Register-Shifted Structures in Base-Flipped Uracil-Damaged DNA.

We report the occurrence of register-shifted structures in simulations of uracil-containing dsDNA. These occur when the 3' base vicinal to uracil is thymine in U:A base-paired DNA. Upon base flipping of uracil, this 3' thymine hydrogen bonds with the adenine across the uracil instead of its complementary base. The register-shifted structure is persistent and sterically blocks re-entry of uracil into the helix stack. Register shifting might be important for DNA repair since the longer exposure of the lesion in register-shifted structures could facilitate enzymatic recognition and repair.

Systematic assessment of the flexibility of uracil damaged DNA.

Uracil is a common DNA lesion which is recognized and removed by uracil DNA-glycosylase (UDG) as a part of the base excision repair pathway. Excision proceeds by base flipping, and UDG efficiency is thought to depend on the ease of deformability of the bases neighboring the lesion. We used molecular dynamics simulations to assess the flexibility of a large library of dsDNA strands, containing all tetranucleotide motifs with U:A, U:G, T:A or C:G base pairs. Our study demonstrates that uracil damaged DNA largely follows trends in flexibility of undamaged DNA. Measured bending persistence lengths, groove widths, step parameters and base flipping propensities demonstrate that uracil increases the flexibility of DNA, and that U:G base paired strands are more flexible than U:A strands. Certain sequence contexts are more deformable than others, with a key role for the 3' base next to uracil. Flexibilities are large when this base is an A or G, and repressed for a C or T. A 5' T adjacent to the uracil strongly promotes flexibility, but other 5' bases are less influential. DNA bending is correlated to step deformations and base flipping, and bending aids flipping. Our study implies that the link between substrate flexibility and UDG efficiency is widely valid, helps explain why UDG prefers to bind U:G base paired strands, and suggests that the DNA bending angle of the UDG-substrate complex is optimal for base flipping.Communicated by Ramaswamy H. Sarma.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

Secondary structure of peptides mimicking the Gly-rich regions of major ampullate spidroin protein 1 and 2.

Spider dragline silk has highly desirable material properties, possessing high extensibility, strength, and biocompatibility. Before it is spun, the constituent proteins are stored in a concentrated dope that is void of fibrils. To investigate the structural properties of the amorphous fiber regions in the dope, computer simulations were performed on model peptides representing the N. clavipes Gly-rich regions. Analysis of the secondary structure found predominantly turns, bends and coils; a small 31-helical population decreased with increasing concentration. Interestingly, the population of 31-helices saw a large increase in octanol. These results indicate that the unusual 31-helical secondary structure of the Gly-rich region of the fiber is a consequence of the spinning process, and that the low dielectric environment of the fiber may assist in favoring this structure.

Conformational Free-Energy Differences of Large Solvated Systems with the Focused Confinement Method.

The focused confinement method (FCM) is a reaction coordinate-free simulation approach for the calculation of conformational free-energy differences in explicit solvent. The method uses reference states for the conformations of interest, partitions the solute into conformationally active and inactive regions, and requires the calculation of desolvation free energies of mixed harmonic-anharmonic states as part of its procedure. The reference states and partitioning affect the speed of convergence of FCM's constituent simulations in opposing manners, but in the thermodynamic limit, they have no effect on calculated conformational free-energy differences. To aid fast convergence of large systems, a general procedure to quickly partition and construct reference states is introduced. With this, two sets of reference states and associated partitionings were constructed for the closed and open conformation of triosephosphate isomerase (TIM). Despite TIM's size, highly converged desolvation free energies were readily obtained from standard free-energy perturbation simulations because the mixed harmonic-anharmonic states are heavily rigidified. FCM-calculated free-energy differences for loop closing matched the experimental value for both reference sets. The insensitivity to reference states and associated partitionings favors reference states that merely reflect main structural differences for which convergence is faster. The calculations demonstrate the accuracy and robustness of FCM for large systems.

Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2.

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

Calculation of Conformational Free Energies with the Focused Confinement Method.

We introduce the focused confinement method, a reaction coordinate-free simulation approach for the calculation of conformational free energies. These are obtained in a series of restrained simulations that transform part of the molecule of interest to independent harmonic oscillators resulting in mixed harmonic-anharmonic states. It is shown that the free energy difference between these mixed states can be readily calculated through the construction of chimeric trajectories. By focusing the confinement to the conformationally active region, the method requires fewer restrained simulations than the traditional confinement method, which eases the treatment of large systems. The accuracy and efficiency of the method is demonstrated for implicitly and explicitly solvated systems.

Orthogonal Halogen-Bonding-Driven 3D Supramolecular Assembly of Right-Handed Synthetic Helical Peptides.

Peptide-mediated self-assembly is a prevalent method for creating highly ordered supramolecular architectures. Herein, we report the first example of orthogonal C-X⋅⋅⋅X-C/C-X⋅⋅⋅π halogen bonding and hydrogen bonding driven crystalline architectures based on synthetic helical peptides bearing hybrids of l-sulfono-γ-AApeptides and natural amino acids. The combination of halogen bonding, intra-/intermolecular hydrogen bonding, and intermolecular hydrophobic interactions enabled novel 3D supramolecular assembly. The orthogonal halogen bonding in the supramolecular architecture exerts a novel mechanism for the self-assembly of synthetic peptide foldamers and gives new insights into molecular recognition, supramolecular design, and rational design of biomimetic structures.

De Novo Left-Handed Synthetic Peptidomimetic Foldamers.

The development of peptidomimetic helical foldamers with a wide repertoire of functions is of significant interest. Herein, we report the X-ray crystal structures of a series of homogeneous l-sulfono-γ-AA foldamers and elucidate their folding conformation at the atomic level. Single-crystal X-ray crystallography revealed that this class of oligomers fold into unprecedented dragon-boat-shaped and unexpected left-handed helices, which are stabilized by the 14-hydrogen-bonding pattern present in all sequences. These l-sulfono-γ-AApeptides have a helical pitch of 5.1 Šand exactly four side chains per turn, and the side chains lie perfectly on top of each other along the helical axis. 2D NMR spectroscopy, computational simulations, and CD studies support the folding conformation in solution. Our results provide a structural basis at the atomic level for the design of novel biomimetics with a precise arrangement of functional groups in three dimensions.

A novel gene expression analytics-based approach to structure aided design of rexinoids for development as next-generation cancer therapeutics.

Rexinoids are powerful ligands that bind to retinoid-X-receptors (RXRs) and show great promise as therapeutics for a wide range of diseases, including cancer. However, only one rexinoid, bexarotene (Targretin TM) has been successfully transitioned from the bench to the clinic and used to treat cutaneous T-cell lymphoma (CTCL). Our goal is to develop novel potent rexinoids with a less untoward side effect profile than bexarotene. To this end, we have synthesized a wide array of rexinoids with EC50 values and biological activity similar to bexarotene. In order to determine their suitability for additional downstream analysis, and to identify potential candidate analogs for clinical translation, we treated human CTCL cells in culture and employed microarray technology to assess gene expression profiles. We analyzed twelve rexinoids and found they could be stratified into three distinct categories based on their gene expression: similar to bexarotene, moderately different from bexarotene, and substantially different from bexarotene. Surprisingly, small changes in the structure of the bexarotene parent compound led to marked differences in gene expression profiles. Furthermore, specific analogs diverged markedly from our hypothesis in expression of genes expected to be important for therapeutic promise. However, promoter analysis of genes whose expression was analyzed indicates general regulatory trends along structural frameworks. Our results suggest that certain structural motifs, particularly the basic frameworks found in analog 4 and analog 9, represent important starting points to exploit in generating additional rexinoids for future study and therapeutic applications.

