Molecular dynamics simulations of cyclosporin A: The crystal structure and dynamic modelling of a structure in apolar solution based on NMR data (original) (raw)
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Spatial structure of cyclosporin A and insight into its flexibility
Journal of Molecular Structure, 2013
h i g h l i g h t s " Structures of cyclosporin A in CHCl 3 and in complex with SDS micelles are determined. " Thermodynamic parameters of conformational exchange in CHCl 3 are found. " Residual dipolar couplings and NOE are used as a source of structural information.
Biopolymers, 1990
The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H,O and in CC1, are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule.
Biochemical Pharmacology, 1990
Cyclophilin (163 amino acids; 17,737 daltons) is a ubiquitous cytosolic protein that specifically binds the potent immunosuppressive drug cyclosporin A (CsA). To characterize the structural details of this interaction, extensive use has been made of two-dimensional (2D) NMR methods. For studies on CsA, these methods are being used to assign the conformational space accessible to CsA by analysis of the spectra from the multiple CsA conformers present in slow exchange in mixed solvent systems. These same 2D NMR methods also have been used for extensive studies of the major bovine thymus cyclophilin (CyP) isoform and its complex with stoichiometric amounts of CsA. In the former case, these studies pave revealed 81% of the 156 expected HFH, crosspeaks. The complete spin-coupled spin systems for one-third of these amide resonances have been assigned according to amino acid type. After exhaustive D,O exchange, there remain 44 amide protons which exhibit 2D NMR features indicative of a hydrophobic domain with &sheet secondary structure. The CsA-complexed form of CyP exhibits a discrete structure and set of resonances in slow exchange with the drug-free CyP. The amino acids that have been specifically identified to be affected by the interaction are limited in number and include three Phe residues, the unique Trp at position 120, and two Ala residues.
Calculating three-dimensional molecular structure from atom-atom distance information: cyclosporin A
International journal of peptide and protein research, 1989
In recent years methods for deriving spatial molecular structure from atom-atom distance information have gained in importance due to the emergence of two-dimensional nuclear magnetic resonance (n.m.r) techniques, which make it possible to obtain such distance information for polypeptides, small proteins, sugars, and DNA fragments in solution. Distance geometry (DG) and restrained molecular dynamics (MD) refinement are applied to a cyclic polypeptide, the immunosuppressive drug cyclosporin A, and the results are compared. Two different procedures, DG followed by restrained MD, and straightforward restrained MD starting from the X-ray structure, both lead to a unique conformation that satisfies the 58 experimentally determined distance constraints. The results nicely show the relative merits of DG and restrained MD techniques for determining spatial molecular structure from distance information.
Structure, 1994
Background: Cyclophilin (CyP) is a ubiquitous intracellular protein that binds the immunosuppressive drug cyclosporin A (CsA). CyP-CsA forms a ternary complex with calcineurin and thereby inhibits T-cell activation. CyP also has enzymatic activity, catalyzing the cis-trans isomerization of peptidyl-prolyl amide bonds. Results: We have determined the structure of human cyclophilin A (CyPA) complexed with CsA to 2.1 A resolution. We also report here the structure of CyPA complexed with an analog of CsA, N-methyl-4-[(E)-2-butenyll]-4,4-dimethylthreonine CsA (MeBm 2 tl-CsA), which binds less well to CyPA, but has increased immunosuppressive activity. Comparison of these structures with previously determined structures of unligated CyPA and CyPA complexed with a candidate substrate for the isomerase activity, the dipeptide AlaPro, reveals that subtle conformational changes occur in both CsA and CyPA on complex formation. Conclusions: MeBm 2 tl-CsA binds to CyPA in an essentially similar manner to CsA. The 100-fold weaker affinity of its binding may be attributable to the close contact between MeBmtl and the active site residue Ala103 of CyPA, which causes small conformational changes in both protein and drug. One change, the slight movement of MeLeu6 in CsA relative to MeBm 2 tl-CsA, may be at least partially responsible for the higher affinity of the CyPA-MeBm 2 tl-CsA complex for calcineurin. Our comparison between CyPA-CsA and CyPA-AlaPro suggests that CsA is probably not an analog of the natural substrate, confirming that the catalytic activity of CyPA is not related to its role in immunosuppression either structurally or functionally.
