American Chemical Society
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NMR Studies of the Secondary Structure in Solution and the Steroid Binding Site of Δ5-3-Ketosteroid Isomerase in Complexes with Diamagnetic and Paramagnetic Steroids

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journal contribution
posted on 1997-03-25, 00:00 authored by Qinjian Zhao, Chitrananda Abeygunawardana, Albert S. Mildvan
Backbone and side chain resonances of steroid-bound Δ5-3-ketosteroid isomerase (EC, a homodimeric enzyme with 125 residues per monomer, have been assigned by heteronuclear NMR methods with the 15N- and 13C-labeled enzyme. The secondary structure in solution of steroid-bound isomerase, based on interproton NOE's and differences in chemical shifts of backbone Hα, Cα, Cβ, and CO resonances from random coil values, consists of two α-helices (residues 5−21, 48−60), one 310 helix (residues 23−30), seven β-strands (residues 34−38, 44−47, 62−67, 71−73, 78−87, 92−104, and 111−116), and five turns (residues 39−42, 74−77, 88−91, 105−108, and 119−122). Thus isomerase consists of 30% helix, 38% β-sheet, and 16% turns. The remaining 20 residues (16%) are assumed to form coils. With the exception of a parallel interaction between β-strands 1 and 7, all β-strand interactions are antiparallel, forming both a β-hairpin (β1, β2) and a four-stranded β-sheet in which the first strand is interrupted (β3−β4, β5, β6, β7). 1H−15N HSQC titrations of the free enzyme with the substrate analog 19-nortestosterone hemisuccinate revealed steroid-induced changes in backbone 15N and NH chemical shifts throughout the enzyme, with maximal effects on helix 1 (Val-15), β-strand 1 of the β-hairpin (Asp-38), the loop between helix 3 and β-strand 3 (Leu-61), β-strand 3 (Ala-64), β-strand 5 (Phe-82, Ser-85, Glu-87), β-strand 6 (Ile-98), and β-strand 7 (Ala-114, Phe-116) of the β-sheet, thus indicating the secondary structural components involved in steroid binding. These effects include regions near the catalytic residues Tyr-14 and Asp-38 which function as the general acid and base, respectively, in the ketosteroid isomerase reaction. Intermolecular NOE's between 19-nortestosterone hemisuccinate and isomerase indicate that the steroid binds near α-helices 1 and 3, which form one wall of the active site, and one end of the four-stranded β-sheet which forms the other wall. Consistent with these observations, doxyldihydrotestosterone, a steroid that is spin-labeled at its solvent-exposed end [Kuliopulos, A., Westbrook, E. M., Talalay, P., & Mildvan, A. S. (1987) Biochemistry 26, 3927−3937], induced the selective attenuation in the 1H−15N HSQC spectra of cross peaks of residues at the end of helix 3 (Ser-58, Leu-59, Lys-60, Leu-61), β-strand 5 (Val-84, Ser-85), and β-strand 6 (Val-95), due to the proximity of the nitroxide radical to the backbone 15N and NH nuclei of these residues, thus confirming the location of the D ring of the bound steroid and defining the mouth of the active site.