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Published online before print October 31, 2005, 10.1110/ps.051622005
Protein Science (2005), 14:2993-3003. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils

Charles Galea1, Prentice Bowman1 and Richard W. Kriwacki1,2

1 Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA
2 Department of Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA

(RECEIVED June 1, 2005; FINAL REVISION August 4, 2005; ACCEPTED August 15, 2005)

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337–Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37°C, a p53 tetramerization domain mutant (p53tet-R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild-type (p53tet-wt) did not. We characterized these proteins by equilibrium denaturation, 13C{alpha} secondary chemical shifts, {1H}-15N heteronuclear NOEs, and H/D exchange. Although p53tet-R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pKa values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet-R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet-R337H stability resulted in an increased propensity to form amyloid fibrils.

Keywords: p53 tetramerization domain; amyloid fibrils; salt bridge; pKa; hydrogen exchange

Abbreviations: H/D exchange, hydrogen-deuterium exchange • p53tet, p53 tetramerization domain • p53tet-wt, wild-type p53 tetramerization domain • p53tet-R337H, p53 tetramerization domain mutant R337H • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser effect spectroscopy • TROSY, transverse relaxation optimized spectroscopy

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051622005.

Reprint requests to: Richard W. Kriwacki, Department of Structural Biology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA; e-mail: richard.kriwacki{at}stjude.org; fax: (901) 495-3032.


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