H+/e-donor reagent (e.g. substituted phenols ArOH) show the reduction of nitrite to NO gas. Detailed kinetic investigations including kinetic isotope effect (KIE), Eyring analyses for determining the activation parameters unfold that reduction of nitrite at copper(II) by Ph3P or ArOH are influenced by the CuII/CuIredox potential. Finally, this study allows mechanism driven development of catalytic nitrite reduction by ArOH in the presence of 10 mol% copper complex (1).

Creation of Metal-Complex-Integrated Tensegrity Triangle DNA Crystals.

Structural DNA nanotechnology is an emerging field and is expected to be used for various applications in materials science. In this study, we designed a DNA tensegrity triangle to accommodate the bipyridine complexes with metal ions (Ni2+ and Fe2+) at the center of the space within the triangle. A metal-bipyridine-incorporated DNA tensegrity triangle was crystalized, and the presence of metals within it was confirmed through X-ray crystal structure analysis. A signal of the anomalous dispersion effect derived from metal was observed in the center of the DNA triangle.

Quantum refinement in real and reciprocal space using the Phenix and ORCA software.

X-ray and neutron crystallography, as well as cryogenic electron microscopy (cryo-EM), are the most common methods to obtain atomic structures of biological macromolecules. A feature they all have in common is that, at typical resolutions, the experimental data need to be supplemented by empirical restraints, ensuring that the final structure is chemically reasonable. The restraints are accurate for amino acids and nucleic acids, but often less accurate for substrates, inhibitors, small-molecule ligands and metal sites, for which experimental data are scarce or empirical potentials are harder to formulate. This can be solved using quantum mechanical calculations for a small but interesting part of the structure. Such an approach, called quantum refinement, has been shown to improve structures locally, allow the determination of the protonation and oxidation states of ligands and metals, and discriminate between different interpretations of the structure. Here, we present a new implementation of quantum refinement interfacing the widely used structure-refinement software Phenix and the freely available quantum mechanical software ORCA. Through application to manganese superoxide dismutase and V- and Fe-nitrogenase, we show that the approach works effectively for X-ray and neutron crystal structures, that old results can be reproduced and structural discrimination can be performed. We discuss how the weight factor between the experimental data and the empirical restraints should be selected and how quantum mechanical quality measures such as strain energies should be calculated. We also present an application of quantum refinement to cryo-EM data for particulate methane monooxygenase and show that this may be the method of choice for metal sites in such structures because no accurate empirical restraints are currently available for metals.

Structural insights into the recognition of purine-pyrimidine dinucleotide repeats by zinc finger protein ZBTB43.

Purine-pyrimidine repeats (PPRs) can form left-handed Z-form DNA and induce DNA double-strand breaks (DSBs), posing a risk for genomic rearrangements and cancer. The zinc finger (ZF) and BTB domain-containing protein 43 (ZBTB43) is a transcription factor containing two Cys2-His2 (C2H2) and one C3H1 zinc fingers and plays a crucial role in maintaining genomic and epigenomic integrity by converting mutagenic Z-form PPRs to the B-form in prospermatogonia. Despite its importance, the molecular mechanism underlying the recognition of PPRs by ZBTB43 remains elusive. In this study, we determined the X-ray crystal structure of the ZBTB43 ZF1-3 in complex with the B-form DNA containing the CA repeats sequence. The structure reveals that ZF1 and ZF2 primarily recognize the CACA sequence through specific hydrogen-bonding and van der Waals contacts via a quadruple center involving Arg389, Met411, His413, and His414. These interactions were further validated by fluorescence-based DNA-binding assays using mutated ZBTB43 variants. Our structural investigation provides valuable insights into the recognition mechanism of PPRs by ZBTB43 and suggests a potential role for ZBTB43 in the transformation of Z-DNA to B-DNA, contributing to the maintenance of genomic stability.

Insoluble Acyclic Cucurbit[n]uril-Type Receptors Capture Iodine from the Vapor Phase.

Nuclear energy makes large contributions toward meeting global energy needs, but societal concerns remain high given the impacts of the intended release of radioactive materials including 129I and 131I. In this paper we explore the use of a homologous series of acyclic CB[n] type hosts (H1 - H4) as adsorbents of iodine from the vapor phase. We find that H2 - H4, but not H1 - perform well in this application with uptake capacities of 2.2 g g-1, 1.5g g-1, and 1.9g g-1, respectively. The chemisorptive uptake process involves partial oxidation of catechol walled H2 to quinone walled host and capture of I3- and I5-. Solid H2 can be regenerated by treatment with Na2S2O4 and reused at least five times. The x-ray crystal structure of H2 is also reported.

New Tricyclic Aryl Quinazoline Derivatives by Suzuki-Miyaura Cross-Coupling.

A number of new deoxyvasicinone (2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one) and mackinazolinone (6,7,8,9-tetrahydro-11H-pyrido[2,1-b]quinazolin-11-one) derivatives with aryl substituents at C7/C8 and at C5 are reported. These compounds are rare representatives of their kind and were prepared in high yields by Suzuki-Miyaura cross-coupling reactions between 7-bromo-2,3-dihydro[2,1-b]quinazoline-9-(1H)-one, 5,7-dibromo-2,3-dihydro[2,1-b]quinazoline-9-(1H)-one or 8-bromomackinazolinone and respective arylboronic acids with palladium acetate as the catalyst. The products were characterized spectroscopically and, in addition, by X-ray crystal structure analyses in six cases.