The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA.
The DH-PH domain tandems of Dbl-homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho-family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH-PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH-PH tandem of RhoA-specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH-PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide-free RhoA exhibits elevated dynamics when in complex with DH-PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.
Cierpicki
Tomasz
T
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, 22908, USA.
Bielnicki
Jakub
J
Zheng
Meiying
M
Gruszczyk
Jakub
J
Kasterka
Marta
M
Petoukhov
Maxim
M
Zhang
Aming
A
Fernandez
Erik J
EJ
Svergun
Dmitri I
DI
Derewenda
Urszula
U
Bushweller
John H
JH
Derewenda
Zygmunt S
ZS
D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry*; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry*; D054731:PDZ Domains; D011487:Protein Conformation; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry*
D020662:Guanine Nucleotide Exchange Factors; D046912:Multiprotein Complexes; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein