Conformational sub-states in B-DNA.

Theoretical studies of the sequence-dependent conformation of B-DNA have been carried out using Jumna, a helicoidal co-ordinate minimization algorithm. The results obtained for a series of six oligomers with repetitive sequences show that, with the exception of the homopolymers (dA)n.(dT)n and (dG)n.(dC)n, all sequences can adopt a variety of conformations characterized by considerable changes in helicoidal parameters and also in sugar puckers which adopt C(2')-endo (falling into 2 classes) or, in the case of pyrimidine nucleotides, O(1')-endo forms. These studies lead to an improved understanding of the role of base sequence on DNA conformation and point to a number of interesting correlations between the various structural parameters describing the double helix.     

Binding of Non-Intercalating Antibiotics to B-DNA: A Theoretical Study Taking Into Account Nucleic Acid Flexibility

Abstract

A detailed theoretical study has been made for five antibiotics which all bind selectively to AT sequences in the minor groove of B-DNA: SN-18071, NSCT-101327, distamycin-2, distamycin-3 and netropsin. The optimal complexes were found for systems in which the flexibility of DNA, as well as that of the antibiotics, was taken into account. Explicit, mobile counterions and a dielectric function modelling aqueous solution were also included. The binding geometries of the most strongly interacting antibiotics, distamycin-3 and netropsin, are compared in considerable detail and it is shown that notable differences exist between them. The results for netropsin are also discussed in the light of recent disagreements concerning its exact binding location within DNA.     

Conformational Flexibility of Choline Derivatives

CHOLINERGIC substrates have been found in a gauche conformation (G), skewed about the C_α–C_β bond of the (CH₃)₃N⁺–CH₂–CH₂–O cholinic fragment in a number of crystal structures^1–11. Sulphur and selenium isologues, on the other hand, with the (CH₃)₃N–CH₂–CH₂–(S,Se) group, are normally in the extended trans conformations (T)^10,12,13. In agreement with the assumption that the reduced spectrum of biological activity of many rigid analogues and C_α or C_β substituted derivatives of acetylcholine can be partially ascribed to the reduction in conformational flexibility^14–17, a theoretical investigation¹⁸ predicted the existence of four almost isoenergetic conformations TTTT, TGTT, TTGT and TGGT about the ψ₀, ψ₁, ψ₂ and ψ₃ internal rotation angles schematically represented in Fig. 1.     

Static and Dynamic Conformational Properties of AT sequences in B-DNA

Abstract

A theoretical study of the optimal conformations of nucleic acid oligomers containing tracts of AT base pairs is presented. The oligomers are studied in isolation and complexed with netropsin, a minor groove binding ligand. The flexibility of the oligomers and of their complexes is calculated by adiabatic mapping with respect to the total winding angle. The results of this study show that in uncomplexed oligomers the dinucleotide junctions AA, AT and TA have very different structural parameters and different responses to winding stress. The TA junction is clearly the most flexible and is the principal site for accommodating the imposed overwinding. Complexation by netropsin leads to two important effects: firstly, the three junctions adopt more uniform structures, the largest changes again being observed for TA, secondly, the differences in flexibility as a function of sequence are strongly attenuated.     

Conformational flexibility of B-DNA at 0.74 A resolution: d(CCAGTACTGG)(2)

The affinity and specificity of a ligand for its DNA site is a function of the conformational changes between the isolated and complexed states. Although the structures of a hydroxypyrrole-imidazole-pyrrole polyamide dimer with 5'-CCAGTACTGG-3' and the trp repressor recognizing the sequence 5'-GTACT-3' are known, the baseline conformation of the DNA site would contribute to our understanding of DNA recognition by these ligands. The 0.74 A resolution structure of a B-DNA double helix, 5'-CCAGTACTGG-3', has been determined by X-ray crystallography. Six of the nine phosphates, two of four bound calcium ions and networks of water molecules hydrating the oligonucleotide have alternate conformations. By contrast, nine of the ten bases have a single, unique conformation with hydrogen atoms visible in most cases. The polyamide molecules alter the geometry of the phosphodiester backbone, and the water molecules mediating contacts in the trp repressor/operator complex are conserved in the unliganded DNA. Furthermore, the multiple conformational states, ions and hydration revealed by this ultrahigh resolution structure of a B-form oligonucleotide are potentially general considerations for understanding DNA-binding affinity and specificity by ligands.     

