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Conformational change upon ligand binding and dynamics of the PDZ domain from leukemia-associated Rho guanine nucleotide exchange factor

¹ Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China
² INSERM, U554, Centre de Biochimie Structurale, F-34090 Montpellier, France
³ CNRS, UMR5048, F-34090 Montpellier, France

(RECEIVED December 19, 2007; FINAL REVISION March 3, 2008; ACCEPTED March 4, 2008)

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in βB/βC and βE/B loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the ¹⁵N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the βB/βC and βE/B loops affects the PDZ–peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding.

Keywords: LARG PDZ; NMR; complex structure; conformational change; internal motions; protein–peptide interaction

Reprint requests to: Yunyu Shi, School of Life Science, University of Science and Technology of China, Hefei, Anhui, 230026, People's Republic of China; e-mail: yyshi{at}ustc.edu.cn; fax: 86-551-3601443; or Jihui Wu, School of Life Science, University of Science and Technology of China, Hefei, Anhui, 230026, People's Republic of China; e-mail: wujihui{at}ustc.edu.cn; fax: 86-551-3601443.

Abbreviations: LARG, leukemia-associated Rho guanine nucleotide exchange factor; GEF, guanine nucleotide exchange factor; PDZ, PSD-95/Disc-large/ZO-1 homology; RMSD, root mean square deviation; ITC, isothermal titration calorimetry.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073416508.

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