Volume 21, Issue 4, Pages (April 2013)

Slides:



Advertisements
Similar presentations
Structure of the Rho Family GTP-Binding Protein Cdc42 in Complex with the Multifunctional Regulator RhoGDI  Gregory R. Hoffman, Nicolas Nassar, Richard.
Advertisements

Bhalchandra Jadhav, Klemens Wild, Martin R. Pool, Irmgard Sinning 
Volume 124, Issue 6, Pages (March 2006)
Crystal Structure of the Tandem Phosphatase Domains of RPTP LAR
Ross Alexander Robinson, Xin Lu, Edith Yvonne Jones, Christian Siebold 
Volume 16, Issue 4, Pages (April 2008)
Structure of the Rab7:REP-1 Complex
Sebastian Meyer, Raimund Dutzler  Structure 
Hierarchical Binding of Cofactors to the AAA ATPase p97
Structural Basis for Vertebrate Filamin Dimerization
The Structure of the Cytoplasmic Domain of the Chloride Channel ClC-Ka Reveals a Conserved Interaction Interface  Sandra Markovic, Raimund Dutzler  Structure 
Volume 124, Issue 1, Pages (January 2006)
Identification of Phe187 as a Crucial Dimerization Determinant Facilitates Crystallization of a Monomeric Retroviral Integrase Core Domain  Meytal Galilee,
Volume 21, Issue 9, Pages (September 2013)
Structural Basis for Dimerization in DNA Recognition by Gal4
Volume 21, Issue 9, Pages (September 2013)
Xiaojing He, Yi-Chun Kuo, Tyler J. Rosche, Xuewu Zhang  Structure 
Volume 40, Issue 4, Pages (November 2010)
Volume 64, Issue 3, Pages (November 2016)
Volume 108, Issue 6, Pages (March 2002)
Structure of RGS4 Bound to AlF4−-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis  John J.G. Tesmer, David M. Berman, Alfred G.
Volume 23, Issue 11, Pages (November 2015)
Volume 23, Issue 7, Pages (July 2015)
Volume 14, Issue 3, Pages (March 2006)
Structure of the Angiopoietin-2 Receptor Binding Domain and Identification of Surfaces Involved in Tie2 Recognition  William A. Barton, Dorothea Tzvetkova,
Volume 19, Issue 1, Pages (January 2011)
Crystal Structure of the Rab9A-RUTBC2 RBD Complex Reveals the Molecular Basis for the Binding Specificity of Rab9A with RUTBC2  Zhe Zhang, Shanshan Wang,
Volume 28, Issue 1, Pages (October 2007)
Crystal Structures of Ral-GppNHp and Ral-GDP Reveal Two Binding Sites that Are Also Present in Ras and Rap  Nathan I. Nicely, Justin Kosak, Vesna de Serrano,
Ross Alexander Robinson, Xin Lu, Edith Yvonne Jones, Christian Siebold 
Volume 16, Issue 10, Pages (October 2008)
The Mitochondrial Fission Receptor MiD51 Requires ADP as a Cofactor
Volume 20, Issue 9, Pages (September 2012)
Volume 18, Issue 8, Pages (August 2010)
Volume 4, Issue 11, Pages (November 1996)
Structural Basis for Vertebrate Filamin Dimerization
Volume 90, Issue 1, Pages (July 1997)
Volume 16, Issue 5, Pages (May 2008)
Crystal Structure of the TAO2 Kinase Domain
Structural Insights into Ligand Recognition by a Sensing Domain of the Cooperative Glycine Riboswitch  Lili Huang, Alexander Serganov, Dinshaw J. Patel 
Elizabeth J. Little, Andrea C. Babic, Nancy C. Horton  Structure 
Volume 56, Issue 6, Pages (December 2007)
Structural Basis of EZH2 Recognition by EED
Volume 7, Issue 4, Pages (April 2005)
Volume 25, Issue 11, Pages e3 (November 2017)
Crystal Structure of the p53 Core Domain Bound to a Full Consensus Site as a Self- Assembled Tetramer  Yongheng Chen, Raja Dey, Lin Chen  Structure  Volume.
