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

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis, a severe emerging infection. BopE is a GEF (guanine-nucleotide-exchange factor) for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1. We have determined the structure of BopE catalytic domain (amino acids 78–261) by NMR spectroscopy and it shows that BopE78–261 comprises two three-helix bundles (α1α4α5 and α2α3α6). This fold is similar to that adopted by the BopE homologues SopE and SopE2, which are GEFs from Salmonella. Whereas the two three-helix bundles of SopE78–240 and SopE269–240 form the arms of a ‘Λ’ shape, BopE78–261 adopts a more closed conformation with substantial interactions between the two three-helix bundles. We propose that arginine and proline residues are important in the conformational differences between BopE and SopE/E2. Analysis of the molecular interface in the SopE78–240–Cdc42 complex crystal structure indicates that, in a BopE–Cdc42 interaction, the closed conformation of BopE78–261 would engender steric clashes with the Cdc42 switch regions. This implies that BopE78–261 must undergo a closed-to-open conformational change in order to catalyse guanine nucleotide exchange. In an NMR titration to investigate the BopE78–261–Cdc42 interaction, the appearance of additional peaks per NH for residues in hinge regions of BopE78–261 indicates that BopE78–261 does undergo a closed-to-open conformational change in the presence of Cdc42. The conformational change hypothesis is further supported by substantial improvement of BopE78–261 catalytic efficiency through mutations that favour an open conformation. Requirement for closed-to-open conformational change explains the 10–40-fold lower kcat of BopE compared with SopE and SopE2.

Download Full-text

Direct Binding of the β1 Adrenergic Receptor to the Cyclic AMP-Dependent Guanine Nucleotide Exchange Factor CNrasGEF Leads to Ras Activation

Molecular and Cellular Biology ◽

10.1128/mcb.22.22.7942-7952.2002 ◽

2002 ◽

Vol 22 (22) ◽

pp. 7942-7952 ◽

Cited By ~ 48

Author(s):

Youngshil Pak ◽

Nam Pham ◽

Daniela Rotin

Keyword(s):

Cyclic Amp ◽

Adrenergic Receptor ◽

Pdz Domain ◽

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Ras Activation

ABSTRACT G-protein-coupled receptors (GPCRs) can indirectly activate Ras primarily through the βγ subunits of G proteins, which recruit c-Src, phosphatidylinositol 3-kinase, and Grb2-SOS. However, a direct interaction between a Ras activator (guanine nucleotide exchange factor [GEF]) and GPCRs that leads to Ras activation has never been demonstrated. We report here a novel mechanism for a direct GPCR-mediated Ras activation. The β1 adrenergic receptor (β1-AR) binds to the PDZ domain of the cyclic AMP (cAMP)-dependent Ras exchange factor, CNrasGEF, via its C-terminal SkV motif. In cells heterologously expressing β1-AR and CNrasGEF, Ras is activated by the β1-AR agonist isoproterenol, and this activation is abolished in β1-AR mutants that cannot bind CNrasGEF or in CNrasGEF mutants lacking the catalytic CDC25 domain or cAMP-binding domain. Moreover, the activation is transduced via Gsα and not via Gβγ. In contrast to β1-AR, the β2-AR neither binds CNrasGEF nor activates Ras via CNrasGEF after agonist stimulation. These results suggest a model whereby the physical interaction between the β1-AR and CNrasGEF facilitates the transduction of Gsα-induced cAMP signal into the activation of Ras. The present study provides the first demonstration of direct physical association between a Ras activator and a GPCR, leading to agonist-induced Ras activation

Download Full-text