The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle.
Calcium sensitization in smooth muscle is mediated by the RhoA GTPase, activated by hitherto unspecified nucleotide exchange factors (GEFs) acting downstream of Galphaq/Galpha(12/13) trimeric G proteins. Here, we show that at least one potential GEF, the PDZRhoGEF, is present in smooth muscle, and its isolated DH/PH fragment induces calcium sensitization in the absence of agonist-mediated signaling. In vitro, the fragment shows high selectivity for the RhoA GTPase. Full-length fragment is required for the nucleotide exchange, as the isolated DH domain enhances it only marginally. We crystallized the DH/PH fragment of PDZRhoGEF in complex with nonprenylated human RhoA and determined the structure at 2.5 A resolution. The refined molecular model reveals that the mutual disposition of the DH and PH domains is significantly different from other previously described complexes involving DH/PH tandems, and that the PH domain interacts with RhoA in a unique mode. The DH domain makes several specific interactions with RhoA residues not conserved among other Rho family members, suggesting the molecular basis for the observed specificity.
Derewenda
Urszula
U
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908, USA.
Oleksy
Arkadiusz
A
Stevenson
Andra S
AS
Korczynska
Justyna
J
Dauter
Zbigniew
Z
Somlyo
Andrew P
AP
Otlewski
Jacek
J
Somlyo
Avril V
AV
Derewenda
Zygmunt S
ZS
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009130:Muscle, Smooth / Q000502:physiology*; D011487:Protein Conformation; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein; D002118:Calcium