Solution structure of cyclosporin A and a nonimmunosuppressive analog bound to fully deuterated cyclophilin.
A simple strategy involving 1H 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% D2O. 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 NMR 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 conformation with no detectable elements of regular secondary structure and no intramolecular hydrogen bonds. A nonactive CsA analog, MeAla6-CsA, was studied using the same CyP deuteration strategy. In addition to structural elucidation of the two bound drugs, we were able to differentiate between the bound and surface-exposed residues of the drugs and also validate our previous hypothesis that the single CyP tryptophan is located in the CsA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Hsu
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Armitage
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D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D016572:Cyclosporine / Q000737:chemistry*; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D015394:Molecular Structure; D019696:Peptidylprolyl Isomerase; D011994:Recombinant Proteins / Q000378:metabolism; D012996:Solutions
D002352:Carrier Proteins; D011994:Recombinant Proteins; D012996:Solutions; D016572:Cyclosporine; D003903:Deuterium; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase