Structure Of Apo Form Research Articles

High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Crystal structure of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1 in complex with chitotetraose

BackgroundChitosanases (EC 3.2.1.132) hydrolyze chitosan which is a polymer of glucosamine (GlcN) linked by β − 1,4 bonds, and show cleavage specificity against partially acetylated chitosan containing N-acetylglucosamine (GlcNAc) residues. Chitosanases' structural underpinnings for cleavage specificity and the conformational switch from open to closed structures are still a mystery. MethodsThe GH-46 subclass III chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), which also catalyzes the hydrolysis of GlcN-GlcNAc bonds in addition to GlcN-GlcN, has had its chitotetraose [(GlcN)4]-complexed crystal structure solved at 1.35 Å resolution. ResultsThe MH-K1 chitosanase's (GlcN)4-bound structure has numerous structural similarities to other GH-46 chitosanases in terms of substrate binding and catalytic processes. However, subsite −1, which is absolutely specific for GlcN, seems to characterize the structure of a subclass III chitosanase due to its distinctive length and angle of a flexible loop. According to a comparison of the (GlcN)4-bound and apo-form structures, the particular binding of a GlcN residue at subsite −2 through Asp77 causes the backbone helix to kink, which causes the upper- and lower-domains to approach closely when binding a substrate. ConclusionsAlthough GH-46 chitosanases vary in the finer details of the subsites defining cleavage specificity, they share similar structural characteristics in substrate-binding, catalytic processes, and potentially in conformational change. General significanceThe precise binding of a GlcN residue to the −2 subsite is essential for the conformational shift that occurs in all GH-46 chitosanases, as shown by the crystal structures of the apo- and substrate-bound forms of MH-K1 chitosanase.

Bacterioferritin nanocage structures uncover the biomineralization process in ferritins.

Iron is an essential element involved in various metabolic processes. The ferritin family of proteins forms nanocage assembly and is involved in iron oxidation, storage, and mineralization. Although several structures of human ferritins and bacterioferritins have been solved, there is still no complete structure that shows both the trapped Fe-biomineral cluster and the nanocage. Furthermore, whereas the mechanism of iron trafficking has been explained using various approaches, structural details on the biomineralization process (i.e. the formation of the mineral itself) are generally lacking. Here, we report the cryo-electron microscopy (cryo-EM) structures of apoform and biomineral bound form (holoforms) of the Streptomyces coelicolor bacterioferritin (ScBfr) nanocage and the subunit crystal structure. The holoforms show different stages of Fe-biomineral accumulation inside the nanocage, in which the connections exist in two of the fourfold channels of the nanocage between the C-terminal of the ScBfr monomers and the Fe-biomineral cluster. The mutation and truncation of the bacterioferritin residues involved in these connections significantly reduced the iron and phosphate binding in comparison with those of the wild type and together explain the underlying mechanism. Collectively, our results represent a prototype for the bacterioferritin nanocage, which reveals insight into its biomineralization and the potential channel for bacterioferritin-associated iron trafficking.

Cryo-EM structures of mitochondrial ABC transporter ABCB10 in apo and biliverdin-bound form

ABCB10, a member of ABC transporter superfamily that locates in the inner membrane of mitochondria, plays crucial roles in hemoglobin synthesis, antioxidative stress and stabilization of the iron transporter mitoferrin-1. Recently, it was found that ABCB10 is a mitochondrial biliverdin exporter. However, the molecular mechanism of biliverdin export by ABCB10 remains elusive. Here we report the cryo-EM structures of ABCB10 in apo (ABCB10-apo) and biliverdin-bound form (ABCB10-BV) at 3.67 Å and 2.85 Å resolution, respectively. ABCB10-apo adopts a wide-open conformation and may thus represent the apo form structure. ABCB10-BV forms a closed conformation and biliverdin situates in a hydrophobic pocket in one protomer and bridges the interaction through hydrogen bonds with the opposing one. We also identify cholesterols sandwiched by BVs and discuss the export dynamics based on these structural and biochemical observations.

SARS-CoV-2 polyprotein substrate regulates the stepwise Mpro cleavage reaction

The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate 10 recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscore the roles of the structure, conformation and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.