Antonina Roll-Mecak, Chune Cao, Thomas E. Dever, Stephen K. Burley 
Shehab A. Ismail, Ingrid R. Vetter, Begona Sot, Alfred Wittinghofer 
Rab35/ACAP2 and Rab35/RUSC2 Complex Structures Reveal Molecular Basis for Effector Recognition by Rab35 GTPase  Lin Lin, Yingdong Shi, Mengli Wang, Chao.
Saccharomyces cerevisiae Ski7 Is a GTP-Binding Protein Adopting the Characteristic Conformation of Active Translational GTPases  Eva Kowalinski, Anthony.
Volume 25, Issue 9, Pages e3 (September 2017)
Volume 17, Issue 10, Pages (October 2009)
A Role for Intersubunit Interactions in Maintaining SAGA Deubiquitinating Module Structure and Activity  Nadine L. Samara, Alison E. Ringel, Cynthia Wolberger 
Volume 15, Issue 3, Pages (March 2007)
Volume 29, Issue 6, Pages (March 2008)
Structure of the Rho Family GTP-Binding Protein Cdc42 in Complex with the Multifunctional Regulator RhoGDI  Gregory R. Hoffman, Nicolas Nassar, Richard.
Volume 34, Issue 3, Pages (May 2009)
Volume 24, Issue 9, Pages (September 2016)
Volume 17, Issue 8, Pages (August 2009)
Structure of the Staphylococcus aureus AgrA LytTR Domain Bound to DNA Reveals a Beta Fold with an Unusual Mode of Binding  David J. Sidote, Christopher.
Volume 19, Issue 7, Pages (July 2011)
Jue Wang, Jia-Wei Wu, Zhi-Xin Wang  Structure 
Karin Kühnel, Stefan Veltel, Ilme Schlichting, Alfred Wittinghofer 
Volume 13, Issue 5, Pages (May 2005)
Volume 14, Issue 3, Pages (March 2006)
Volume 25, Issue 11, Pages e3 (November 2017)
Structural and Biochemical Analysis of the Obg GTP Binding Protein
Petra Hänzelmann, Hermann Schindelin  Structure 
Structure of the Mtb CarD/RNAP β-Lobes Complex Reveals the Molecular Basis of Interaction and Presents a Distinct DNA-Binding Domain for Mtb CarD  Gulcin.
Volume 15, Issue 6, Pages (September 2004)
Presentation transcript:

Volume 21, Issue 4, Pages 550-559 (April 2013) Structural Insights into the Mechanism of GTPase Activation in the GIMAP Family  David Schwefel, B. Sivanandam Arasu, Stephen F. Marino, Björn Lamprecht, Karl Köchert, Eva Rosenbaum, Jenny Eichhorst, Burkhard Wiesner, Joachim Behlke, Oliver Rocks, Stephan Mathas, Oliver Daumke  Structure  Volume 21, Issue 4, Pages 550-559 (April 2013) DOI: 10.1016/j.str.2013.01.014 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 GIMAP2 and GIMAP7 Colocalize at the Surface of Lipid Droplets (A) Localization of N-terminally mCherry-tagged GIMAP7 (red) in living Jurkat cells. Lipid droplets were costained with BODIPY 493/503 (green). All scale bars represent 10 μm. (B) The localization of endogenous GIMAP2 (red) in Jurkat cells was determined by antibody staining and immunofluorescence analysis. Lipid droplets were costained with BODIPY 493/503 (green). (C) N-terminally EGFP-tagged GIMAP7 (green) and N-terminally mCherry-tagged GIMAP2 (red) were coexpressed and visualized in living Jurkat cells. See also Figure S1. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Biochemical Characterization of GIMAP7 (A) Nucleotide-binding affinities for GIMAP7 L100Q were determined using ITC. The following values were obtained from the fits: GIMAP7-GTP-γ-S (□): Kd = 10 ± 2 μM (n = 0.9), GIMAP7-GDP (○): Kd = 32 ± 2 μM (n = 0.8). (B) Equilibrium sedimentation analytical ultracentrifugation experiments were performed to determine apparent molecular masses at the indicated GIMAP7 concentrations; 200 μM GMPPNP (▪) or 500 μM GDP (○) were added