Hydrogen-Bonding-Driven 3D Supramolecular Assembly of Peptidomimetic Zipper.

Hydrogen-bonding-driven three-dimensional (3D) assembly of a peptidomimetic zipper has been established for the first time by using an α/AApeptide zipper that assembles into a de novo lattice arrangement through two layers of hydrogen-bonded linker-directed interactions. Via a covalently bridged 1D 413-helix, drastic enhancement in stability has been achieved in the formed 3D crystalline supramolecular architecture as evidenced by gas-sorption studies. As the first example of an unnatural peptidic zipper, the dimensional augmentation of the zipper differs from metal-coordinated strategies, and may have general implications for the preparation of peptidic functional materials for a variety of future applications.

Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation.

During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner.

One-Bead-Two-Compound Thioether Bridged Macrocyclic γ-AApeptide Screening Library against EphA2.

Identification of molecular ligands that recognize peptides or proteins is significant but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce, and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented one-bead-two-compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound that tightly bound to EphA2 (Kd = 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform for biomacromolecular surface recognition and function modulation.

Free Energy Coupling between DNA Bending and Base Flipping.

Free energy simulations are presented to probe the energetic coupling between DNA bending and the flipping of a central thymine in double stranded DNA 13mers. The energetics are shown to depend on the neighboring base pairs, and upstream C or T or downstream C tended to make flipping more costly. Flipping to the major groove side was generally preferred. Bending aids flipping, by pushing the system up in free energy, but for small and intermediate bending angles the two were uncorrelated. At higher bending angles, bending and flipping became correlated, and bending primed the system for base flipping toward the major groove. Flipping of the 6-4 pyrimidine-pyrimidone and pyrimidine dimer photoproducts is shown to be more facile than for undamaged DNA. For the damages, major groove flipping was preferred, and DNA bending was much facilitated in the 6-4 pyrimidine-pyrimidone damaged system. Aspects of the calculations were verified by structural analyses of protein-DNA complexes with flipped bases.

Right-Handed Helical Foldamers Consisting of De Novo d-AApeptides.

New types of foldamer scaffolds are formidably challenging to design and synthesize, yet highly desirable as structural mimics of peptides/proteins with a wide repertoire of functions. In particular, the development of peptidomimetic helical foldamers holds promise for new biomaterials, catalysts, and drug molecules. Unnatural l-sulfono-γ-AApeptides were recently developed and shown to have potential applications in both biomedical and material sciences. However, d-sulfono-γ-AApeptides, the enantiomers of l-sulfono-γ-AApeptides, have never been studied due to the lack of high-resolution three-dimensional structures to guide structure-based design. Herein, we report the first synthesis and X-ray crystal structures of a series of 2:1 l-amino acid/d-sulfono-γ-AApeptide hybrid foldamers, and elucidate their folded conformation at the atomic level. Single-crystal X-ray crystallography indicates that this class of oligomers folds into well-defined right-handed helices with unique helical parameters. The helical structures were consistent with data obtained from solution 2D NMR, CD studies, and molecular dynamics simulations. Our findings are expected to inspire the structure-based design of this type of unique folding biopolymers for biomaterials and biomedical applications.

[KCl] Dependence of B-DNA Groove Bending Anisotropy.

The energetics of B-DNA bending toward the major and minor grooves were quantified by free energy simulations at four different KCl concentrations. Increased [KCl] led to more flexible DNA, with persistence lengths that agreed well with experimental values. At all salt concentrations, major groove bending was preferred, although preferences for major and minor groove bending were similar for the A-tract containing sequence. Since the phosphate repulsions and DNA internal energy favored minor groove bending, the preference for major groove bending was thought to originate from differences in solvation. Water in the minor groove was tighter bound than water in the major groove, and harder to displace than major groove water, which favored the compression of the major groove upon bending. Higher [KCl] decreased the persistence length for both major and minor groove bending but did not greatly affect the free energy spacing between the minor and major groove bending curves. For sequences without A-tracts, salt affected major and minor bending to nearly the same degree, and did not change the preference for major groove bending. For the A-tract containing sequence, an increase in salt concentration decreased the already small energetic difference between major and minor groove bending. Since salts did not significantly affect the relative differences in bending energetics and hydration, it is likely that the increased bending flexibilities upon salt increase are simply due to screening.

