Structural basis for recruitment of traffic proteins by small GTPases

STRUCTURAL BASIS FOR RECRUITMENT OF TRAFFIC PROTEINS BY SMALL GTPASES WU MOUSHENG NATIONAL UNIVERSITY OF SINGAPORE 2006 STRUCTURAL BASIS FOR RECRUITMENT OF TRAFFIC PROTEINS BY SMALL GTPASES WU MOUSHENG (B.Sc., M.Sc) Xiamen University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 To My Parents i Table of Contents Table of Contents ……………………………………………………………….i Abstract …………………………………………………………………………vi Acknowledgements ……………………………………………………………viii Lists of Figures …………………………………………………………………ix Lists of Tables ………………………………………………………………….xi Lists of Abbreviations …………………………………………………………xii Chapter Introduction 1.1 Vesicular trafficking ……………………………………………………… .1 1.2 GTP-binding proteins (G proteins) ……………………………………… .4 1.3 Ras superfamily …………………………………………………………… 1.3.1 Ras superfamily ………………………………………………………….7 1.3.2 Tertiary structures ………………………………………………………7 1.3.3 Molecular switches and regulations ………………………………… 11 1.3.3.1 GTP hydrolysis and GAPs ………………………………………….13 1.3.3.2 Nucleotide exchange by GEF ……………………………………….14 1.3.3.3 Guanine dissociation inhibitor (GDI) ………………………………16 1.3.4 Localization and lipid modification ……………………………………18 1.3.5 Functional and structural studies of Ras-like superfamily ………… 19 1.3.5.1 Ras family ……………………………………………………………19 1.3.5.2 Rho family ……………………………………………………………23 1.3.5.3 Ran family ……………………………………………………………27 i ii 1.3.5.4 Arf and Rab family ………………………………………………….32 1.4 Arf, Ar1 and GRIP domain ……………………………………………….32 1.4.1 Arf family ……………………………………………………………….32 1.4.2 Arl1 and GRIP domain ……………………………………………… .36 1.5 Rab family, Rab7 and RILP ………………………………………………38 1.5.1 Rab family ………………………………………………………………38 1.5.2 Rab7 and RILP ……………………………………………………… .40 1.6 The aims of the projects ………………………………………………… .43 1.6.1 Arl1-GRIP domain project ………………………………………… 43 1.6.2 Rab7-RILP effector domain project ………………………………… 43 Chapter Materials and Methods 2.1 Materials ………………………………………………………………… 44 2.1.1 Bacteria ………………………………………………………………….44 2.1.2 Culture media ………………………………………………………… 44 2.1.3 Vector ………………………………………………………………… .44 2.1.4 Constructs ………………………………………………………………44 2.1.5 Primers ………………………………………………………………….44 2.1.6 Enzymes and nucleotides ………………………………………………45 2.1.7 Kits ………………………………………………………………………45 2.1.8 Column Materials ………………………………………………………46 2.1.9 Chemicals ……………………………………………………………….46 2.1.10 Crystallization kits and tools ……………………………………… 46 2.2 Molecular cloning ………………………………………………………….46 2.2.1 Polymerase chain reaction (PCR) …………………………………… 46 ii iii 2.2.2 Agarose gel electrophoresis ………………………………………… 47 2.2.3 Purification of PCR products ………………………………………….47 2.2.4 Enzyme digestion, dephosphorylation and purification …………… 47 2.2.5 Ligation and transformation ………………………………………… 48 2.2.6 Postive clone selection and plamid preparation …………………… .48 2.2.7 DNA sequencing ……………………………………………………… 49 2.2.8 BL21 Star transformation ……………………………………………. 49 2.3 Testing expression of cloned genes ………………………………………. 49 2.4 SDS-PAGE ………………………………………………………………… 50 2.5 Cell storage ………………………………………………………………… 50 2.6 Large-scale cell culture …………………………………………………… 50 2.7 Site-directed mutagenesis ………………………………………………… 51 2.8 Yeast two-hybrid assays ………………………………………………… .51 2.9 Cell culture and transfection ………………………………………………51 2.10 Indirect immunofluorescence microscopy ………………………………52 2.10.1 Paraformaldehyde fixation ……………………………………….… .52 2.10.2 Methanol fixation ………………………………………………………52 2.10.3 Indirect immunofluorescence labeling ………………………… .… .52 2.11 Proteins purification ………………………………………………………53 2.11.1 Cell lysis and Glutathione Sepharose Affinity Chromatograhy … .53 2.11.2 Desalting and removal of GST ……………………………………….54 2.11.3 Purification of Arl1-GRIP domain (Arl-GRIP) complex ……… . 54 2.11.3.1 Purification of Arl1 (residues 14-181) ……………………………54 2.11.3.2 Purification of GRIP domain (2171-2230) of golgin-245 ……… 56 2.11.3.3 Conversion of Arl1 from GDP- to GTP-bound form ……………57 iii iv 2.11.3.4 Purification of Arl1-GRIP domain complex …………………… 57 2.11.4 Purification of Rab7Q67L-RILP effector domain (denoted hereafter as Rab7-RILPe) complex ……………………….