Structure of human NaV1.6 channel reveals Na+ selectivity and pore blockade by 4,9-anhydro-tetrodotoxin

The sodium channel NaV1.6 is widely expressed in neurons of the central and peripheral nervous systems, which plays a critical role in regulating neuronal excitability. Dysfunction of NaV1.6 has been linked to epileptic encephalopathy, intellectual disability and movement disorders. Here we present cryo-EM structures of human NaV1.6/β1/β2 alone and complexed with a guanidinium neurotoxin 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), revealing molecular mechanism of NaV1.6 inhibition by the blocker. The apo-form structure reveals two potential Na+ binding sites within the selectivity filter, suggesting a possible mechanism for Na+ selectivity and conductance. In the 4,9-ah-TTX bound structure, 4,9-ah-TTX binds to a pocket similar to the tetrodotoxin (TTX) binding site, which occupies the Na+ binding sites and completely blocks the channel. Molecular dynamics simulation results show that subtle conformational differences in the selectivity filter affect the affinity of TTX analogues. Taken together, our results provide important insights into NaV1.6 structure, ion conductance, and inhibition.

Structural and Dynamics Studies of the Spcas9 Variant Provide Insights into the Regulatory Role of the REC1 Domain

CRISPR-Cas9 from Streptococcus pyogenes has been harnessed as a powerful biotechnological tool for DNA sequence modification. The gRNA binding the recognition lobe (REC lobe) of Cas9 is the initial step of Cas9-mediated DNA cleavage and is a prerequisite to trigger the subsequent conformational transition of Cas9. The functions of REC1–3 domains have been summarized as “sense” nucleic acids, “regulate” the HNH conformational transition, and finally “lock” the HNH domain at the cleavage site. Therefore, understanding the dynamic mechanism of the REC lobe is important to design and engineer better Cas9 enzymes. Here, we solved the apo form structure of a SpCas9 variant (xCas9 P411T) that harbors seven mutations in the SpCas9 REC region and one mutation in the C-terminal domain. This structure reveals that the mutations in the REC lobe resulted in a distinct conformation of the REC1 domain. Together with the biochemical results, we hypothesized that the dynamic properties of the REC1 domain are associated with Cas9 PAM specificity. Furthermore, using molecular dynamics simulations to model the conformational activation process of xCas9 P411T, we found that REC1 acts as a hub that modulates multiple domain motion during Cas9 activation and target site binding. These results lay the foundation for engineering an improved CRISPR-Cas9 system.

Structural analysis of LpqY, a substrate-binding protein from the SugABC transporter of Mycobacterium tuberculosis, provides insights into its trehalose specificity.

The LpqY-SugABC transporter of Mycobacterium tuberculosis (Mtb) salvages residual trehalose across the cell membrane, which is otherwise lost during the formation of cell-wall glycoconjugates in the periplasm. LpqY, a substrate-binding protein from the SugABC transporter, acts as the primary receptor for the recognition of trehalose, leading to its transport across the cell membrane. Since trehalose is crucial for the survival and virulence of Mtb, trehalose receptors should serve as important targets for novel drug design against tuberculosis. In order to comprehend the detailed architecture and substrate specificity, the first crystal structures of both apo and trehalose-bound forms of M.tuberculosis LpqY (Mtb-LpqY) are presented here at 2.2 and 1.9 Å resolution, respectively. The structure exhibits an N-lobe and C-lobe and is predominantly composed of a globular α/β domain connected by a flexible hinge region concealing a deep binding cleft. Although the trehalose-bound form of Mtb-LpqY revealed an open ligand-bound conformation, the glucose moieties of trehalose are seen to be strongly held in place by direct and water-mediated hydrogen bonds within the binding cavity, producing a Kd of 6.58 ± 1.21 µM. These interactions produce a distinct effect on the stereoselectivity for the α-1,1-glycosidic linkage of trehalose. Consistent with the crystal structure, molecular-dynamics simulations further validated Asp43, Asp97 and Asn151 as key residues responsible for strong and stable interactions throughout a 1 µs time frame, thus capturing trehalose in the binding cavity. Collectively, the results provide detailed insights into how the structure and dynamics of Mtb-LpqY enable it to specifically bind trehalose in a relaxed conformation state.

α-synuclein enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis.