Biochemistry, 1992
A simple strategy involving I H nuclear magnetic resonance (NMR) spectroscopy and complete protein deuteration was used to determine the structures of two receptor-bound drugs. A potent immunosuppressive, cyclosporin A (CsA) binds tightly to the ubiquitous and highly conserved 17.7-kDa immunophilin, cyclophilin (CyP). Fully deuterated CyP was produced by overexpressing the human CyP gene in Escherichia coli grown on deuterated algal hydrolysate in 98% DzO. As only the CsA molecule is protonated in the CsA-CyP complex, we were able to make a complete sequential assignment of the bound drug using standard two-dimensional proton N M R experiments. The structure determination was accomplished using dynamical simulated annealing calculations with a total of 124 NMR-derived distance and torsion angle restraints. Aside from binding CsA, CyP also acts as a peptidyl-prolyl cis-trans isomerase. Thus, much importance had been ascribed to the cis peptide bond present in the structures reported for free CsA in organic solvents and in crystal studies. Interestingly, CyP-bound CsA exists in an all-trans This work was supported in part by grants from the NIH (GM 40660 and DK 18778) and a James Hudson Brown-Alexander B. Coxe Fellowship (to V.L.H.) NMR instrumentation and computational facilities wereprovided by grantsfromtheNIH (RR03475), NSF(DMB 8610557), and the American Cancer Society (RD 259).
Novel backbone conformation of cyclosporin A: the complex with lithium chloride
The complexation of cyclosporin A (CsA) with lithium chloride (LiC1) in THF-d8 has been examined by NMR at different concentrations of LiC1. With 3 equiv of LiCl both forms of CsA, complexed and uncomplexed, are visible while at higher concentrations of lithium chloride only one conformation is observed. Two-dimensional NMR methods were used to assign the IH and 13C NMR spectra of cyclosporin A in THF-ds with and without addition of excess LiCl (30.9 equiv). For the lithium-complexed CsA (Li-CsA) NOE buildup rates were measured at five mixing times at 600 MHz. The conformation has been determined by restrained molecular dynamics calculations in vacuo and an iterative relaxation matrix approach to take spin-diffusion effects into account. The lithium-complexed CsA adopts a conformation completely different from the uncomplexed CsA in THF-d,. The latter is almost identical to the known conformation of CsA in CDCl,. The configuration of the peptide bond between MeLeug and MeLeu'O in the lithium complex has changed from cis to trans and all transannular hydrogen bonds are disrupted in the complex, similar in this regard to the recently published conformation of CsA bound to its natural receptor cyclophilin (CyP) even though differences in the backbone conformation exist. The conformations of CsA in CDCI,, complexed to LiCl in THF, and bound to the receptor (CsACyP) in H 2 0 are compared and discussed in light of the biological activity of this important drug. 35, 48 1-494. 1991, 66, 807-815.
Journal of chromatography. B, Biomedical applications, 1994
The immunosuppressant drug cyclosporin A exists as various conformers in water. Up to 1 h is needed to reach maximum complex formation after mixing the drug with its receptor, cyclophilin, or an antibody, indicating that only a fraction of the conformers in aqueous solution adopts a conformation suitable for binding. In the present study we compare the binding behavior of cyclosporin to that of two analogs, using a biosensor instrument (BIAcore, Pharmacia). The amount of complex formation was measured as a function of time after adding the peptides to cyclophilin. The equilibrium affinity constants of cyclophilin for these analogs have been measured. The slow binding of cyclosporin to cyclophilin compared to the instant binding of the cyclosporin analogs supports the hypothesis that cyclophilin recognizes a well defined conformation of cyclosporin that exists in water prior to binding.