…………59 2.11.4.1 Purification of Rab7Q67L-GTP (denoted hereafter as Rab7-GTP) ………………………………………… ……………59 2.11.4.2 Purification of RILPe (residues 241-320) …………………… … .60 2.11.4.3 Purification of Rab7-RILPe complex …………………………… 61 2.12 Crystallization ………………………………………………………………62 2.13 Data collection ………………………………………………………………62 2.14 Structure determination ……………………………………………………65 2.14.1 Structure of the Arl1-GRIP complex …………………………….…….65 2.14.2 The structure of Rab7-GTP … …………………………………………67 2.14.3 Structure of the Rab7-RILPe complex …………………………………67 Chapter Structure of the Arl1-GRIP complex 3.1 Results ……………………………………………………………………… 70 3.1.1 Structural overview of the Arl1-GRIP complex ……………………… 70 3.1.2 Arl1-GRIP domain interface …………………………………………….71 3.1.3 GRIP-GRIP domain interface ………………………………………… .78 3.1.4 Conformational switch of Arl1 and GRIP domain binding ……………83 3.2 Discussion …………………………………………………………………….87 3.2.1 Structural comparison of Arf proteins in complex with their effectors ………………………………………………….………… 87 3.2.2 Specific interaction between Arl1 and GRIP domain ………………….90 3.2.3 Mechanism of recruitment of Golgins containing GRIP domain iv v to the Golgi …………………………………………………………………91 Chapter Structures of Rab7-GTP and Rab7-RILPe complex 4.1 Results ……………………………………………………………………… 94 4.1.1 Overall structure of Rab7-GTP …………………………………………94 4.1.2 Structure determination of Rab7-RILPe complex ………………… …95 4.1.3 Overall structure of the Rab7-RILPe complex … ……………………97 4.1.4 Rab7-RILP interaction ………………………………………………… 100 4.1.5 RILP dimer interface …………………………………………………….105 4.1.6 Mutagenesis and cellular localization ………………………………… .106 4.1.7 Structural diversity of Rab-effector recognition ……………………….112 4.2 Discussion …………………………………………………………………….115 Coordinates .………………………………………………………………… .119 References …………………………………………………………………… 120 Publication …………………………………………………………………….145 v vi Abstract In eukaryotic cells, vesicular trafficking plays significant roles in exocytosis, endocytosis and cell component recycling. Vesicular trafficking includes vesicle budding, targeting, docking and fusion with the targeting apparatus. Small GTPases are involved in all stages of vesicular trafficking by recruiting their effectors. This thesis focuses on two small GTPases, Arl1 and Rab7, and their respective effectors, the Golgins containing GRIP domain and the Rab-interacting lysosomal protein (RILP). The aims of these two projects are to investigate the molecular mechanism by which Arl1 and Rab7 recruit their respective effectors by X-ray crystallography. The structure of the Arl1-GRIP domain complex showed that the GRIP domain consisting of three twisted helices forms a tight homodimer with each subunit binding to one Arl1-GTP on opposite sides respectively. Arl1-GTP interacts with helices α1 and α2 of GRIP domain predominantly in a hydrophobic manner with the switch II region conferring the main recognition surface. The involvement of the switch and the interswitch regions in the Arl1-GTP:GRIP domain interaction explains the specificity of GRIP domain for Arl1-GTP. The most significant finding is that GRIP domain is a homodimer with all three α-helices involved in dimerization. Based on structural data, functional studies on the GRIP domain showed that mutations disrupting the GRIP domain dimerization also abrogated their Golgi targeting, strongly suggesting that the dimeric form of the GRIP domain is a functional unit. In the structure of Rab7-RILP effector domain complex, Rab7 interacts with RILP specifically via two distinct areas, with the first one involving the switch and interswitch regions, and the second one consisting of the hypervariable regions vi vii RabSF1 and RabSF4. Disruption of these interactions by mutations abrogates the late endosomal/lysosomal targeting of Rab7 and RILP. 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Structural insight into poly(A) binding and catalytic mechanism of human PARN. EMBO J. 24(23):4082-4093. 145 [...]