Conformational Flexibility of A Highly Conserved Helix Controls Cryptic Pocket Formation in FtsZ

Mycobacterium tuberculosis is responsible for more than 1.6 million deaths each year. One potential antibacterial target in M. tuberculosis is filamentous temperature sensitive protein Z (FtsZ), which is the bacterial homologue of mammalian tubulin, a validated cancer target. M. tuberculosis FtsZ function is essential, with its inhibition leading to arrest of cell division, elongation of the bacterial cell and eventual cell death. However, the development of potent inhibitors against FtsZ has been a challenge owing to the lack of structural information. Here we report multiple crystal structures of M. tuberculosis FtsZ in complex with a coumarin analogue. The 4-hydroxycoumarin binds exclusively to two novel cryptic pockets in nucleotide-free FtsZ, but not to the binary FtsZ-GTP or GDP complexes. Our findings provide a detailed understanding of the molecular basis for cryptic pocket formation, controlled by the conformational flexibility of the H7 helix, and thus reveal an important structural and mechanistic rationale for coumarin antibacterial activity.     

A Model of Sequence-Dependent Protein Diffusion Along DNA

We introduce a probabilistic model for protein sliding motion along DNA during the search of a target sequence. The model accounts for possible effects due to sequence-dependent interaction between the nonspecific DNA and the protein. Hydrogen bonds formed at the target site are used as the main sequence-dependent interaction between protein and DNA. The resulting dynamical properties and the possibility of an experimental verification are discussed in details. We show that, while at large times the process reaches a linear diffusion regime, it initially displays a sub-diffusive behavior. The sub-diffusive regime can last sufficiently long to be of biological interest.     

Conformational Flexibility of the Peptide Hormone Ghrelin in Solution and Lipid Membrane Bound: A Molecular Dynamics Study

Abstract

Human ghrelin is a peptide hormone of 28 aminoacid residues, in which the Ser3 is modified by an octanoyl group. Ghrelin has a major role in the energy metabolism of the human body stimulating growth hormone release as well as food intake. Here we perform molecular dynamics simulations in explicit water and in a DMPC-lipid bilayer/water system in order to structurally characterize this highly flexible peptide and its lipid binding properties. We find a loop structure with residues Glu17 to Lys 20 in the bending region and a short α-helix from residues Pro7 to Glu13. The presence of a lipid membrane does not influence these structural features, but reduces the overall flexibility of the molecule as revealed by reduced root mean square fluctuations of the atom coordinates. The octanoyl-side chain does not insert into the lipid membrane but points into the water phase. The peptide binds to the lipid membrane with its bending region involving residues Arg15, Lys16, Glu17, and Ser18. The implications of these results for the binding pocket of the ghrelin receptor are discussed.     

Conformational Behavior and Flexibility of Terminally Blocked Cysteine and Cystine

Conformational potential energy hypersurfaces, PES, for the terminally blocked L-Cysteine, L,L-Cystine and D,L-Cystine have been analyzed by means of molecular mechanics in combination with the programs ROSE, CICADA, PANIC and COMBINE. Low energy conformations and conformational transitions, conformational channels, have been located. Global and fragmental flexibility and conformational softness have been calculated for each conformer as well as for the entire molecule. The PES analyses were used for simulation of conformational movement based on Boltzmann probability of the points obtained on the PES. Boltzmann travelling revealed interesting correlated conformational movement where three or even more dihedral angles changed simultaneously. It could be shown that conformational behavior and flexibility were strongly influenced by the absolute configurations of the amino acids in the peptides.     

Conformational Behavior and Flexibility of Terminally Blocked Cysteine and Cystine

Conformational potential energy hypersurfaces, PES, for the terminally blocked L-Cysteine, L,L-Cystine and D,L-Cystine have been analyzed by means of molecular mechanics in combination with the programs ROSE, CICADA, PANIC and COMBINE. Low energy conformations and conformational transitions, conformational channels, have been located. Global and fragmental flexibility and conformational softness have been calculated for each conformer as well as for the entire molecule. The PES analyses were used for simulation of conformational movement based on Boltzmann probability of the points obtained on the PES. Boltzmann travelling revealed interesting correlated conformational movement where three or even more dihedral angles changed simultaneously. It could be shown that conformational behavior and flexibility were strongly influenced by the absolute configurations of the amino acids in the peptides.     

Conformational Flexibility and Peptide Interaction of the Translocation ATPase SecA

The SecA ATPase forms a functional complex with the protein-conducting SecY channel to translocate polypeptides across the bacterial cell membrane. SecA recognizes the translocation substrate and catalyzes its unidirectional movement through the SecY channel. The recent crystal structure of the Thermotoga maritima SecA-SecYEG complex shows the ATPase in a conformation where the nucleotide-binding domains (NBDs) have closed around a bound ADP-BeFx complex and SecA's polypeptide-binding clamp is shut. Here, we present the crystal structure of T. maritima SecA in isolation, determined in its ADP-bound form at 3.1 Å resolution. SecA alone has a drastically different conformation in which the nucleotide-binding pocket between NBD1 and NBD2 is open and the preprotein cross-linking domain has rotated away from both NBDs, thereby opening the polypeptide-binding clamp. To investigate how this clamp binds polypeptide substrates, we also determined a structure of Bacillus subtilis SecA in complex with a peptide at 2.5 Å resolution. This structure shows that the peptide augments the highly conserved β-sheet at the back of the clamp. Taken together, these structures suggest a mechanism by which ATP hydrolysis can lead to polypeptide translocation.     

Discrimination Between BI and BII Conformational Substates of B-DNA Based on Sugar-base Interproton Distances

Abstract

Molecular dynamics (MD) simulations of four water-solvated DNA duplexes were used to generate a database of ～27000 dinucleotide conformations. Analyzing this database, we investigated the relationship between so-called BI-BII transitions and short-range interproton distances. Four H-H distances were found particularly sensitive to BI-BII transitions: inter- nucleotide H1′(n)-H68(n+1), H2′(n)-H68(n+1), and H2″(n)-H68(n+1), and intranucleotide H2″(n)-H68(n). Determination of these distances using classical NOESY spectroscopy can thus provide valuable indications on the existence of BII substates, complementing the existing method based on ³¹P chemical shifts and ³¹P-¹H spin-spin coupling constants.     

Flexibility of the B-DNA backbone: effects of local and neighbouring sequences on pyrimidine-purine steps.

The structurally correlated dihedral angles epsilon and zeta are known for their large variability within the B-DNA backbone. We have used molecular modelling to study both energetic and mechanical features of these variations which can produce BI/BII transitions. Calculations were carried out on DNA oligomers containing either YpR or RpY dinucleotides steps within various sequence environments. The results indicate that CpA and CpG steps favour the BI/BII transition more than TpA or any RpY step. The stacking energy and its intra- and inter-strand components explain these effects. Analysis of neighbouring base pairs reveals that BI/BII transitions of CpG and CpA are easiest within (Y)n(R)n sequences. These can also induce a large vibrational amplitude for TpA steps within the BI conformation.     

Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility

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Crystal Structure of Prothrombin Reveals Conformational Flexibility and Mechanism of Activation

The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.     

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation.     

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation.