Molecular dynamics simulations to the bidirectional adhesion signaling pathway of integrin αV ß3.

The bidirectional force transmission process of integrin through the cell membrane is still not well understood. Several possible mechanisms have been discussed in literature on the basis of experimental data, and in this study, we investigate these mechanisms by free and steered molecular dynamics simulations. For the first time, constant velocity pulling on the complete integrin molecule inside a dipalmitoyl-phosphatidylcholine membrane is conducted. From the results, the most likely mechanism for inside-out and outside-in signaling is the switchblade model with further separation of the transmembrane helices.

Phosphorylation of Fibronectin Influences the Structural Stability of the Predicted Interchain Domain.

As a key player in cell adhesion, the glycoprotein fibronectin is involved in the complex mechanobiology of the extracellular matrix. Although the function of many modules in the fibronectin molecule has already been understood, the structure and biological relevance of the C-terminal cross-linked region (CTXL) still remains unclear. It is known that fibronectin is only phosphorylated in the CTXL domain, but no results have been presented to date, which indicate a biological function based on this phosphorylation. For the first time, we introduce a structural model of the CTXL region in fibronectin, which we obtained by exhaustive replica exchange molecular dynamics simulations (TIGER2hs). The sampling revealed a conformational landscape of the dimerization module, and the global minimum state showed an umbrella-like module body and conspicuous structural region with two feet. We observed that the CTXL foot region exhibits a structural stability in its physiological state, which disappears upon changes in the phosphorylation state. Thus, our in silico studies enabled us to show that the flexibility of the CTXL region is guided by phosphorylation. These results indicate an in vivo function of the CTXL domain in protein binding and cell adhesion, which is controlled by phosphorylation.

Molecular dynamics simulations on networks of heparin and collagen.

Synthetic scaffolds containing collagen (Type I) are of increasing interest for bone tissue engineering, especially for highly porous biomaterials in combination with glycosaminoglycans. In experiments the integration of heparin during the fibrillogenesis resulted in different types of collagen fibrils, but models for this aggregation on a molecular scale were only tentative. We conducted molecular dynamic simulations investigating the binding of heparin to collagen and the influence of the telopeptides during collagen aggregation. This aims at explaining experimental findings on a molecular level. Novel structures for N- and C-telopeptides were developed with the TIGER2 replica exchange algorithm and dihedral principle component analysis. We present an extended statistical analysis of the mainly electrostatic interaction between heparin and collagen and identify several binding sites. Finally, we propose a molecular mechanism for the influence of glycosaminoglycans on the morphology of collagen fibrils. Proteins 2017; 85:1119-1130. © 2017 Wiley Periodicals, Inc.

Simulation of adhesion forces and energies of peptides on titanium dioxide surfaces.

Force field molecular dynamics simulations on a decapeptide in contact with a rutile (100) surface in aqueous solution are reported. The peptide sequence is part of α(1)-collagen. Force-distance curves yield discrete peaks to the rupture of charged lysine and glutamate side chains from the surface according to the model of contact points. The rupture forces are 0.2-2.2 nN, and the values strongly depend on the charges of surface hydroxyl groups. Adhesion energies are evaluated from the areas of the rupture peaks. For proton charges of 0.4 and 1, adhesion energies between 40 and 190 kJ/mol were found being comparable to recent ab initio molecular dynamics results. Flips in the torsional angles of the peptide are observed during restrained desorption. The partial charges of hydroxyproline are revised, and the polarization of the C(ß)-C(γ) bond as well as the ring pucker conformations are taken into account. It is shown that transition from collagen to helix fold is more likely for hydroxyproline than for proline and might be relevant for differences in the properties of POG (proline, hydroxyproline, glycine) and PPG collagen sequences.

NMR structural studies on the covalent DNA binding of a pyrrolobenzodiazepine-naphthalimide conjugate.

The DNA binding of a naphthalimide drug conjugated through a piperazine containing linker with a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) to d(AACAATTGTT)(2) was studied by a combination of high-resolution (1)H and (31)P 2D NMR spectroscopy and restrained molecular dynamics calculations in explicit solvent. The bifunctional hybrid binds with its PBD moiety covalently linked within the minor groove to a guanine with an S stereochemistry at its covalent linkage site at C11 and a 5'-orientation of its A-ring carrying the linker with the naphthalimide ligand. The latter inserts from the minor groove between an A-A.T-T base pair step resulting in an opposite buckling of the base pairs at the intercalation site and duplex unwinding at adjacent internucleotide steps. There is NMR spectroscopic evidence that the naphthalimide undergoes a ring-flip motion with exchange rates slow to intermediate on the chemical shift time scale at ambient temperatures.