to saturate GIMAP7 with the respective nucleotide. Monomer-dimer equilibria were fitted to the data obtained in the presence of GDP (Kd = 110 ± 20 μM) and GMPPNP (Kd = 9 ± 1 μM). Dashed lines indicate the molecular mass of the GIMAP7 monomer and dimer. (C) Single turnover GTP hydrolysis reactions for GIMAP7 (▪) were performed at 20°C, using a nucleotide and protein concentration of 50 μM. Plotted is the remaining GTP concentration versus time, determined as [GTP]/([GDP]+[GTP])×[GTP]initial. Data points represent mean value ± SD of three independent experiments. An exponential decay was fitted to the data. For comparison, data of the cytosolic domain of GIMAP2 (residues 1–260, ▪) are shown. (D) Initial observed rates from multiple turnover GTP hydrolysis reactions in the presence of 500 μM GTP (○) were determined for GIMAP7 at the indicated protein concentrations (at least two independent measurements per data point). Data were fitted to a monomer-dimer equilibrium (Praefcke et al., 1999). A kmax value of 3.2 ± 0.2 min−1 and a Kd value of 1.2 ± 0.4 μM were obtained from the fit. See also Figure S2. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Structure of GIMAP7 (A) Schematic representation of the domain structure of GIMAP7 and GIMAP2 with amino acid positions indicated. HS, hydrophobic segment. (B) Cartoon representation of the GMPPNP-bound GIMAP7 L100Q monomer. The G domain is shown in green, switch I and switch II in blue, the P loop in red, and the conserved box in cyan. Secondary structure elements differing from the core G domain of H-Ras (helix α3∗ and the C-terminal helices α6 and α7) are shown in orange. The nucleotide is shown in ball-and-stick representation. (C) Detailed view of the C-terminal extension and its contact to switch II and the G domain. Selected residues are shown in stick representation. (D) Comparison of the C-terminal extensions of GIMAP7 L100Q (orange) and GIMAP2 (magenta). The G domain of GIMAP7 is colored green and superimposed on the GIMAP2 G domain (magenta). The solvent-accessible surface of the GIMAP7 G domain is rendered semitransparent. See also Figure S3. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 The G-Interface Dimer of GIMAP7 (A) Cartoon representation of the GIMAP7 L100Q dimer, with one protomer shown in the same colors as in Figure 3B and the other protomer shown in cyan/orange. The pseudo 2-fold dimer axis is indicated by a dashed line. (B) Superposition of the GIMAP7 L100Q (green) and GIMAP2 (magenta, Protein Data Bank code 2XTN) G-domain dimers. The pseudo 2-fold dimer axis is indicated by an ellipse. (C) Detailed view of the GIMAP7 dimer interface. To the right, a 180° rotation is shown. Selected residues are shown in stick representation. See also Figure S4. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 Dimerization-Dependent GTPase Activity of GIMAP7 Employs a Catalytic Arginine Finger (A) 2FO-FC density, contoured at 1 σ, is shown for Arg103 in chain B and the water molecule connecting the arginine side chain with the opposing GMPPNP molecule via hydrogen bonding. Chain A is shown in green and chain B in cyan; the P loop of chain A is colored in red. The magnesium ion is shown as a gray sphere. (B) Nucleotide-binding affinities of GIMAP7 mutants to GTP-γ-S were determined using ITC, as in Figure 2A. The following values were obtained from the fits: R103D (○), Kd = 14 ± 4 μM (n = 0.8); E136W (□), Kd = 19 ± 3 μM (n = 1.1). (C) Sedimentation equilibrium ultracentrifugation experiments for GIMAP7 R103D (▴) and E136W (□) in the presence of 200 μM GMPPNP, as in Figure 2B. The following values for a monomer-dimer equilibrium were obtained from the data fits: GIMAP7 E136W: Kd = 47 ± 6 μM, GIMAP7 R103D: Kd = 8 ± 1 μM. Data for GIMAP7 (○, Figure 2B) in the presence of 200 μM GMPPNP are shown for comparison. (D) Nucleotide hydrolysis of the E136W (▴) and R103D (□) mutants of GIMAP7 were measured by HPLC in a single turnover assay (using a protein and nucleotide concentration of 50 μM), as in Figure 2C. GTP hydrolysis of GIMAP7 (○, Figure 2C) is shown for comparison. Data points are mean values ± SD of three independent experiments. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 6 GTPase Enhancement in Mixtures of GIMAP2 and GIMAP7 (A) Nucleotide hydrolysis was measured by HPLC, as in Figure 2C, employing 50 μM GIMAP2 (○), 5 μM GIMAP7 (▵), and a mixture of 50 μM GIMAP2 together with 5 μM GIMAP7 (□) at a GTP concentration of 50 μM (complete nucleotide loading of GIMAP2). Further control experiments were conducted using mixtures of 50 μM GST and 5 μM GIMAP7 (▿) as well as 50 μM GIMAP2 and 0.2 U alkaline phosphatase (♢, AP). This amount of AP hydrolyzes 50 μM free GTP in less than a minute (data not shown). Data points are mean values ± SD of three independent experiments. Note that under these conditions, GIMAP7 is not fully saturated with nucleotide, resulting in a lower GTPase rate than in multiple turnover assays. (B) Mutational analysis of the GTPase rate enhancement under single turnover conditions for GIMAP2. Mixtures of 50 μM GIMAP2 and 5 μM GIMAP7 R103D (♢), 50 μM GIMAP2, and 5 μM GIMAP7 E136W (▿), 50 μM GIMAP2 R117D, and 5 μM GIMAP7 (○) as well as 50 μM GIMAP2 S54A and 5 μM GIMAP7 (▵) were analyzed for their GTPase activities. The hydrolysis reaction for 50 μM GIMAP2 together with 5 μM GIMAP7 (□) is shown for comparison (see Figure 6A). Data points are mean values ± SD of three independent experiments. (C) Analysis of the GTPase rate enhancement of GIMAP2/GIMAP2 R117D and GIMAP7 using multiple turnover conditions (500 μM GTP). A constant GIMAP7 concentration of 2.5 μM and increasing concentrations of GIMAP2 (○) or GIMAP2 R117D (□) were used. Rates were calculated by normalizing the reaction velocity to the GIMAP7 concentration. Data points represent mean values ± range of two independent experiments. (D) The GIMAP7 R103D mutant at a concentration of 2.5 μM is catalytically inactive and can be stimulated only to a minor extent by 50 μM GIMAP2. For comparison, the GTPase activity of 2.5 μM GIMAP7 is shown (see Figure 6C). Data points represent mean values ± range of two independent experiments. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 7 Altered Expression of Human GIMAPs in ALCL-Derived Lymphoma Cell Lines mRNA expression of the seven human GIMAP family members was determined by semiquantitative RT-PCR in purified CD3+ and CD4+ T cells from the peripheral blood of two healthy donors (#1, #2; primary T cells), a panel of T cell leukemia cell lines (T cell lines) and a panel of eight ALCL-derived lymphoma cell lines (ALCL). mRNA expression of GAPDH was analyzed as a control. Structure 2013 21, 550-559DOI: (10.1016/j.str.2013.01.014) Copyright © 2013 Elsevier Ltd Terms and Conditions