Inhibitory Effect on RT-PCR and Restriction Enzyme Activity by Ommochrome and Its Mechanism.

To explore the physiological role and/or pharmacological effects of ommochrome, which is a natural organic pigment widely distributed in Protostomia, we attempted to investigate the influence of ommochrome on RT-PCR and activities of restriction enzymes. It was found that ommin, an ommochrome purified from the diapause eggs of Bombyx mori, inhibited the RT-PCR and restriction enzyme activities. The mechanism of these inhibitory reactions is assumed to be the direct binding of ommochrome to DNA rather than acting against the enzymes because, similarly to actinomycin D, there is a phenoxazine ring in the structure of ommin that is known to be intercalated to DNA. To reveal the ommin/DNA interaction, it was investigated by computational approaches such as molecular docking, molecular dynamics simulation, and free energy calculation. From the computational analyses, it was expected that ommin would bind to DNA with almost the same strength as actinomycin D and intercalate into DNA. This is the first report on the pharmacological effect of ommochrome and its inhibitory mechanism obtained from biochemical and computational analyses.

RNAapt3D: RNA aptamer 3D-structural modeling database.

RNA aptamers are oligonucleotides with high binding affinity and specificity for target molecules and are expected to be a new generation of therapeutic molecules and targeted delivery materials. The tertiary structure of RNA molecules and RNA-protein interaction sites are increasingly important as potential targets for new drugs. The pathological mechanisms of diseases must be understood in detail to guide drug design. In developing RNA aptamers as drugs, information about the interaction mechanisms and structures of RNA aptamer-target protein complexes are useful. We constructed a database, RNA aptamer 3D-structural modeling (RNAapt3D), consisting of RNA aptamer data that are potential drug candidates. The database includes RNA sequences and computationally predicted RNA tertiary structures based on secondary structures and implements methods that can be used to predict unknown structures of RNA aptamer-target molecule complexes. RNAapt3D should enable the design of RNA aptamers for target molecules and improve the efficiency and productivity of candidate drug selection. RNAapt3D can be accessed at https://rnaapt3d.medals.jp.

Importin α2 association with chromatin: Direct DNA binding via a novel DNA-binding domain.

The nuclear transport of proteins is important for facilitating appropriate nuclear functions. The importin α family proteins play key roles in nuclear transport as transport receptors for copious nuclear proteins. Additionally, these proteins possess other functions, including chromatin association and gene regulation. However, these nontransport functions of importin α are not yet fully understood, especially their molecular-level mechanisms and consequences for functioning with chromatin. Here, we report the novel molecular characteristics of importin α binding to diverse DNA sequences in chromatin. We newly identified and characterized a DNA-binding domain-the Nucleic Acid Associating Trolley pole domain (NAAT domain)-in the N-terminal region of importin α within the conventional importin ß binding (IBB) domain that is necessary for nuclear transport of cargo proteins. Furthermore, we found that the DNA binding of importin α synergistically coupled the recruitment of its cargo protein to DNA. This is the first study to delineate the interaction between importin α and chromatin DNA via the NAAT domain, indicating the bifunctionality of the importin α N-terminal region for nuclear transport and chromatin association.

Evaluation of protein-ligand affinity prediction using steered molecular dynamics simulations.

In computational drug design, ranking a series of compound analogs in a manner that is consistent with experimental affinities remains a challenge. In this study, we evaluated the prediction of protein-ligand binding affinities using steered molecular dynamics simulations. First, we investigated the appropriate conditions for accurate predictions in these simulations. A conic harmonic restraint was applied to the system for efficient sampling of work values on the ligand unbinding pathway. We found that pulling velocity significantly influenced affinity predictions, but that the number of collectable trajectories was less influential. We identified the appropriate pulling velocity and collectable trajectories for binding affinity predictions as 1.25 Å/ns and 100, respectively, and these parameters were used to evaluate three target proteins (FK506 binding protein, trypsin, and cyclin-dependent kinase 2). For these proteins using our parameters, the accuracy of affinity prediction was higher and more stable when Jarzynski's equality was employed compared with the second-order cumulant expansion equation of Jarzynski's equality. Our results showed that steered molecular dynamics simulations are effective for predicting the rank order of ligands; thus, they are a potential tool for compound selection in hit-to-lead and lead optimization processes.

