Elucidation of binding mechanism, affinity, and complex structure between mWT1 tumor-associated antigen peptide and HLA-A*24:02.

We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved x-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the major histocompatibility complex (MHC) molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine.

Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.

The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca2+ -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca2+ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca2+ to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS.

Development of novel lithocholic acid derivatives as vitamin D receptor agonists.

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.

Mutational effects of Cys113 on structural dynamics of Pin1.

Pin1 is a peptidyl-prolyl isomerase (PPIase) which catalyzes cis/trans isomerization of pS/pT-P bond. Its activity is related to various cellular functions including suppression of Alzheimer's disease. A cysteine residue C113 is known to be important for its PPIase activity; a mutation C113A reduced the activity by 130-fold. According to various nuclear magnetic resonance experiments for mutants of C113 and molecular dynamics (MD) simulation of wild-type Pin1, the protonation sate of Sγ of C113 regulates the hydrogen-bonding network of the dual-histidine motif (H59, H157) whose dynamics may affect substrate binding ability. However, it was still unclear why such local dynamic changes altered the PPIase activity of Pin1. In this study, we performed 500 ns of MD simulations of full-length wild-type Pin1 and C113A mutant in order to elucidate why the mutation C113A drastically reduced the PPIase activity of Pin1. The principal component analysis for both MD trajectories clearly elucidated that the mutation C113A suppressed the dynamics of Pin1 because it stabilized a hydrogen-bond between Nɛ of H59 and Oγ of S115. In the dynamics of wild-type protein, the phosphate binding loop (K63-S71) as well as the interdomain hinge showed the closed-open dynamics which correlated with the change of the hydrogen-bonding network of the dual-histidine motif. In contrast, in the dynamics of C113A mutant, the phosphate binding loop took only the closed conformation together with the interdomain hinge. Such closed-open dynamics must be essential for the PPIase activity of Pin1.

The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.

CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP.

Toll-like receptor 7 (TLR7) plays an essential role in development of systemic lupus erythematosus by co-stimulating B cells reactive to the endogenous TLR7 ligand Sm/ribonucleoprotein (RNP), a crucial lupus self-antigen. However, how the TLR7-mediated autoimmune response is regulated is not yet known. In this study, we demonstrate that CD72, an inhibitory B cell co-receptor known to prevent development of lupus, recognizes Sm/RNP at the extracellular C-type lectin-like domain (CTLD) and specifically inhibits B cell response to Sm/RNP. Moreover, the CTLD of CD72c, a lupus-susceptible allele, binds to Sm/RNP less strongly than that of lupus-resistant CD72a Reduced binding of CD72c is supported by x-ray crystallographic analysis that reveals a considerable alteration in charge at the putative ligand-binding site. Thus, CD72 appears to specifically inhibit B cell response to the endogenous TLR7 ligand Sm/RNP through CTLD-mediated recognition of Sm/RNP, thereby preventing production of anti-Sm/RNP antibody crucial for development of lupus.

Modeling and experimental assessment of a buried Leu-Ile mutation in dengue envelope domain III.

Envelope protein domain III (ED3) of the dengue virus is important for both antibody binding and host cell interaction. Here, we focused on how a L387I mutation in the protein core could take place in DEN4 ED3, but cannot be accommodated in DEN3 ED3 without destabilizing its structure. To this end, we modeled a DEN4_L387I structure using the Penultimate Rotamer Library and taking the DEN4 ED3 main-chain as a fixed template. We found that three out of seven Ile(387) conformers fit in DEN4 ED3 without introducing the severe atomic clashes that are observed when DEN3 serotype's ED3 is used as a template. A more extensive search using 273 side-chain rotamers of the residues surrounding Ile(387) confirmed this prediction. In order to assess the prediction, we determined the crystal structure of DEN4_L387I at 2 Å resolution. Ile(387) indeed adopted one of the three predicted rotamers. Altogether, this study demonstrates that the effects of single mutations are to a large extent successfully predicted by systematically modeling the side-chain structures of the mutated as well as those of its surrounding residues using fixed main-chain structures and assessing inter-atomic steric clashes. More accurate and reliable predictions require considering sub-angstrom main-chain deformation, which remains a challenging task.

Synthesis, Biological Activities, and X-ray Crystal Structural Analysis of 25-Hydroxy-25(or 26)-adamantyl-17-[20(22),23-diynyl]-21-norvitamin D Compounds.

Novel 19-norvitamin D analogues (ADYW1-4, 5a-d) in which an adamantyl diyne side chain is attached directly to the 17-position of the D ring are designed and stereoselectively synthesized. The adamantane ring of these analogues was expected to interfere with helix 12 (H12, activation function 2) of the vitamin D receptor (VDR) to modulate its activities. The analogue 5b binds to the VDR (7% of the natural hormone) and shows significant partial agonistic activity in transactivation assay. Compound 5b showed considerable selectivity in VDR target genes expressions in vitro, it was taken up by target cells 2-3 times more readily, and its lifetime was three times longer than the natural hormone. The X-ray crystal structure of 5b in complex with VDR reveals that the ligand binds similarly to the natural hormone, but the diyne moiety is slightly bent (angles around the diyne 5° to 8°) with respect to the original diyne vitamin D compound 6 in VDR (<1°) due to steric hindrance with helix 12.

Design and synthesis of a potent inhibitor of class 1 DYRK kinases as a suppressor of adipogenesis.

Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer's disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARγ and C/EBPα. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome.

Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants.

Dengue fever is a re-emerging tropical disease and its severe form is caused by cross-reactivity between its four serotypes (DEN1, DEN2, DEN3 and DEN4). The third domain of the viral envelope protein (ED3) contains the two major putative epitopes and is a highly suitable model protein for examining the molecular determinants of a virus' sero-specificity. Here we examine d the sero-specificity and cross-reactivity of the immune response against DEN3 and DEN4 ED3 using six epitope grafted ED3 variants where the surface-exposed epitope residues from DEN3 ED3 were switched to those of DEN4 ED3 and vice versa. We prepared anti-DEN3 and anti-DEN4 ED3 serum by immunizing Swiss albino mice and measured their reactivities against all six grafted mutants. As expected, both sera exhibited strong reactivity against its own serotype's ED3, and little cross-reactivity against their counterpart serotype's ED3s. E2 played a major role in the sero-specificity of anti-DEN3 serum, whereas E1 was important for DEN4 ED3's sero-specificity. Next, the reactivity patterns corroborated our working hypothesis that sero-specificity could be transferred by grafting the surface exposed epitope residues from one serotype to the other. To analyze the above results from a structural viewpoint, we determined the crystal structure of a DEN4 ED3 variant, where E2 was grafted from DEN3 ED3, at 2.78Å resolution and modeled the structures of the five remaining grafted variants by assuming that the overall backbone remained unchanged. The examination of the electrostatic and molecular surfaces of the variants suggested some further rationale for the sero-specificity of the immune responses.

Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice.

Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.

CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses.

A wide range of antiviral drugs is currently available; however, drug-resistant viruses have begun to emerge and represent a potential public health risk. Here, we explored the use of compounds that inhibit or interfere with the action of essential host factors to prevent virus replication. In particular, we focused on the cyclin-dependent kinase 9 (CDK9) inhibitor, FIT-039, which suppressed replication of a broad spectrum of DNA viruses through inhibition of mRNA transcription. Specifically, FIT-039 inhibited replication of herpes simplex virus 1 (HSV-1), HSV-2, human adenovirus, and human cytomegalovirus in cultured cells, and topical application of FIT-039 ointment suppressed skin legion formation in a murine HSV-1 infection model. FIT-039 did not affect cell cycle progression or cellular proliferation in host cells. Compared with the general CDK inhibitor flavopiridol, transcriptome analyses of FIT-039-treated cells revealed that FIT-039 specifically inhibited CDK9. Given at concentrations above the inhibitory concentration, FIT-039 did not have a cytotoxic effect on mammalian cells. Importantly, administration of FIT-039 ameliorated the severity of skin lesion formation in mice infected with an acyclovir-resistant HSV-1, without noticeable adverse effects. Together, these data indicate that FIT-039 has potential as an antiviral agent for clinical therapeutics.

Combination of triple bond and adamantane ring on the vitamin D side chain produced partial agonists for vitamin D receptor.

Vitamin D receptor (VDR) ligands are therapeutic agents that are used for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism and have immense potential as therapeutic agents for autoimmune diseases, cancers, and cardiovascular diseases. However, the major side effect of VDR ligands, the development of hypercalcemia, limits their expanded use. To develop tissue-selective VDR modulators, we have designed vitamin D analogues with an adamantane ring at the side chain terminal, which would interfere with helix 12, the activation function 2, and modulate the VDR potency. Here we report 25- or 26-adamantyl-23,23,24,24-tetradehydro-19-norvitamin D derivatives (ADTK1-4, 4b,a and 5a,b). These compounds showed high VDR affinities (90% at maximum), partial agonistic activities (EC50 10(-9)-10(-8) M with 40-80% efficacy) in transactivation, and tissue-selective activity in target gene expressions. We investigate the structure-activity relationships of these compounds on the basis of their X-ray crystal structures.

Peptidyl-prolyl isomerase activity of FK506 binding protein 12 prevents tau peptide from aggregating.

The Alzheimer's disease-related protein, tau, aggregates into neurofibrillary tangles when it is hyperphosphorylated. The amino acid sequence included in the third repeat (R3) of the microtubule-binding region is suspected to be the main factor for tau aggregation. Here, we synthesized a 31-residue oligopeptide, corresponding to the R3 region, and characterized its aggregation propensity under various conditions. This peptide aggregated even in the absence of an aggregation-inducing molecule at a low salt concentration, while it did not form any aggregates at a high salt concentration. This suggests that hydrophilic interactions are the main cause of aggregation. We then investigated the function of FK506-binding protein (FKBP) 12, which is known to accumulate in neurofibrillary tangles in vivo, on aggregation of the R3 peptide and found that FKBP12 completely prevented the peptide from aggregating at a concentration ratio of 1 : 4 (peptide:FKBP12). FKBP12 also restored the oligomer of the peptide to its monomeric status. Mutational studies on the catalytic center of FKBP12 indicated that peptidyl-prolyl isomerase activity of FKBP12 was essential for prevention of aggregation. Assuming that the propensity of aggregation of the peptide is different in each cis-/trans-isomer, we propose that the aggregation behavior of the R3 peptide can be theoretically described with a simple kinetic scheme, in which only the cis-isomer can aggregate and FKBP12 catalyzes isomerization of the peptide in both the monomeric and aggregative states.

22S-butyl-1alpha,24R-dihydroxyvitamin D3: recovery of vitamin D receptor agonistic activity.

We recently reported that 22S-butyl-1alpha,24R-dihydroxyvitamin D(3)3 recovers the agonistic activity for vitamin D receptor (VDR), although its 25,26,27-trinor analog 2 is a potent VDR antagonist. To investigate the structural features involved in the recovery of agonism, we crystallized the ternary complex of VDR-ligand-binding domain, ligand 3 and coactivator peptide, and conducted X-ray crystallographic analysis of the complex. Compared with the complex with 2, the complex with 3 recovered the following structural features: a pincer-type hydrogen bond between the 24-hydroxyl group and VDR, the conformation of Leu305, the positioning of His301 and His393, the stability of the complex, and intimate hydrophobic interactions between the ligand and helix 12. In addition, we evaluated the potency of both compounds for recruiting RXR and coactivator. The results indicate that the complex with 3 generates a suitable surface for coactivator recruitment. These studies suggest that the action of 2 as an antagonist is caused by the generation of a surface not suitable for coactivator recruitment due to the lack of hydrophobic interactions with helix 12 as well as insufficient hydrogen bond formation between the 24-hydroxyl group and VDR. We concluded that the action of 3 as an agonist is based on the elimination of these structural defects in the complex with 2.

A new class of vitamin D analogues that induce structural rearrangement of the ligand-binding pocket of the receptor.

To identify novel vitamin D receptor (VDR) ligands that induce a novel architecture within the ligand-binding pocket (LBP), we have investigated eight 22-butyl-1alpha,24-dihydroxyvitamin D(3) derivatives (3-10), all having a butyl group as the branched alkyl side chain. We found that the 22S-butyl-20-epi-25,26,27-trinorvitamin D derivative 5 was a potent VDR agonist, whereas the corresponding compound 4 with the natural configuration at C(20) was a potent VDR antagonist. Analogues with the full vitamin D(3) side chain were less potent agonist, and whether they were agonists or antagonists depended on the 24-configuration. X-ray crystal structures demonstrated that the VDR-LBD accommodating the potent agonist 5 has an architecture wherein the lower side and the helix 11 side of the LBP is simply expanded relative to the canonical active-VDR situation; in contrast, the potent antagonist 4 induces an extra cavity to accommodate the branched moiety. This is the first report of a VDR antagonist that generates a new cavity to alter the canonical pocket structure of the ligand occupied VDR.

Requirements for peptidyl-prolyl isomerization activity: a comprehensive mutational analysis of the substrate-binding cavity of FK506-binding protein 12.

Peptidyl-prolyl isomerase (PPIase) activity is exhibited by many proteins belonging to the PPIase family. However, the catalytic mechanism of this activity remains to be completely elucidated. Here, we selected human FK506-binding protein 12 (FKBP12) as the model PPIase and investigated the nature of amino acid residues essential for the activity. The crystal structures of several complexes of PPIase with short peptides revealed that the residues Asp37, Arg42, Phe46, Val55, Trp59, and Tyr82 in the substrate-binding cavity of FKBP12 appear to play key roles in the PPIase activity. Each of these six residues was substituted by 20 common amino acid residues. The activity of each mutant protein was measured using a peptide analog by the chymotrypsin digestion assay and then compared with wild-type FKBP12. It was found that site-specific interactions by the side chains of amino acid residues constituting the substrate-binding cavity were not essential for the PPIase activity, although the 37th, 55th, and 82nd amino acid residues significantly contributed to the activity. This suggests that the PPIase activity requires only the hydrophobic cavity that captures the Pro-containing peptide.

Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii.

Archaeal/eukaryotic primases form a heterodimer consisting of a small catalytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N-terminal domain (NTD) of PriL (PriL(NTD); residues 1-222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii, at 2.9 A resolution. The PriL(NTD) structure consists of two subdomains, the helix-bundle and twisted-strand domains. The latter is structurally flexible, and is expected to contain a PriS interaction site. Pull-down and surface plasmon resonance analyses of structure-based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155-Tyr156-Ile157 region near the flexible region is the PriS-binding site, as the Y155A/Y156A/I157A mutation markedly reduces PriS binding, by 1000-fold. These findings and a structural comparison with a previously reported PriL(NTD)-PriS complex suggest that the presented alternative conformations of the twisted-strand domain facilitate the heterodimer assembly.