Solution structure of a covalently bound pyrrolo[2,1-c][1,4]benzodiazepine-benzimidazole hybrid to a 10mer DNA duplex.

A pyrrolo[2,1-c][1,4]benzodiazepine-benzimidazole hybrid (PBD-BIMZ) derived from the tricyclic anticancer PBD antibiotics can covalently bind to a guanine base at its exocyclic 2-amino group in double-helical DNA. Through the formation of stable DNA adducts, these hybrids have previously been shown to have significant anticancer activity in a number of cell lines. Here, the three-dimensional solution structure of the complex formed between the self-complementary DNA decamer 5'-AACAATTGTT-3' and PBD-BIMZ has been investigated by two-dimensional NMR spectroscopy and NOE distance restraint molecular dynamics simulations. Refinements using an explicit solvation model yielded a complex structure that is in good agreement with the NMR structural data. Successful convergence is indicated by an average mutual root-mean-square deviation of <1 A for three final representative structures selected by clustering methods from the molecular dynamics trajectories at 300 K. The ligand binds in an (11S,11aS) configuration to one of the two symmetrically located guanine bases of the duplex and is oriented with its benzimidazole moiety toward the 5'-end of the modified guanine. It is accommodated within the minor groove covering the centrally located 6 bp. Conformational and helical parameters of the DNA adduct are typical of a B-like duplex, and more significant helical distortions by the covalent binding of PBD-BIMZ are mostly confined to the covalent binding site and the junction between complexed and noncomplexed DNA segments. In contrast to the overall well-determined conformation of the bound hybrid, its terminal N-methylpiperazine ring appears to adopt various conformations associated with increased flexibility.

Replica-Based Protein Structure Sampling Methods II: Advanced Hybrid Solvent TIGER2hs.

In many cases, native states of proteins may be predicted with sufficient accuracy by molecular dynamics simulations (MDSs) with modern force fields. Enhanced sampling methods based on MDS are applied for exploring the phase space of a protein sequence and to overcome barriers on rough conformational energy landscapes. The minimum free energy state is obtained with sampling algorithms providing sufficient convergence and accuracy. A reliable but computationally very expensive method is replica exchange molecular dynamics, with many modifications to this approach presented in the past. Recently, we demonstrated how our temperature intervals with global exchange of replicas hybrid (TIGER2h) solvent sampling algorithm made a good compromise between efficiency and accuracy. There, all states are sampled under full explicit solvent conditions with a freely chosen number of replicas, whereas an implicit solvent is used during the swap decisions. This hybrid method yielded a much better approximation to the agreement with calculations in an explicit solvent than fully implicit solvent simulations. Here, we present an extension of TIGER2h and add a few layers of explicit water molecules around the peptide for the energy calculations, whereas the dynamics in fully explicit water is maintained. We claim that these water layers better reproduce steric effects, the polarization of the solvent, and the resulting reaction field energy than typical implicit solvent models. By investigating the protein-solvent interactions across comprehensive thermodynamic state ensembles, we found a strong conformational dependence of this reaction field energy. All simulations were performed with nanoscale molecular dynamics on two peptides, the α-helical peptide (AAQAA)3 and the ß-hairpin peptide HP7. A production-ready TIGER2hs implementation is supplied, approaching the accuracy of full explicit solvent sampling at a fraction of computational resources.

Drug-induced activation of integrin alpha IIb beta 3 leads to minor localized structural changes.

Integrins are transmembrane proteins involved in hemostasis, wound healing, immunity and cancer. In response to intracellular signals and ligand binding, integrins adopt different conformations: the bent (resting) form; the intermediate extended form; and the ligand-occupied active form. An integrin undergoing such conformational dynamics is the heterodimeric platelet receptor αIIbß3. Although the dramatic rearrangement of the overall structure of αIIbß3 during the activation process is potentially related to changes in the protein secondary structure, this has not been investigated so far in a membrane environment. Here we examine the Mn2+- and drug-induced activation of αIIbß3 and the impact on the structure of this protein reconstituted into liposomes. By quartz crystal microbalance with dissipation monitoring and activation assays we show that Mn2+ induces binding of the conformation-specific antibody PAC-1, which only recognizes the extended, active integrin. Circular dichroism spectroscopy reveals, however, that Mn2+-treatment does not induce major secondary structural changes of αIIbß3. Similarly, we found that treatment with clinically relevant drugs (e.g. quinine) led to the activation of αIIbß3 without significant changes in protein secondary structure. Molecular dynamics simulation studies revealed minor local changes in the beta-sheet probability of several extracellular domains of the integrin. Our experimental setup represents a new approach to study transmembrane proteins, especially integrins, in a membrane environment and opens a new way for testing drug binding to integrins under clinically relevant conditions.

Tetrahydroindoles as Multipurpose Screening Compounds and Novel Sirtuin Inhibitors.

Indoles are privileged structures in medicinal and bioorganic chemistry that are particularly well suited to serve as platforms for diversity. Among many other therapeutic areas, the indole scaffold has been used to design aromatic compounds useful to interfere with enzymes engaged in the regulation of substrate acylation status, such as sirtuins. However, the planarity of the indole ring is not necessarily optimal for all target enzymes, especially when functionalization with aromatic side chains is required. Replacement of flat scaffolds by nonplanar molecular cores dominated by sp3 hybridization is a common strategy to avoid the disadvantages associated with poor solubility and high promiscuity, while covering less-well-explored areas of chemical space. Thus, we synthesized fragment-like tetrahydroindoles suitable for fragment-based drug discovery as well as a well-characterized small library intended as multipurpose screening compounds. For proof of principle, these compounds were screened against sirtuins 1-3, enzymes known to be addressable by indoles. We found that 2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxamides are potent and selective SIRT2 inhibitors. Compound 16 t displayed an IC50 value of 0.98 µm and could serve as exquisite starting point for hit-to-lead profiling.

Replica-Based Protein Structure Sampling Methods: Compromising between Explicit and Implicit Solvents.

The structure of a protein is often not completely accessible by experiments. In silico, replica exchange molecular dynamics (REMD) is the standard sampling method for predicting the secondary and tertiary structures from the amino acid sequence, but it is computationally very expensive. Two recent adaptations from REMD, temperature intervals with global exchange of replicas (TIGER2) and TIGER2A, have been tested here in implicit and explicit solvents. Additionally, explicit, implicit, and hybrid solvent REMD are compared. On the basis of the hybrid REMD (REMDh) method, we present a new hybrid TIGER2h algorithm for faster structural sampling, while retaining good accuracy. The implementations of REMDh, TIGER2, TIGER2A, and TIGER2h are provided for nanoscale molecular dynamics (NAMD). All the methods were tested with two model peptides of known structure, (AAQAA)3 and HP7, with helix and sheet motifs, respectively. The TIGER2 methods and REMDh were also applied to the unknown structure of the collagen type I telopeptides, which represent bigger proteins with some degree of disorder. We present simulations covering more than 180 µs and analyze the performance and convergence of the distributions of states between the particular methods by dihedral principal component and secondary structure analysis.

Tracing Effects of Fluorine Substitutions on G-Quadruplex Conformational Changes.

A human telomere sequence that folds into an intramolecular (3 + 1)-hybrid G-quadruplex was modified by the incorporation of 2'-fluoro-2'-deoxyriboguanosines (FG) into syn positions of its outer tetrad. A circular dichroism and NMR spectral analysis reveals a nearly quantitative switch of the G-tetrad polarity with concerted syn↔anti transitions of all four G residues. These observations follow findings on a FG-substituted (3 + 1)-hybrid quadruplex with a different fold, suggesting a more general propensity of hybrid-type quadruplexes to undergo a tetrad polarity reversal. Two out of the three FG analogs in both modified quadruplexes adopt an S-type sugar pucker, challenging a sole contribution of N-type sugars in enforcing an anti glycosidic torsion angle associated with the tetrad flip. NMR restrained three-dimensional structures of the two substituted quadruplexes reveal a largely conserved overall fold but significant rearrangements of the overhang and loop nucleotides capping the flipped tetrad. Sugar pucker preferences of the FG analogs may be rationalized by different orientations of the fluorine atom and its resistance to be positioned within the narrow groove with its highly negative electrostatic potential and spine of water molecules.

Molecular dynamics simulations and in vitro analysis of the CRMP2 thiol switch.

The collapsin response mediator protein CRMP2 (gene: DPYSL2) is crucial for neuronal development. The homotetrameric CRMP2 complex is regulated via two mechanisms: first by phosphorylation and second by the reduction and oxidation of the Cys504 residues of two adjacent subunits. Here, we have analysed the effects of this redox switch on the protein in vitro combined with force field molecular dynamics (MD). Earlier X-ray data reveal the structure of the rigid body of the molecule but lack the flexible C-terminus with the important sites for phosphorylation and redox regulation. An in silico model for this part was established by replica exchange simulations and homology modelling, which is consistent with the CD spectroscopy results of the recombinant protein. Thermofluor data indicated that the protein aggregates at bivalent ion concentrations below 200 mM. In simulations the protein surface was covered under these conditions by a large number of ions, which most likely prevent aggregation. A tryptophan residue (Trp295) in close proximity to the forming disulphide allowed the measurement of the structural relaxation of the rigid body upon reduction by fluorescence quenching. We were also able to determine the second-order rate constant of CRMP2 oxidation by H2O2. The simulated solvent accessible surface of the hydroxyl group of Ser518 significantly increased upon reduction of the disulphide bond. Our results give the first detailed insight into the profound structural changes of the tetrameric CRMP2 due to oxidation and indicate a tightly connected regulation by phosphorylation and redox modification.

Atomistic modeling of peptide adsorption on rutile (100) in the presence of water and of contamination by low molecular weight alcohols.

Previous models for the interface between titanium implants and biosystems take into account the oxide passivation layer and the hydroxylation, but omit the hydrocarbon contamination on air-exposed samples. The authors develop a consistent model for the contamination of the rutile (100) surface by small alcohols, which are known to be present in ambient atmosphere, and use this approach in molecular dynamics calculations. Contact angle evaluation reveals that hydrophobic surfaces can be generated. During molecular dynamics simulations with three peptides (RPRGFGMSRERQ, WFCLLGCDAGCW, and RKLPDA), polar side chains penetrate the hydrocarbons and become immobilized on the titanium dioxide. In the carbon layer, the peptide recognizes a hydrophobic environment, which was not present on the clean surface, and the authors attribute changes in the secondary structure in one case to this interaction. The authors further include the popular Matsui-Akaogi approach [M. Matsui and M. Akaogi, Mol. Simul. 6, 239 (1991)] into the frame of the AMBER force field and quote van der Waals parameters for fitting the original Buckingham part. With the new potential, the authors evaluated lattice parameters, thermal fluctuation, and bulk modulus. Translational diffusion coefficients and dipole autocorrelation functions of water on the surface are discussed in relation to surface properties, and it is shown that the water layers are more rigid than on earlier titanium dioxide models, and that contacts between peptide and surface are less direct.

Poly (hexamethylene biguanide) adsorption on hydrogen peroxide treated Ti-Al-V alloys and effects on wettability, antimicrobial efficacy, and cytotoxicity.

An effective amount of the antiseptic agent PHMB cannot simply be placed on the surface of titanium alloys where hydrocarbons were removed by different purification procedures. Pre-treatment of Ti6Al4V specimen with 5% H2O2 in 24 h results in extra introduced -OH and -COOH groups as well as an adsorbed water film on the surface, which provide the base for the subsequent formation of a relatively stable and multi-layered PHMB film. The superficially adhering PHMB film produces no adverse effects on MG63 cells within a 48 h-cell culture, but promotes the initial attachment and spreading of the osteoblasts on the modified Ti6Al4V surface within 15 min. After direct bacterial inoculation of the active sample, the PHMB film reacts antimicrobially against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative strains (Pseudomonas aeruginosa, Escherichia coli) after surface contact. The bactericidal efficacy is only slightly reduced after using of the same specimen for re-testing the antibacterial activity. MG63 cells adhere and proliferate within 48 h on a PHMB film-containing Ti6Al4V surface, which has been pre-contaminated with S. aureus. Bacterial biofilms were only revealed in controls without PHMB.

Simulation on the structure of pig liver esterase.

A homology model for pig liver esterase was generated on the basis of human carboxyl esterase (hCE) and subjected to extensive molecular dynamics simulations. By virtual mutations the isoenzymes PLE1-6 and APLE were obtained, and the PLE1 trimer was built from the respective model of hCE. Stable structures for all systems were attained after simulations in solution for 12-18 ns, and contact zones between the monomers in the trimer are described. By evaluation of RMSD values of the residues in the monomer a rigid backplane with a number of ß-strands and a flexible front part containing several α helices are distinguished. All mutations are concentrated in the soft part, and significant differences in the folding states of the helices were distinguished between the isoenzymes. Substrate access to the active site passes through two helices whose structures are affected by mutations. Variations in substrate specificity between the isoenzymes are ascribed to the structure of the entrance channel rather than to the conformation of the active site itself. The assignment of the residue with a negative side chain stabilizing the histidine protonation in the catalytic triad was revised, being GLU 452 in some isoenzymes rather than GLU 336, which would be the correspondent to most hydrolases. Arguments for this new assignment are given on the basis of simulations and statistics from the 3DM database for hydrolases.

Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block.

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).