The origin of the stability of isolated β-hairpins in aqueous
solution is unclear with contrasting
opinions as to the relative importance of interstrand hydrogen bonding,
hydrophobic interactions, and
conformational preferences, the latter being associated largely with
the turn sequence. We have designed an
unconstrained 16-residue peptide that we show folds autonomously in
water to form a β-hairpin that mimics
the two-stranded anti-parallel β-sheet DNA binding motif of the
met repressor dimer. The designed peptide,
with a type I‘ turn (INGK), is shown by CD and a range of NMR
parameters to be appreciably folded (≈50%
at 303 K) in aqueous solution with the predicted alignment of the
peptide backbone. We show that the folding
transition approximates to a two-state model. The hairpin has a
marked temperature-dependent stability, reaching
a maximum value at 303 K in water with both lower and higher
temperatures destabilizing the folded structure.
Van't Hoff analysis of Hα chemical shifts, reveals that folding
is endothermic and entropy-driven in aqueous
solution with a large negative ΔC
p, all of
which are reminiscent of proteins with hydrophobic cores,
pointing
to the hydrophobic effect as the dominant stabilizing interaction in
water. We have examined the conformational
properties of the C-terminal β-strand (residues 9−16) in isolation
and have shown that
3
J
α
N values and
backbone
intra- and inter-residue Hα-NH NOE intensities deviate from those
predicted for a random coil, indicating
that the β-strand has a natural predisposition to adopt an extended
conformation in the absence of secondary
structure interactions. A family of β-hairpin structures
calculated from 200 (distance and torsion angle)
restraints
using molecular dynamics shows that the conformation of the hairpin
mimics closely the DNA binding face
of the met repressor dimer (backbone RMSD between
corresponding β-strands of 1.0 ± 0.2 Å).
We describe a method for docking a ligand into a protein receptor while allowing flexibility of the protein binding site. The method employs a multistep procedure that begins with the generation of protein and ligand conformations. An initial placement of the ligand is then performed by computing binding site hotspots. This initial placement is followed by a protein side-chain refinement stage that models protein flexibility. The final step of the process is an energy minimization of the ligand pose in the presence of the rigid receptor. Thus the algorithm models flexibility of the protein at two stages, before and after ligand placement. We validated this method by performing docking and cross docking studies of eight protein systems for which crystal structures were available for at least two bound ligands. The resulting rmsd values of the 21 docked protein-ligand complexes showed values of 2 A or less for all but one of the systems examined. The method has two critical benefits for high throughput virtual screening studies. First, no user intervention is required in the docking once the initial binding site selection has been made in the protein. Second, the initial protein conformation generation needs to be performed only once for a given binding region. Also, the method may be customized in various ways depending on the particular scenario in which dockings are being performed. Each of the individual steps of the method is fully independent making it straightforward to explore different variants of the high level workflow to further improve accuracy and performance.
NMR and circular dichroism (CD) spectroscopy shows that an unconstrained 16 residue linear peptide folds autonomously in water into a b-hairpin: the designed peptide adopts a conformation that mimics the anti-parallel b-sheet DNA binding motif of the met repressor protein dimer with key residues for DNA recognition presented in the same positions and orientations principally on one face of the b-hairpin template.
Respinomycin D is a member of the anthracycline family of antitumour antibiotics that interact with double stranded DNA through intercalation. The clinical agents daunomycin and doxorubicin are the most well-studied of this class but have a relatively simple molecular architecture in which the pendant daunosamine sugar resides in the DNA minor groove. Respinomycin D, which belongs to the nogalamycin group of anthracyclines, possesses additional sugar residues at either end of the aglycone chromophore that modulate the biological activity but whose role in molecular recognition is unknown. We report the NMR structure of the respinomycin D-d(AGACGTCT)2 complex in solution derived from NOE restraints and molecular dynamics simulations. We show that the drug threads through the DNA double helix forming stabilising interactions in both the major and minor groove, the latter through a different binding geometry to that previously reported. The bicycloaminoglucose sugar resides in the major groove and makes specific contacts with guanine at the 5'-CpG intercalation site, however, the disaccharide attached at the C4 position plays little part in drug binding and DNA recognition and is largely solvent exposed.
Generating a pharmacophore is often the first step towards understanding the interactions between a receptor and a ligand and can be pivotal to a successful drug discovery project. The pharmacophore tools at Accelrys have been used to assist in many different projects over the years, such as lead generation, scaffold hopping, mining ligand databases as well as many more. In this article, we will review the pharmacophore tools that have been developed at Accelrys. These will include the often used and well validated ligand based algorithms, HipHop and HypoGen and as well as extensions of these algorithms, HipHopRefine and HypoGenRefine. Recently we also developed new pharmacophore tools in the area of structure based design - deriving pharmacophores from the receptor as well as the receptor-ligand complex - which will also be discussed in this paper.
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