Parkinson's disease (PD) results from degeneration of dopamine and norepinephrine neurons due to α-synuclein aggregates that likely have their origin in the gut. Tyrosine hydroxylase (TH) catalyses the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A second enzyme, DOPA decarboxylase (DDC), catalyzes the conversion of L-DOPA to dopamine. A third enzyme, dopamine ß-hydroxylase (DBH), catalyzes the conversion of dopamine to norepinephrine. To analyze possible interactions of α-synuclein with TH, DDC and DBH, we performed in silico protein-protein docking. Protein data bank (pdb) entries were searched on the RCSB Protein Data Bank. We identified four structures that allowed us to examine the relationship of α-synuclein with TH, DDC, and DBH: (1) Human micelle-bound alpha-synuclein, (2) solution structure of the regulatory domain of tyrosine hydroxylase (Rattus norvegicus), (3) crystal structure of human aromatic L-amino acid decarboxylase (DOPA decarboxylase) in the apo form and (4) crystal structure of human dopamine ß-hydroxylase at 2.9 angstrom resolution. We used the ClusPro server (https://cluspro.org) for protein-protein docking. The protein structures were visualized with PyMOL v 2.3.4. α-synuclein partially enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis. α-synuclein may dock too far away from DOPA decarboxylase to affect its function directly. Our in silico finding of α-synuclein partly enfolding tyrosine hydroxylase and dopamine ß-hydroxylase suggests that α-synuclein docking inhibition could increase dopamine and norepinephrine biosynthesis, ameliorating PD symptoms. Small molecules that bind to α-synuclein have already been identified. Further studies may lead to new small molecule drugs that block α-synuclein enfolding of tyrosine hydroxylase and dopamine ß-hydroxylase.

Prerequisite Binding Modes Determine the Dynamics of Action of Covalent Agonists of Ion Channel TRPA1.

Transient receptor potential ankyrin 1 (TRPA1) is a transmembrane protein channeling the influx of calcium ions. As a polymodal nocisensor, TRPA1 can be activated by thermal, mechanical stimuli and a wide range of chemically damaging molecules including small volatile environmental toxicants and endogenous algogenic lipids. After activation by such compounds, the ion channel opens up, its central pore widens allowing calcium influx into the cytosol inducing signal transduction pathways. Afterwards, the calcium influx desensitizes irritant evoked responses and results in an inactive state of the ion channel. Recent experimental determination of structures of apo and holo forms of TRPA1 opened the way towards the design of new agonists, which can activate the ion channel. The present study is aimed at the elucidation of binding dynamics of agonists using experimental structures of TRPA1-agonist complexes at the atomic level applying molecular docking and dynamics methods accounting for covalent and non-covalent interactions. Following a test of docking methods focused on the final, holo structures, prerequisite binding modes were detected involving the apo forms. It was shown how reversible interactions with prerequisite binding sites contribute to structural changes of TRPA1 leading to covalent bonding of agonists. The proposed dynamics of action allowed a mechanism-based forecast of new, druggable binding sites of potent agonists.

Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation

The circadian clock is a biological timekeeper that operates through transcription-translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations ("out" for CRY1 and "in" for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic "out" and "in" conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

Structural analysis and insight into effector binding of the niacin-responsive repressor NiaR from Bacillus halodurans

The niacin-responsive repressor, NiaR, is transcriptional repressor of certain nicotinamide adenine dinucleotide (NAD) biosynthetic genes in response to an increase in niacin levels. NAD is a vital molecule involved in various cellular redox reactions as an electron donor or electron acceptor. The NiaR family is conserved broadly in the Bacillus/Clostridium group, as well as in the Fusobacteria and Thermotogales lineages. The NiaR structure consists of two domains: an N-terminal DNA-binding domain, and a C-terminal regulation domain containing a metal-binding site. In this paper, we report the crystal structures of apo and niacin-bound forms of NiaR from Bacillus halodurans (BhNiaR). The analysis of metal-binding and niacin-binding sites through the apo and niacin-bound structures is described. Each N- and C-terminal domain structure of BhNiaR is almost identical with NiaR from Thermotoga maritima, but the overall domain arrangement is quite different. A zinc ion is fully occupied in each subunit with well-conserved residues in the C-terminal domain. Niacin is also located at a hydrophobic pocket near the zinc ion in the C-terminal domain.

Crystal structure of barley agmatine coumaroyltransferase, an N-acyltransferase from the BAHD superfamily.

The enzymes of the BAHD superfamily, a large group of acyl-CoA-dependent acyltransferases in plants, are involved in the biosynthesis of diverse secondary metabolites. While the structures of several O-acyltransferases from the BAHD superfamily, such as hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase, have been elucidated, no structural information on N-acyltransferases is available. Hordeum vulgare agmatine coumaroyltransferase (HvACT) is an N-acyltransferase from the BAHD superfamily and is one of the most important enzymes in the secondary metabolism of barley. Here, an apo-form structure of HvACT is reported as the first structure of an N-acyltransferase from the BAHD superfamily. HvACT crystals diffracted to 1.8 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 57.6, b = 59.5, c=73.6 Å, α = 90, β = 91.3 , γ = 90°. Like other known BAHD superfamily structures, HvACT contains two domains that adopt a two-layer αβ-sandwich architecture and a solvent-exposed channel that penetrates the enzyme core.

