Structural Basis For The Specific Recognition Of RhoA By ARAP3 And Specific Recognition Of 18S Ribosomal RNA By APUM23

In the first chapter,we introduce the structural basis for the specific recognition of RhoA by the dual GTPase-activating protein ARAP3.ARAP3,a multi-domain protein,belongs to the ARAP(ArfGAP with RhoGAP domain,ANK repeat and PH domain-containing protein)superfamily proteins and contains both functional ArfGAP and RhoGAP domains,a SAM(sterile a motif)domain,five PH(pleckstrin homology)domains and an RA(Ras-associating)domain.ARAP3 is unique for its dual specificity GAPs(GTPase activating protein)activity for Arf6 and RhoA regulated by PtdIns(3,4,5)P3(phosphatidylinositol(3,4,5)-trisphosphate)and Rap1-GTP,respectively.It is known that ARAP3 regulates many cell functions through its RhoGAP activity,such as formation of lamellipodia,development of angiogenesis,inhibition of peritoneal dissemination of scirrhous gastric carcinoma cells,chemotaxis and adhesion-dependent processes in neutrophils,neurite outgrowth,and so on.However,the molecular interface between the ARAP3-RhoGAP domain and RhoA is unknown,as is the substrates specificity of the RhoGAP domain.To determine the structure and function of the ARAP3-RhoGAP domain,we solved the X-ray crystal structures of the RhoGAP domain of human ARAP3 and of the RhoGAP·RhoA complex with a substrate transition-state analogue GDP·AlF4-.The structure of the complex presented a clear interface between the RhoGAP domain and RhoA.The overall structure of the ARAP3-RhoGAP domain exhibits a canonical RhoGAP domain structure,which consists of nine long a-helices and two short a-helices packed in an anti-parallel way,and with an extra a-helix at the C-terminus.By analyzing the crystal structure and in combination with in vitro GTPase activity assays and ITC(isothermal titration calorimetry)experiments,we found that single point mutations of Arg-942,Arg-949,Arg-982 or Arg-985 can dramatically reduce the binding affinity and the RhoGAP activity of the ARAP3-RhoGAP domain toward RhoA in vitro,and residues Asp-90 and Glu-97 in the ?3 helix of RhoA are responsible for the substrate-specific GAP activity of the ARAP3-RhoGAP domain.In the second chapter,we introduce the structural basis for the specific recognition of 11-mer RNA sequence 5'-GGAAUUGACGG-3' in 18S ribosomal RNA(rRNA)by APUM23,a Pumilio/fem-3 mRNA binding factor(PUF)protein in Arabidopsis.PUF proteins have been extensively investigated,but little is known about these proteins in plants.APUM23,a new family of PUF protein in Arabidopsis,located in the nucleolus,is involved in pre-rRNA processing and is distinct from classical PUF family proteins,which are located in the cytoplasm and bind to 3'UTRs of mRNA to modulate mRNA expression and localization.We found that APUM23 recognizes eleven nucleotides in 18S rRNA,and solved the crystal structure of APUM23-RNA complex.APUM23 has ten Puf repeats with one pseudo-repeat at each terminus,and assembles into a C-shape with an insert region located in the inner concave surface.We also solved a crystal structure of APUM23Dedete-RNA complex with the insert region removed.Surprisingly,Go in the APUM23Delete-RNA structure was not recognized by the tenth Puf repeat;instead,it bent over to stack with A+3.The existence of insert region would block from Go bending over via steric clashes,indicating that the insert region is involved in both RNA base recognition and stabilizing the RNA conformation.Sequence alignment shows that the insert region of APUM23 and Nop9 homologs is conserved in different species,further indicating the importance of this insert region.Our study illuminates the structural basis for the specific recognition of 18S rRNA by APUM23.