Journal of Biochemistry Advance Access originally published online on May 18, 2009
Journal of Biochemistry 2009 146(3):317-325; doi:10.1093/jb/mvp075
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NMR Structure of the Heterodimer of Bem1 and Cdc24 PB1 Domains from Saccharomyces Cerevisiae
1Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo 060-0812; 2Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561; and 3Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
To whom correspondence should be addressed. Tel: +81-11-706-9011, Fax: +81-11-706-9012, E-mail: finagaki{at}pharm.hokudai.ac.jp
Received April 2, 2009; Accepted April 30, 2009
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Abstract |
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Bem1 and Cdc24 of the budding yeast Saccharomyces cerevisiae interact with each other through PB1–PB1 heterodimer formation to regulate the establishment of cell polarity. Here we present the tertiary structure of the heterodimer of Bem1 and Cdc24 PB1 domains determined by NMR spectroscopy. To avoid ambiguity in the NMR spectral analysis, we first prepared a mutant of the Cdc24 PB1 domain that had truncated loops. The mutant provided well dispersed spectra without spectral overlapping, thus allowing unambiguous spectral assignments for structure determination. We confirmed that the loop deletion-mutant was quite similar to the wild-type in both 3D structure and binding affinity. The NMR structure of the heterodimer of the deletion-mutant of Cdc24 PB1 and Bem1 PB1 was determined using a variety of isotope labelled samples including perdeuteration. The interface between the Bem1/Cdc24 PB1 heterodimer was analysed at atomic resolution. Through a comparison with the tertiary structures of other PB1–PB1 heterodimers, we found that conserved electrostatic properties on the molecular surface were commonly used for PB1–PB1 interaction, but hydrophobic interactions were important for cognate interaction in Bem1/Cdc24 PB1 heterodimer formation.
Key Words: Bem1, Cdc24, PB1 domain, heterodimer, NMR, perdeutration, residual dipolar coupling, solution structure
Abbreviations: Bem1, Bud emergence mediator 1; Cdc24, Cell division control protein 24; PB1, Phox and Bem homology 1; OPCA, OPA, PC and AID; PAR6, partitioning defective 6; phox, phagocyte oxidase; PKC, Protein kinase C; NBR1, neighour of Brca1 gene 1; GST, glutathione S-transferase; NOESY, Nuclear Overhauser enhancement spectroscopy; RDC, residual dipolar coupling; IPAP-HSQC, In-phase and anti-phase heteronuclear single quantum coherence spectroscopy
*These authors contributed equally to this work.