Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

Abstract

Background: Rho family GTPases regulate Entamoeba histolytica pathogenesis. Results: Despite Ras-like structural features and fast intrinsic nucleotide exchange, EhRho1 engages classical Rho effectors and regulates actin. Conclusion: EhRho1 is a true Rho family GTPase with a unique mode of nucleotide interaction. Significance: Possibly representing an early Rho subfamily divergence from the Ras superfamily, EhRho1 likely regulates actin polymerization in E. histolytica.

Entamoeba histolytica Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

“…In previous work (7), we demonstrated that EhRho1 binds the GBD-FH3 domain tandem of EhFormin1 (a.a. 69-445) selectively in its GTPγS-bound, activated conformation. Since some minor degradation of that particular GBD-FH3 fragment was reported during expression and purification ( e.g.…”

“…A recent proteomic characterization of E. histolytica cysts indicated that EhFormin1 is expressed during both the encysted and trophozoite life cycle stages (22). We recently showed (7) that the GBD-FH3 domain tandem of EhFormin1 binds EhRho1 in a nucleotide state-dependent fashion, which is typical of Rho GTPase/effector interactions. Furthermore, expression of constitutively active EhRho1 in fibroblasts induced stress fiber formation (7), suggesting that EhRho1 might regulate actin filament formation in E. histolytica trophozoites through EhFormin1 or other effectors.…”

“…We recently showed (7) that the GBD-FH3 domain tandem of EhFormin1 binds EhRho1 in a nucleotide state-dependent fashion, which is typical of Rho GTPase/effector interactions. Furthermore, expression of constitutively active EhRho1 in fibroblasts induced stress fiber formation (7), suggesting that EhRho1 might regulate actin filament formation in E. histolytica trophozoites through EhFormin1 or other effectors. Crystal structures of EhRho1 in different nucleotide states highlighted a lack of the signature Rho insert helix (7), suggesting that the EhRho1/EhFormin1 interaction might differ from that of RhoC/mDia1 (20).…”

Structural Determinants of Ubiquitin Conjugation in Entamoeba histolytica

“…Open reading frames of Ehubiquitin (AmoebaDB accession EHI_083410), Ehuba1 (EHI_020270), Ehubc5 (EHI_083560), Ehring1 (EHI_020100), Ehhect1 (EHI_011530), and Ehhect2 (EHI_124600) were PCR amplified from genomic DNA and subcloned as hexahistidine fusions into a pET vector-based ligation-independent cloning vector, pLIC-His, as described previously (19). PCR primer sequences were: Ehubiquitin, 5Ј-ATGCAAATATTTGTTAA-GAC-3Ј and 5Ј-TTAATATCCTCCTCTTAATC-3Ј; Ehuba1, 5Ј-ATGACACAACAAATTGATGAAGCCGTATTG-3Ј and 5Ј-TTAGAAATTCAAAAGAACATCTGGAAATTC-3Ј; Ehubc5, 5Ј-ATGGCTATGCGTAGAATTCAAAAAG-3Ј and 5Ј-TTAT-GGTCGAGCATACATAC-3Ј; Ehring1, 5Ј-ATGTCAAGAGA-AGATTGTG-3Ј and 5Ј-TTAGTGATAAATAATTCGGTG-3Ј; Ehhect1, 5Ј-ATGCGGAAACACCTCAAATAACAAATAA-3Ј and 5Ј-TTAAATTAAACCAAATCCATTTGTATT-3Ј; Ehhect2, 5Ј-ATGAGACCGGCTTGGAGACTTA-3Ј and 5Ј-TTAAGA-AAATGCAAATCCTGATTTTGATG-3Ј.…”

“…Surface Plasmon Resonance (SPR) Assays-SPR-based measurements of protein-protein interactions were performed on a Biacore 3000 (GE Healthcare), essentially as described previously (19). Briefly, purified His 6 -EhUbc5 and His 6 -EhUbc5(F62A) proteins were separately immobilized on an NTA biosensor chip using covalent capture coupling (27).…”