Molecular dynamics simulations and molecular flooding studies of the retinoid X-receptor ligand binding domain.

Bexarotene is an FDA approved retinoid X-receptor (RXR) agonist for the treatment of cutaneous T-cell lymphoma, and its use in other cancers and Alzheimer's disease is being investigated. The drug causes serious side effects, which might be reduced by chemical modifications of the molecule. To rationalize known agonists and to help identify sites for potential substitutions we present molecular simulations in which the RXR ligand-binding domain was flooded with a large number of drug-like molecules, and molecular dynamics simulations of a series of bexarotene-like ligands bound to the RXR ligand-binding domain. Based on the flooding simulations, two regions of interest for ligand modifications were identified: a hydrophobic area near the bridgehead and another near the fused ring. In addition, positional fluctuations of the phenyl ring were generally smaller than fluctuations of the fused ring of the ligands. Together, these observations suggest that the fused ring might be a good target for the design of higher affinity bexarotene-like ligands, while the phenyl ring is already optimized. In addition, notable differences in ligand position and interactions between the RXRα and RXRß were observed, as well as differences in hydrogen bonding and solvation, which might be exploited in the development of subspecies-specific ligands.

Computer Simulations of the Retinoid X Receptor: Conformational Dynamics and Allosteric Networks.

As the heterodimerization partner for a large number of nuclear receptors, the retinoid X receptor (RXR) is important for a large and diverse set of biochemical pathways. Activation and regulation of RXR heterodimers is achieved by complex allosteric mechanisms, which involve the binding of ligands, DNA, coactivators and corepressors, and entail large and subtle conformational motions. Complementing experiments, computer simulations have provided detailed insights into the origins of the allostery by investigating the changes in structure, motion, and interactions upon dimerization, ligand and cofactor binding. This review will summarize a number of simulation studies that have furthered the understanding of the conformational dynamics and the allosteric activation and control of RXR complexes. While the review focuses on the RXR and RXR heterodimers, relevant simulation studies of other nuclear receptors will be discussed as well.

Biasing Simulations of DNA Base Pair Parameters with Application to Propellor Twisting in AT/AT, AA/TT, and AC/GT Steps and Their Uracil Analogs.

An accurate and efficient implementation of the six DNA base pair parameters as order parameters for enhanced sampling simulations is presented. The parameter definitions are defined by vector algebra operations on a reduced atomic set of the base pair, and correlate very well with standard definitions. Application of the model is illustrated by umbrella sampling simulations of propeller twisting within AT/AT, AA/TT, and AC/GT steps and their uracil analogs. Strong correlations are found between propeller twisting and a number of conformational parameters, including buckle, opening, BI/BII backbone configuration, and sugar puckering. The thymine methyl group is observed to notably alter the local conformational free energy landscape, with effects within and directly upstream of the thymine containing base pair.

Secondary Structure Adopted by the Gly-Gly-X Repetitive Regions of Dragline Spider Silk.

Solid-state NMR and molecular dynamics (MD) simulations are presented to help elucidate the molecular secondary structure of poly(Gly-Gly-X), which is one of the most common structural repetitive motifs found in orb-weaving dragline spider silk proteins. The combination of NMR and computational experiments provides insight into the molecular secondary structure of poly(Gly-Gly-X) segments and provides further support that these regions are disordered and primarily non-ß-sheet. Furthermore, the combination of NMR and MD simulations illustrate the possibility for several secondary structural elements in the poly(Gly-Gly-X) regions of dragline silks, including ß-turns, 310-helicies, and coil structures with a negligible population of α-helix observed.