Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI.

The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45-56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein.

[1]  H. Dyson,et al.  Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. , 1999, Journal of molecular biology.

[2]  G Vriend,et al.  WHAT IF: a molecular modeling and drug design program. , 1990, Journal of molecular graphics.

[3]  K. Fiebig,et al.  Folding intermediates of SNARE complex assembly , 1999, Nature Structural Biology.

[4]  G. Wagner,et al.  The Cap-binding Protein eIF4E Promotes Folding of a Functional Domain of Yeast Translation Initiation Factor eIF4G1* , 1999, The Journal of Biological Chemistry.

[5]  Y. Takai,et al.  Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein. , 1990, The Journal of biological chemistry.

[6]  M. Rosen,et al.  C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases , 1997, Nature.

[7]  S. Jones,et al.  Principles of protein-protein interactions. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[8]  A. Doig,et al.  Side-chain structures in the first turn of the alpha-helix. , 1999, Journal of molecular biology.

[9]  G M Bokoch,et al.  GDP dissociation inhibitor prevents intrinsic and GTPase activating protein-stimulated GTP hydrolysis by the Rac GTP-binding protein. , 1993, The Journal of biological chemistry.

[10]  C. Dobson,et al.  NMR analysis of main-chain conformational preferences in an unfolded fibronectin-binding protein. , 1997, Journal of molecular biology.

[11]  Bis(1,10-phenanthrolin-1-ium) chlorodiiodide(1-) dichloroiodide(1-) , 1999 .

[12]  G. Roberts,et al.  A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm. , 1997, Structure.

[13]  K. Kaibuchi,et al.  Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. , 1990, Oncogene.

[14]  G. Bokoch,et al.  Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1. , 2000, Structure.

[15]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[16]  R. S. Spolar,et al.  Coupling of local folding to site-specific binding of proteins to DNA. , 1994, Science.

[17]  C. Chothia,et al.  Principles of protein–protein recognition , 1975, Nature.

[18]  P. Wright,et al.  Role of Secondary Structure in Discrimination between Constitutive and Inducible Activators , 1999, Molecular and Cellular Biology.

[19]  P E Wright,et al.  Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Lewis,et al.  Calculation of the free energy of association for protein complexes , 1992, Protein science : a publication of the Protein Society.

[21]  B. Matthews,et al.  Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala-->Ser and Val-->Thr substitutions in T4 lysozyme. , 1993, Biochemistry.

[22]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[23]  Kevin Struhl,et al.  Folding transition in the DMA-binding domain of GCN4 on specific binding to DNA , 1990, Nature.

[24]  C. Dermardirossian,et al.  Structure-activity relationships in flexible protein domains: regulation of rho GTPases by RhoGDI and D4 GDI. , 2001, Journal of molecular biology.

[25]  C. Chothia,et al.  Hydrophobic bonding and accessible surface area in proteins , 1974, Nature.

[26]  Michael K. Rosen,et al.  Autoinhibition and activation mechanisms of the Wiskott–Aldrich syndrome protein , 2000, Nature.

[27]  A. Fersht,et al.  Structural response to mutation at a protein-protein interface. , 1998, Journal of molecular biology.

[28]  K. Scheffzek,et al.  The Rac–RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI , 2000, Nature Structural Biology.

[29]  L. Kay,et al.  Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer. , 1995, Biochemistry.

[30]  L. Kay,et al.  Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins , 1992 .

[31]  K. Longenecker,et al.  How RhoGDI binds Rho. , 1999, Acta crystallographica. Section D, Biological crystallography.

[32]  Jeffrey W. Peng,et al.  Mapping of Spectral Density Functions Using Heteronuclear NMR Relaxation Measurements , 1992 .

[33]  A. Fersht,et al.  Cold denaturation of barstar: 1H, 15N and 13C NMR assignment and characterisation of residual structure. , 1996, Journal of molecular biology.

[34]  Gregory R. Hoffman,et al.  Structure of the Rho Family GTP-Binding Protein Cdc42 in Complex with the Multifunctional Regulator RhoGDI , 2000, Cell.

[35]  H. Nelson,et al.  Yeast heat shock transcription factor N‐terminal activation domains are unstructured as probed by heteronuclear NMR spectroscopy , 1996, Protein science : a publication of the Protein Society.

[36]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[37]  A. Ridley,et al.  Rho: theme and variations , 1996, Current Biology.

[38]  Paul A. Keifer,et al.  Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity , 1992 .

[39]  L. Kay,et al.  Spectral density function mapping using 15N relaxation data exclusively , 1995, Journal of biomolecular NMR.