Molecular Basis for PI(3,5)P2 Recognition by SNX11, a Protein Involved in Lysosomal Degradation and Endosome Homeostasis Regulation

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is an essential phosphoinositide required for endosome homeostasis and sorting for lysosomal degradation; however, the underlying mechanisms, especially in mammals, remain elusive or unexplored. Here we determined a structure of PI(3,5)P2 bound to Sorting Nexin 11 (SNX11) with an opened PPII-C loop. We also obtained an SNX11 structure with its PPII-C in “closed” form that serves as a potential PI3P-binding model. In addition, our results reveal that SNX11 can interact with the V1D subunit of vacuolar H+-ATPase (V-ATPase), which provides a link between PI(3,5)P2 and human V-ATPase and further evidence for their roles in the endosome homeostasis regulation. Lastly, a new apo-form structure of SNX11, combined with molecular dynamics (MD) studies, indicates that the α5 helix can unfold from the PX domain of SNX11 when targeting the membrane or interacting with its partner. Taken together, these findings identify a novel PI(3,5)P2 effector, which will shed light on the PIs recognizing mechanism and the understanding of the downstream sorting events triggered by different PI binding.

Photosynthetic Phosphoribulokinase Structures: Enzymatic Mechanisms and the Redox Regulation of the Calvin-Benson-Bassham Cycle.

The Calvin-Benson-Bassham (CBB) cycle is responsible for CO2 assimilation and carbohydrate production in oxyphototrophs. Phosphoribulokinase (PRK) is an essential enzyme of the CBB cycle in photosynthesis, catalyzing ATP-dependent conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate. The oxyphototrophic PRK is redox-regulated and can be further regulated by reversible association with both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidized chloroplast protein CP12. The resulting GAPDH/CP12/PRK complex is central in the regulation of the CBB cycle; however, the PRK-CP12 interface in the recently reported cyanobacterial GAPDH/CP12/PRK structure was not well resolved, and the detailed binding mode of PRK with ATP and Ru5P remains undetermined, as only apo-form structures of PRK are currently available. Here, we report the crystal structures of cyanobacterial (Synechococcus elongatus) PRK in complex with ADP and glucose-6-phosphate and of the Arabidopsis (Arabidopsis thaliana) GAPDH/CP12/PRK complex, providing detailed information regarding the active site of PRK and the key elements essential for PRK-CP12 interaction. Our structural and biochemical results together reveal that the ATP binding site is disrupted in the oxidized PRK, whereas the Ru5P binding site is occupied by oxidized CP12 in the GAPDH/CP12/PRK complex. This structure-function study greatly advances the understanding of the reaction mechanism of PRK and the subtle regulations of redox signaling for the CBB cycle.

Characterization of a Schistosoma mansoni NDPK expressed in sexual and digestive organs

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.

The preliminary efficacy evaluation of the CTLA-4-Ig treatment against Lupus nephritis through in-silico analyses

The humanized cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA-4-Ig) has been used to treat Lupus nephritis (LN) based on CTLA-4s negative regulation of T-cell activation through competent to binding with CD80/CD86, the inherent genetic factors influencing the CTLA-4-Ig treatment efficacy are widely unknown. Here, 62 nonsynonymous single nucleotide variants (nsSNVs) of CTLA-4 gene, 184 of CD80 and 201 of CD86 were identified and validated within both EMBL-EBI and dbSNP databases. Next, the nsSNVs rs1466152724 in CTLA-4, rs1196816748, rs765515058, rs1157880125, rs1022857991, and rs142547094 in CD80 and rs1203132714 in CD86 were consistently suggested to be deleterious by SIFT, PolyPhen-2, PROVEAN and meta LR. Based on the 3D structure stability analysis, the variant rs765515058 causing G167V in CD80 was found to reduce the protein's stability through changing the characters of constructed structure of complete CD80 apo form and stabilizing amino acid residues of CD80 holo form in a great degree. Furthermore, the interaction energy analysis results suggested that rs1022857991 causing C50F may reduce the binding energy of CTLA-4 with CD80. Along with the increasing variants, these nsSNVs’ effects on the interaction of CTLA-4 with CD80/CD86 will increase, and thus influence the CTLA-4-Ig treatment efficacy against LN.

Structural and kinetic analyses of penicillin-binding protein 4 (PBP4)-mediated antibiotic resistance in Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) causes serious community-acquired and nosocomial infections worldwide. MRSA strains are resistant to a variety of antibiotics, including the classic penicillin and cephalosporin classes of β-lactams, making them intractable to treatment. Although β-lactam resistance in MRSA has been ascribed to the acquisition and activity of penicillin-binding protein 2a (PBP2a, encoded by mecA), it has recently been observed that resistance can also be mediated by penicillin-binding protein 4 (PBP4). Previously, we have shown that broad-spectrum β-lactam resistance can arise following serial passaging of a mecA-negative COL strain of S. aureus, creating the CRB strain. This strain has two missense mutations in pbp4 and a mutation in the pbp4 promoter, both of which play an instrumental role in β-lactam resistance. To better understand PBP4's role in resistance, here we have characterized its kinetics and structure with clinically relevant β-lactam antibiotics. We present the first crystallographic PBP4 structures of apo and acyl-enzyme intermediate forms complexed with three late-generation β-lactam antibiotics: ceftobiprole, ceftaroline, and nafcillin. In parallel, we characterized the structural and kinetic effects of the PBP4 mutations present in the CRB strain. Localized within the transpeptidase active-site cleft, the two substitutions appear to have different effects depending on the drug. With ceftobiprole, the missense mutations impaired the Km value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. Our findings provide evidence that S. aureus CRB has at least two PBP4-mediated resistance mechanisms.

Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis

MarR family proteins are transcriptional regulators that control expression of bacterial proteins involved in metabolism, virulence, stress responses and multi-drug resistance, mainly via ligand-mediated attenuation of DNA binding. Greater understanding of their underlying regulatory mechanism may open up new avenues for the effective treatment of bacterial infections. To gain molecular insight into the mechanism of Rv2887, a MarR family protein in M. tuberculosis, we first showed that it binds salicylate (SA) and para-aminosalicylic acid (PAS), its structural analogue and an antitubercular drug, in a 1:1 stoichiometry with high affinity. Subsequent determination and analysis of Rv2887 crystal structures in apo form, and in complex with SA, PAS and DNA showed that SA and PAS bind to Rv2887 at similar sites, and that Rv2887 interacts with DNA mainly by insertion of helix α4 into the major groove. Ligand binding triggers rotation of the wHTH domain of Rv2887 toward the dimerization domain, causing changes in protein conformation such that it can no longer bind to a 27 bp recognition sequence in the upstream region of gene Rv0560c. The structures provided here lay a foundation for the design of small molecules that target Rv2887, a potential new approach for the development of anti-mycobacterials.

Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand-Binding Domain Revealed by a Combination Andlysis of MD Simulations and SAXS Experiments

Vitamin D receptor (VDR) is a member of the nuclear receptor (NR) family and regulates the expression of genes related to calcium homeostasis , immunomodulation, and cell differentiation and proliferation. The VDR and other NRs are involved in human diseases, therefore, understanding of their regulation by ligand binding is a pharmaceutical demand for structure-based drug design. According to X-ray crystallographic analysis of NRs and other experimental results, the ligand-binding domain (LBD) undergoes conformational change upon ligand binding, and the local conformational change around helix 12 is key to regulating agonism/antagonism.Many crystal structures of agonist/antagonist-binding VDR-LBD have been solved so far. However, all the crystal structures are almost identical, regardless of agonist/antagonist binding, and those crystal structures are considered as the agonist form. Because the conformation of helix 12 is key, the crystal structures do not provide structural insight into the mechanism of antagonist activity. In addition, no crystal structures of apo form has been reported, and the exact conformation of apo form remains unknown.To reveal the apo and antagonist-binding forms of VDR-LBD, we analyze them by a combination approach of small-angle X-ray scattering (SAXS) and molecular dynamics (MD). We obtained SAXS profiles of both forms, however, the profiles were different from the theoretical profiles calculated from crystal structures. To clarify the solution structures at atomic resolution, we conducted MD and collected each structural ensemble. Comparing the SAXS profiles and the MD results, we report a reliable structure for each form. In both forms, helix 12 is partially unraveled, and does not adopt the active form, preventing co-activator binding for transactivation. In apo form, helix 11 bends outward by a kink-centered hinge-bending motion, and the motion creates wide entrance of the ligand-binding pocket. In antagonist-binding form, loop 11-12 remarkably flexible compared to that in apo form.