W0474

Crystal Structures of a GTPase-dependent Cell Cycle Regulator ERA in Complex with GDP and with a GTP Analog. Yijun Gu, Xin Chen, Wei Guo, Donald Court and Xinhua Ji, Center for Cancer Research, National Cancer Inst., Frederick, MD 21702.

The conformational change of GTPase upon GTP hydrolysis has been widely exploited by biological systems as switches to an array of essential biological processes. Despite the rapid expanding of structural information on selected families of GTPases, such as Ras proteins and G-proteins, our knowledge on how many pathways GTPase are involved, how the information is passed on and how they are regulated are still quite limited. ERA proteins are a family of essential GTPases found initially in bacteria, but later on were shown to be conserved in higher organisms, such as plants and human. While the ERA family show high sequence conservation in both the N-terminal GTPase domain and the C-terminal domain, their similarity to other GTPase proteins are restricted to only the N-terminal domain. The function of ERA in bacteria, as indicated from genetic studies, appears to be associated with cell cycle control at a point after DNA replication but prior to septum formation. We have previous published the crystal structure of apo-ERA (Chen, X., Court, D.L. & Ji, X. Proc. Natl. Acad. Sci. USA 96, 8396-8410, 1999), which reveals that the N-terminal GTPase domain is similar to Ras, whereas the C-terminal RNA-binding motif represents a new type of KH domain. To elucidate the conformational change of ERA upon GTP hydrolysis, we grew the crystals of ERA in complex with GDP and with a GTP analog, GMPPNP. The crystals of ERA•GDP belong to space group P2₁ with unit cell dimensions a=86.627 Å, b=67.939 Å, c=87.123 Å, and β=114.146º. The structure was solved with molecular replacement and refined to 2.8 Å. The final R_free=26.18% and R=22.84%. There are two ERA, two GDP, and 51 water molecules in the asymmetric unit. The crystals of ERA•GMPPNP also belong to space group P2₁ with unit cell dimensions similar to those of ERA•GDP. There are also two ERA molecules in the asymmetric unit. However, a GMPPNP molecule is found in one subunit but a GDP molecule in the other. Comparative analysis of the structures of apo-ERA, ERA•GDP, and ERA•GMPPNP will shed light on the conformational change of ERA upon GTP hydrolysis and therefore the intra molecular signaling in the functional cycle of ERA.