Functional profiling of asymmetrically-organized human CCT/TRiC chaperonin.

Molecular organization of the eukaryote chaperonin known as CCT/TRiC complex was recently clarified. Eight distinct subunits are uniquely organized, providing a favorable folding cavity for specific client proteins such as tubulin and actin. Because of its heterogeneous subunit composition, CCT complex has polarized inner faces, which may underlie an essential part of its chaperonin function. In this study, we structurally characterized the closed and open states of CCT complex, using molecular dynamics analyses. Our results showed that the inter-subunit interaction energies were asymmetrically distributed and were remodeled during conformational changes of CCT complex. In addition, exploration of redox related characteristics indicated changes in inner surface properties, including electrostatic potential, pKa and exposure of inner cysteine thiol groups, between the closed and open states. Cysteine activation events were experimentally verified by interaction analyses, using tubulin as a model substrate. Our data highlighted the importance of dynamics-based structural profiling of asymmetrically oriented chaperonin function.

All-atom molecular dynamics analysis of multi-peptide systems reproduces peptide solubility in line with experimental observations.

In order to investigate the contribution of individual amino acids to protein and peptide solubility, we carried out 100 ns molecular dynamics (MD) simulations of 10(6) Å(3) cubic boxes containing ~3 × 10(4) water molecules and 27 tetra-peptides regularly positioned at 23 Å from each other and composed of a single amino acid type for all natural amino acids but cysteine and glycine. The calculations were performed using Amber with a standard force field on a special purpose MDGRAPE-3 computer, without introducing any "artificial" hydrophobic interactions. Tetra-peptides composed of I, V, L, M, N, Q, F, W, Y, and H formed large amorphous clusters, and those containing A, P, S, and T formed smaller ones. Tetra-peptides made of D, E, K, and R did not cluster at all. These observations correlated well with experimental solubility tendencies as well as hydrophobicity scales with correlation coefficients of 0.5 to > 0.9. Repulsive Coulomb interactions were dominant in ensuring high solubility, whereas both Coulomb and van der Waals (vdW) energies contributed to the aggregations of low solubility amino acids. Overall, this very first all-atom molecular dynamics simulation of a multi-peptide system appears to reproduce the basic properties of peptide solubility, essentially in line with experimental observations.

Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.

Experimental identification and theoretical analysis of a thermally stabilized green fluorescent protein variant.

In this study, we aim to relate experimentally measured macroscopic properties to dynamic and structural changes as calculated by molecular dynamics (MD) simulations. We performed the analysis on four GFP (green fluorescent protein) variants, which have amino acid replacements or insertion in a flexible region on the protein surface and which resulted from a previous protein splicing reaction optimization experiment. The variants are a reference GFP (CEGFP), GFP-N144C, GFP-N144C/Y145F, and a GFP with five residues inserted between Y145 and N146 (GFP-5ins). As a result, we identified a single Y145F mutation that increased the thermal stability of GFP-N144C/Y145F by 3-4 °C. Because circular dichroism measurements indicated that the overall GFP ß-barrel fold was maintained in all variants, we presumed that the fluorescence activity and thermal stability related to local changes that could be detected by standard MD simulations. The 60 ns MD simulations indicated that the Y145's hydroxyl group, which is straight and buried in the crystal structure, was bent avoiding the hydrophobic core during the simulation in both CEGFP and GFP-N144C. This local strain was relieved in GFP-N144C/Y145F, where the tyrosine's hydroxyl group was replaced with the F145 hydrophobic aliphatic carbon. F145 remained indeed buried during the simulation maintaining local compactness, which presumably reflected the improved thermal stability of GFP-N144C/Y145F. Furthermore, the analysis of internal water molecules localized within the GFP's ß-barrel suggested that a change in the local hydrogen bonding pattern around the chromophore correlated with a strong fluorescence activity decrease in GFP-5ins. Although relating experimental observation with calculated molecular features proved to be delicate, this study suggested that some microscopic features could be useful reporters for redesigning GFPs and other proteins. The newly identified GFP-N144C/Y145F was among the most stable GFP variant and demonstrates the potential of such computer-aided design.

An efficient computational method for calculating ligand binding affinities.

Virtual compound screening using molecular docking is widely used in the discovery of new lead compounds for drug design. However, the docking scores are not sufficiently precise to represent the protein-ligand binding affinity. Here, we developed an efficient computational method for calculating protein-ligand binding affinity, which is based on molecular mechanics generalized Born/surface area (MM-GBSA) calculations and Jarzynski identity. Jarzynski identity is an exact relation between free energy differences and the work done through non-equilibrium process, and MM-GBSA is a semimacroscopic approach to calculate the potential energy. To calculate the work distribution when a ligand is pulled out of its binding site, multiple protein-ligand conformations are randomly generated as an alternative to performing an explicit single-molecule pulling simulation. We assessed the new method, multiple random conformation/MM-GBSA (MRC-MMGBSA), by evaluating ligand-binding affinities (scores) for four target proteins, and comparing these scores with experimental data. The calculated scores were qualitatively in good agreement with the experimental binding affinities, and the optimal docking structure could be determined by ranking the scores of the multiple docking poses obtained by the molecular docking process. Furthermore, the scores showed a strong linear response to experimental binding free energies, so that the free energy difference of the ligand binding (ΔΔG) could be calculated by linear scaling of the scores. The error of calculated ΔΔG was within ≈ ± 1.5 kcal.mol(-1) of the experimental values. Particularly, in the case of flexible target proteins, the MRC-MMGBSA scores were more effective in ranking ligands than those generated by the MM-GBSA method using a single protein-ligand conformation. The results suggest that, owing to its lower computational costs and greater accuracy, the MRC-MMGBSA offers efficient means to rank the ligands, in the post-docking process, according to their binding affinities, and to compare these directly with the experimental values.

Calculating the Na⁺ translocating V-ATPase catalytic site affinity for substrate binding by homology modeled NtpA monomer using molecular dynamics/free energy calculation.

Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble catalytic domain (V1; NtpA3-B3-D-G) and an integral membrane domain (V0; NtpI-K10) connected by a central and two peripheral stalks (NtpC, NtpD-G and NtpE-F). Recently nucleotide binding of catalytic NtpA monomer has been reported (Arai et al.). In the present study, we calculated the nucleotide binding affinity of NtpA by molecular dynamics (MD) simulation/free energy calculation using MM-GBSA approach based on homology modeled structure of NtpA monomer docked with ATP analogue, adenosine 5'-[ß, γ-imido] triphosphate (AMP-PNP). The calculated binding free energies showed qualitatively good agreement with experimental data. The calculation was cross-validated further by the rigorous method, thermodynamic integration (TI) simulation. Finally, the interaction between NtpA and nucleotides at the atomic level was investigated by the analyses of components of free energy and the optimized model structures obtained from MD simulations, suggesting that electrostatic contribution is responsible for the difference in nucleotide binding to NtpA monomer. This is the first observation and suggestion to explain the difference of nucleotide binding properties in V-ATPase NtpA subunit, and our method can be a valuable primary step to predict nucleotide binding affinity to other subunits (NtpAB, NtpA3B3) and to explore subunit interactions and eventually may help to understand energy transduction mechanism of E. hirae V-ATPase.

Blockade of inflammatory responses by a small-molecule inhibitor of the Rac activator DOCK2.

Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.

Ligand Diffusion on Protein Surface Observed in Molecular Dynamics Simulation.

The process of binding of small ligands to dihydrofolate reductase protein has been investigated using all-atom molecular dynamics simulations. The existence of a mechanism that facilitates the search of the binding site by the ligand is demonstrated. The mechanism consists of ligand diffusing on the protein's surface. It has been discussed in the literature before, but has not been explicitly confirmed for realistic molecular systems. The strength of this nonspecific binding is roughly estimated and found to be essential for the binding kinetics.

Reversible hydrogel formation driven by protein-peptide-specific interaction and chondrocyte entrapment.

We developed a hydrogel self-assembling method driven by the interaction between recombinant tax-interactive protein-1 (TIP1) with the PDZ domain in a molecule, which is fused to each end of the triangular trimeric CutA protein (CutA-TIP1), and a PDZ domain-recognizable peptide which is covalently bound to each terminus of four-armed poly(ethylene glycol) (PDZ-peptide-PEG). Genetic manipulation based on molecular-dynamic simulation generated a cell-adhesive RGD tripeptidyl sequence in the CutA loop region [CutA(RGD)-TIP1]. Spontaneous viscoelastic hydrogel formation occurred when either CutA-TIP1- or CutA(RGD)-TIP1-containing buffer solution and PDZ-peptide-PEG-containing buffer solutions were stoichiometrically mixed. Dynamic viscoelasticity measurement revealed shear stress-dependent reversible-phase transformation: a spontaneous viscoelastic hydrogel was formed at low shear stress, but it was transformed into a sol at high shear stress. Upon the cessation of shear, hydrogel was restored. When chondrocytes were pre-mixed with one of these two components containing buffer solutions, the stoichiometric mixed solution was also spontaneously gelled. Individual rounded cells and multicellular aggregates were entrapped within both hydrogels without substantial cellular impairment regardless of the presence or absence of RGD motif in the CutA-TIP1 molecule. The potential use of such a shear-sensitive hydrogel for injectable cell delivery into diseased or lost cartilage tissue is discussed.

High-performance drug discovery: computational screening by combining docking and molecular dynamics simulations.

Virtual compound screening using molecular docking is widely used in the discovery of new lead compounds for drug design. However, this method is not completely reliable and therefore unsatisfactory. In this study, we used massive molecular dynamics simulations of protein-ligand conformations obtained by molecular docking in order to improve the enrichment performance of molecular docking. Our screening approach employed the molecular mechanics/Poisson-Boltzmann and surface area method to estimate the binding free energies. For the top-ranking 1,000 compounds obtained by docking to a target protein, approximately 6,000 molecular dynamics simulations were performed using multiple docking poses in about a week. As a result, the enrichment performance of the top 100 compounds by our approach was improved by 1.6-4.0 times that of the enrichment performance of molecular dockings. This result indicates that the application of molecular dynamics simulations to virtual screening for lead discovery is both effective and practical. However, further optimization of the computational protocols is required for screening various target proteins.

Mutation of epidermal growth factor receptor is associated with MIG6 expression.

Controlled activation of epidermal growth factor receptor (EGFR) is systematically guaranteed at the molecular level; however, aberrant activation of EGFR is frequently found in cancer. Transcription induced by EGFR activation often involves the coordinated expression of genes that positively and negatively regulate the original signaling pathway; therefore, alterations in EGFR kinase activity may reflect changes in gene expression associated with the pathway. In the present study, we investigated transcriptional changes after EGF stimulation with or without the EGFR kinase inhibitor Iressa in H1299 human non-small-cell lung cancer cells [parental H1299, H1299 cells that overexpress wild-type EGFR (EGFR-WT) and mutant H1299 cells that overexpress EGFR where Leu858 is substituted with Arg (L858R)]. The results obtained clearly demonstrate differences in transcriptional activity in the absence or presence of EGFR kinase activity, with genes sharing the same molecular functions showing distinct expression dynamics. The results show the particular enrichment of EGFR/ErbB signaling-related genes in a differentially expressed gene set, and significant protein expression of MIG6/RALT(ERRFI1), an EGFR negative regulator, was confirmed in L858R. High MIG6 protein expression was correlated with basal EGFR phosphorylation and inversely correlated with EGF-induced extracellular signal-regulated protein kinase phosphorylation levels. Investigation of the NCI-60 cell lines showed that ERRFI1 expression was correlated with EGFR expression, regardless of tissue type. These results suggest that cells accumulate MIG6 as an inherent negative regulator to suppress excess EGFR activity when basal EGFR kinase activity is considerably high. Taking all the above together, an EGFR mutation can cause transcriptional changes to accommodate the activation potency of the original signaling pathway at the cellular level.

Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks.

Self-assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self-assembling proteins, named "Nanolego." Nanolego consists of "structural elements" of a structurally stable symmetrical homo-oligomeric protein and "binding elements," which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self-assembly process. The stabilization method is mediated by disulfide bonds between Cysteine-residues incorporated into the binding elements, and the termination method uses "capping Nanolegos," in which some of the binding elements in the Nanolego are absent for the self-assembled ends. With these technologies, we successfully constructed timing-controlled and size-regulated filament-shape complexes via Nanolego self-assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.

Molecular dynamics simulations reveal that Tyr-317 phosphorylation reduces Shc binding affinity for phosphotyrosyl residues of epidermal growth factor receptor.

The Src homology 2 (SH2) and collagen domain protein Shc plays a pivotal role in signaling via tyrosine kinase receptors, including epidermal growth factor receptor (EGFR). Shc binding to phospho-tyrosine residues on activated receptors is mediated by the SH2 and phospho-tyrosine binding (PTB) domains. Subsequent phosphorylation on Tyr-317 within the Shc linker region induces Shc interactions with Grb2-Son of Sevenless that initiate Ras-mitogen-activated protein kinase signaling. We use molecular dynamics simulations of full-length Shc to examine how Tyr-317 phosphorylation controls Shc conformation and interactions with EGFR. Our simulations reveal that Shc tyrosine phosphorylation results in a significant rearrangement of the relative position of its domains, suggesting a key conformational change. Importantly, computational estimations of binding affinities show that EGFR-derived phosphotyrosyl peptides bind with significantly more strength to unphosphorylated than to phosphorylated Shc. Our results unveil what we believe is a novel structural phenomenon, i.e., tyrosine phosphorylation of Shc within its linker region regulates the binding affinity of SH2 and PTB domains for phosphorylated Shc partners, with important implications for signaling dynamics.

Structural and functional basis of a role for CRKL in a fibroblast growth factor 8-induced feed-forward loop.

The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics. By taking advantage of structural simulations, we performed in silico analysis of the interactions of the autophosphorylation sites of FGR receptor 1 (FGFR1) with the SH2 domain of CRKL or a highly related protein, CRK. As predicted by simulations, we confirm the specific physical interaction of phosphorylated Y463 (pY463) in FGFR1 with the CRKL SH2 domain at an affinity approximately 30-fold stronger than that of CRK. We also provide evidence that interactions outside of the core YXXP motif have a significant impact on physical association, which is consistent with predictions from molecular-dynamics simulations. Furthermore, we identify CRKL as an essential component of an FGF8-induced feed-forward loop permissive for efficient activation of the mitogen-activated protein kinase Erk1/2, as well as FGF8-induced anchorage-independent cell growth, using Crkl-deficient cells or a pY463 synthetic peptide. Although many cells generally require cell-matrix adhesion, our results demonstrate that CRKL permits cells to bypass the strict need for adhesion in response to FGF8 through direct interaction with receptor.

Folding dynamics of 10-residue beta-hairpin peptide chignolin.

Short peptides that fold into beta-hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 micros) of the 10-residue beta-hairpin peptide chignolin, which is the smallest beta-hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen-bond network and the complete hydrophobic core as well as the arrangement of side-chain-side-chain interactions occur at approximately the same time. This three-step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the beta-hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism.

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.