... the dissociation of GDP from the inactive form of small GTPases and replaces the GDP with GTP GTP binding induces the conformational changes, thus causing the dissociation of GEF from small GTPases and activating the small GTPases to allow the interaction with downstream effectors There are many varieties of GEFs of small GTPases Small GTPases are recognized by one or more homologs of a distinct and... activity of GTPase and locks the small GTPase in its active form, resulting in a dominant-positive mutant (Zhong et al., 1995; Wittinghofer and Valencia, 1995) 1.3.3 Molecular switches and regulations Structural and biochemical studies of small GTPases showed that there are two interconvertible forms of small GTPases: a GDP-bound inactive form and a GTPbound active form In the GDP-bound form, small GTPases. .. activities of small G proteins are variable but all are relatively slow and are stimulated by GAPs Most GAPs are specific for each member or subfamily of small GTPases, but some GAPs show wider substrate specificity (Takai et al., 2001) Figure 1-5 Regulation of small G protein activity (Adapted from Takai et al., 2001) 12 13 1.3.3.1 GTP hydrolysis and GAPs The mechanism of GTP hydrolysis by small GTPases. .. the Rho and Rab proteins are further regulated by another type of regulator, named Rho GDP-dissociation inhibitor (RhoGDI) or Rab GDP-dissociation inhibitor (RabGDI) (Figure 1-5) GDIs bind the GDP-bound form of small GTPases and function as negative regulators by inhibiting both the basal and GEF-stimulated dissociation of GDP Therefore, they keep the small G proteins in the inactive form RhoGDIs and... into the newly formed coated transport vesicle As a clathrin-coated bud grows, other cytosolic proteins including dymanin, a GTPase, assemble around the neck of each bud to regulate the rate of the bud pinching-off to form a vesicle With the help of these proteins around the neck, the two noncytosolic leaflets of the membrane are brought close to each other, fuse and then pinch off to form the vesicle... Figure 3-5 Structural comparisons of Arl1-GTP in the Arl1-GRIP complex with other Arls ……………….…………………….… 85 Figure 3-6 Structural comparisons of the Arf proteins in complex With their effectors …………………………………… ……… 89 Figure 3-7 A schematic model of recruitment of Golgin-245 to the Golgi membrane by Arl1 …………………………………………93 Figure 4-1 Rab7-GTP structure and structural comparisons with other Rab proteins. .. Mutation of the catalytic glutamine in small GTPases completely abolishes the GTP hydrolysis activity and keeps them in the active form This has allowed the creation of the constitutive active small GTPases, which are helpful to study the characteristics of small GTPases and their downstream effectors In the heterotrimeric G protein, an arginine in the G protein stabilizes the negative charge of the... effectors The conformational change then occurs when the GDP is replaced by GTP, which allows the small GTPases to bind the downstream targets The GTP-bound form is converted by the intrinsic GTPase activity or GTPase-activating proteins (GAPs) to the GDP-bound form, which then releases the bound downstream effectors In this way, one cycle of activation and inactivation is achieved, and small G proteins serve... to stabilize the conformations of the altered P-loop and the nucleotide-free protein Thus, a GTP molecule can bind the exposed GTPbinding site of the small GTPase (Cherfils et al., 1999) 1.3.3.3 Guanine dissociation inhibitor (GDI) GDI binds GDP-bound small GTPases and inhibits the GDP release, thus functioning as negative regulator to prevent the activation of small GTPases Small GTPases are commonly... involvement of the GTPases in vesicle trafficking has been well discussed (Nuoffer et al., 1994; Rothman, 1994; Takai et al., 2001; Zerial and McBride, 2001) The following part of this introduction will focus on the GTP-binding proteins (G proteins) and more specifically on the small GTP-binding proteins, Ras superfamily 1.2 GTP-binding proteins (G proteins) G proteins are regulatory GTP hydrolases . STRUCTURAL BASIS FOR RECRUITMENT OF TRAFFIC PROTEINS BY SMALL GTPASES WU MOUSHENG NATIONAL UNIVERSITY OF SINGAPORE 2006 STRUCTURAL BASIS FOR RECRUITMENT OF TRAFFIC. RECRUITMENT OF TRAFFIC PROTEINS BY SMALL GTPASES WU MOUSHENG (B.Sc., M.Sc) Xiamen University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES. with the targeting apparatus. Small GTPases are involved in all stages of vesicular trafficking by recruiting their effectors. This thesis focuses on two small GTPases, Arl1 and Rab7, and their