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NMR studies of the secondary structure in solution and the steroid binding site of delta5-3-ketosteroid isomerase in complexes with diamagnetic and paramagnetic steroids.
Biochemistry. 1997 Mar 25; 36(12):3458-72.B

Abstract

Backbone and side chain resonances of steroid-bound delta5-3-ketosteroid isomerase (EC 5.3.3.1), 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 H alpha, C alpha, C beta, and CO resonances from random coil values, consists of two alpha-helices (residues 5-21, 48-60), one 3(10) helix (residues 23-30), seven beta-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% beta-sheet, and 16% turns. The remaining 20 residues (16%) are assumed to form coils. With the exception of a parallel interaction between beta-strands 1 and 7, all beta-strand interactions are antiparallel, forming both a beta-hairpin (beta1, beta2) and a four-stranded beta-sheet in which the first strand is interrupted (beta3-beta4, beta5, beta6, beta7). 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 I (Val-15), beta-strand 1 of the beta-hairpin (Asp-38), the loop between helix 3 and beta-strand 3 (Leu-61), beta-strand 3 (Ala-64), beta-strand 5 (Phe-82, Ser-85, Glu-87), beta-strand 6 (Ile-98), and beta-strand 7 (Ala-114, Phe-116) of the beta-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 alpha-helices 1 and 3, which form one wall of the active site, and one end of the four-stranded beta-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), beta-strand 5 (Val-84, Ser-85), and beta-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.

Authors+Show Affiliations

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9131995

Citation

Zhao, Q, et al. "NMR Studies of the Secondary Structure in Solution and the Steroid Binding Site of Delta5-3-ketosteroid Isomerase in Complexes With Diamagnetic and Paramagnetic Steroids." Biochemistry, vol. 36, no. 12, 1997, pp. 3458-72.
Zhao Q, Abeygunawardana C, Mildvan AS. NMR studies of the secondary structure in solution and the steroid binding site of delta5-3-ketosteroid isomerase in complexes with diamagnetic and paramagnetic steroids. Biochemistry. 1997;36(12):3458-72.
Zhao, Q., Abeygunawardana, C., & Mildvan, A. S. (1997). NMR studies of the secondary structure in solution and the steroid binding site of delta5-3-ketosteroid isomerase in complexes with diamagnetic and paramagnetic steroids. Biochemistry, 36(12), 3458-72.
Zhao Q, Abeygunawardana C, Mildvan AS. NMR Studies of the Secondary Structure in Solution and the Steroid Binding Site of Delta5-3-ketosteroid Isomerase in Complexes With Diamagnetic and Paramagnetic Steroids. Biochemistry. 1997 Mar 25;36(12):3458-72. PubMed PMID: 9131995.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - NMR studies of the secondary structure in solution and the steroid binding site of delta5-3-ketosteroid isomerase in complexes with diamagnetic and paramagnetic steroids. AU - Zhao,Q, AU - Abeygunawardana,C, AU - Mildvan,A S, PY - 1997/3/25/pubmed PY - 1997/3/25/medline PY - 1997/3/25/entrez SP - 3458 EP - 72 JF - Biochemistry JO - Biochemistry VL - 36 IS - 12 N2 - Backbone and side chain resonances of steroid-bound delta5-3-ketosteroid isomerase (EC 5.3.3.1), 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 H alpha, C alpha, C beta, and CO resonances from random coil values, consists of two alpha-helices (residues 5-21, 48-60), one 3(10) helix (residues 23-30), seven beta-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% beta-sheet, and 16% turns. The remaining 20 residues (16%) are assumed to form coils. With the exception of a parallel interaction between beta-strands 1 and 7, all beta-strand interactions are antiparallel, forming both a beta-hairpin (beta1, beta2) and a four-stranded beta-sheet in which the first strand is interrupted (beta3-beta4, beta5, beta6, beta7). 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 I (Val-15), beta-strand 1 of the beta-hairpin (Asp-38), the loop between helix 3 and beta-strand 3 (Leu-61), beta-strand 3 (Ala-64), beta-strand 5 (Phe-82, Ser-85, Glu-87), beta-strand 6 (Ile-98), and beta-strand 7 (Ala-114, Phe-116) of the beta-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 alpha-helices 1 and 3, which form one wall of the active site, and one end of the four-stranded beta-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), beta-strand 5 (Val-84, Ser-85), and beta-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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9131995/NMR_studies_of_the_secondary_structure_in_solution_and_the_steroid_binding_site_of_delta5_3_ketosteroid_isomerase_in_complexes_with_diamagnetic_and_paramagnetic_steroids_ L2 - https://doi.org/10.1021/bi962844u DB - PRIME DP - Unbound Medicine ER -