A Peptide Fragment of Human DNA Topoisomerase II α Forms a Stable Coiled-coil Structure in Solution

Valérie Frère,Frédéric Sourgen,Monique Monnot,Frédéric Troalen,Serge Fermandjian

doi:10.1074/jbc.270.29.17502

Abstract

Results are presented on a peptide fragment (1013-1056) from human DNA topoisomerase II alpha. This was selected using the procedure of Lupas et al. (Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) for its potential to adopt a stable coiled-coil structure. The same theoretical treatment rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling, L. A., and Perry, W. M. (1989) Mol. Endocrinol. 3, 603-604) as a possible core for leucine-zipper formation. Our experimental studies combine cross-linking and CD analysis. Cross-linking establishes that the 1013-1056 fragment forms a stable homodimer in solution. Effects of increasing peptide concentration on CD spectra confirm that only the 1013-1056 fragment can undergo a coiled-coil stabilization from an isolated alpha-helix. Unfolding experiments further show that the coiled-coil is more stable in guanidium chloride than in urea. Values of -6.8 and -7.4 kcal/mol for the dimerization free energy are determined by thermal and urea unfolding, respectively. These are strikingly similar to the value recently found for the dissociation/reassociation of the entire yeast topoisomerase II from sedimentation equilibrium experiments (Lamhasni, S., Larsen, A. K., Barray, M., Monnot, M., Delain, E., and Fermandjian, S. (1995) Biochemistry 34, 3632-3639), although their significance relatively to topoisomerase II undoubtedly requires further analysis.

Highlights

From the Wepartement de Biologie et Pharmacologie Structurales, URA 147 CNRS, Institut Gustave Roussy, Villejuif 94805 Cedex, France and §Service d'Immunologie Moleculaire, Institut Gustave Roussy, Villejuif94805 Cedex, France
The most striking feature of coiled-coil helices is the 4-3 or 3-4 hydrophobic repeat. This repeat, firstly ing, respectively. These are strikingly similar to the identified by Hodges et al [19], consists of a repeating heptad value recently found for the dissociation/reassociation sequence designated by the letters a-g, in which the positions of the entire yeast topoisomerase II from sedimentation a and d usually present hydrophobic residues
We estimated the capacity of this sequence to adopt an a-helical structure using the procedure of Biou et at. [23] based on the combination of two prediction methods: one by homology and the other by Garnier Osguthorpe Robson (GOR) III [22]

Summary

Introduction

From the Wepartement de Biologie et Pharmacologie Structurales, URA 147 CNRS, Institut Gustave Roussy, Villejuif 94805 Cedex, France and §Service d'Immunologie Moleculaire, Institut Gustave Roussy, Villejuif94805 Cedex, France. Values of -6.8 and -7.4 kcallmol for the dimerization free energy are determined by thermal and urea unfoldpoisomerase II a, as suggested by Caron and Wang [8] This structure belongs to the widespread coiled-coil motif initially characterized by Crick in 1953 [9] and consisting of several amphipathic a-helices wrapped around each other in a slightly left-handed supercoil [10,11,12,13]. These are strikingly similar to the identified by Hodges et al [19], consists of a repeating heptad value recently found for the dissociation/reassociation sequence designated by the letters a-g, in which the positions of the entire yeast topoisomerase II from sedimentation a and d usually present hydrophobic residues. Tions of adjacent heptads could stabilize the dimer and contribute to the specificity of dimerization through electrostatic in-

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