Crystal Structure of IIGP1 Agnidipta Ghosh, Revathy Uthaiah, Jonathan Howard, Christian Herrmann, Eva Wolf  Molecular Cell  Volume 15, Issue 5, Pages 727-739 (September 2004) DOI: 10.1016/j.molcel.2004.07.017

Figure 1 Sequence Alignment of p47 Family GTPases Structure based sequence alignment of IIGP1 (Swissprot AccNo. Q9QZ85) with the mouse p47 GTPases TGTP (Swissprot AccNo. Q62293), IRG-47 (Swissprot AccNo. Q61635), GTPI (Swissprot AccNo. Q9Z1M2), IGTP (Swissprot AccNo. Q8C2M8), and LRG-47 (Swissprot AccNo. Q60766). The sequence of human H-Ras (Swissprot AccNo.P01112) is aligned with the IIGP1 G domain. Secondary structure elements were assigned from the IIGP1 structure. Residues involved in dimer formation are marked with an asterisk, dimer interface residues subjected to site-directed mutagenesis with a shaded asterisk, and nucleotide interacting residues with a “+”. A distance cutoff of 3.6 Å was used to define interacting residues. Conserved residues are colored according to residue type (hydrophobic core, yellow; polar, gray; acidic, red; basic, blue). The GTPase motifs (G1–G5) and switch regions (SWI/II) are annotated and boxed. The P loop lysine/methionine is highlighted in magenta. T35Ras, T102IIGP1, T108IIGP1, and Q61Ras are printed in bold letters. Molecular Cell 2004 15, 727-739DOI: (10.1016/j.molcel.2004.07.017)

Figure 2 3D Structure of IIGP1 Molecule 1 of the IIGP1-GDP dimer (ribbon presentation) is shown with the G domain (S1-H5) colored in light-blue and the N- and C-terminal helical regions colored in cyan (αA-αC) and dark-blue (αF-αL). The linker helix αE connecting the G domain and C-terminal helical region is shown in gray. GDP and Mg2+ are shown as atomic stick figure and yellow sphere. The topology is shown schematically using the same color code. The approximate dimensions of the IIGP1 molecule are 70 × 60 × 45 Å. Molecular Cell 2004 15, 727-739DOI: (10.1016/j.molcel.2004.07.017)

Figure 3 The IIGP1 Dimer (A) Structure of the IIGP1-GDP dimer. Subdomains are color coded as in Figure 2. Secondary structure elements involved in the dimer interfaces I and II are labeled. The 2-fold noncrystallographic symmetry axis is shown. (B) Interface I (stereoview). Interface I is formed by αB and Ser18 (αA) in the N-terminal helical region. αB of molecules 1 and 2 are depicted in dark blue (1) and light blue (2). Leu44 and Lys48, which were mutated within our interface analysis, are highlighted in red. Ser18 is omitted for clarity. (C) Interface II (stereoview). Interface II is formed by H2, H3, S5, and H4 of the G domain. H3 of molecule 1 (dark blue) is shown together with interface residues in H3, S5, and H4 of molecule 2 (light blue). Ser172 and Met173 (H3), which have been mutated within this study, are highlighted in red. Ile165, Ala168, and Lys169 in the Met173 binding pocket of molecule 2 as well as Lys175, Glu177, Arg218, and H2 are omitted for clarity. Molecular Cell 2004 15, 727-739DOI: (10.1016/j.molcel.2004.07.017)

Figure 4 Effect of Dimer Interface Mutations on Cooperativity and Oligomerization of IIGP1 (A) Cooperativity of IIGP1. The specific activity of IIGP1wt and the dimer interface mutants was measured at enzyme concentrations of 2–100 μM IIGP1 in the presence of 700 μM GTP. Whereas IIGP1wt (open squares) showed a 7-fold increase of activity with rising protein concentrations, all mutants except for K48E (asterisks) showed no cooperativity. Mutants L44R (open triangles) and K48A (open circles) in the helical interface I have specific activities of 0.1 min−1 comparable to the basal activity of IIGP1wt. Mutants S172R (closed triangles) and M173A (closed circles) in the G domain interface II have lower specific activities of 0.03 min−1. The double mutant L44R/S172R (closed rhombuses) has a higher specific activity of 0.3 min−1. (B) Light scattering of wild-type and mutant IIGP1: GTP-dependent oligomerization and GTP hydrolysis. The time course of scattered light was monitored in a solution of wild-type or mutant IIGP1 (50 μM) and GTP (400 μM). GTP-dependent oligomerization shows reversibility upon GDP formation. All dimer interface mutants display reduced oligomerization compared to IIGP1wt, but a time-dependent decrease of oligomerization upon GTP hydrolysis. Symbols used for wild-type and mutant IIGP1 are identical to (A). (C) Time-dependent oligomerization of GTP-bound wild-type and mutant IIGP1. The formation of oligomers was analyzed by dynamic light scattering (DLS) upon addition of GTP (1 mM) to wild-type or mutant IIGP1 (50 μM). Oligomer formation is monitored as time-dependent increase of the hydrodynamic radius Rh. Except for K48A, which shows a slight tendency to form oligomers, the mutants exhibit Rh values of 3.1 nm corresponding to the monomer size of 47 kDa. In contrast, IIGP1wt shows a rapid time-dependent increase in oligomer size. Molecular Cell 2004 15, 727-739DOI: (10.1016/j.molcel.2004.07.017)

Figure 5 Nucleotide Complexes of IIGP1 (A) Comparison of nucleotide-free, GDP-, and GppNHp-bound IIGP1. Nucleotide-free (cyan)-, GDP-bound (dark blue)- and GppNHp-bound (light blue) IIGP1wt, and the mutant complexes K48A-GDP (crimson) and M173A-GppNHp (dark orange) are superimposed on their G domains. GDP and Mg2+ are shown as atomic stick figure and yellow sphere. In this figure and in Figure 6, molecule 2 of the IIGP1wt dimer is shown. (B) Binding of GDP and GppNHp to the IIGP1 active site. The GDP-Mg2+ complex (colored by atom type) and GppNHp (black) of IIGP1wt are superimposed. Gly81/Lys82 (P loop/G1), Asp126 (126DLPG129/G3), Asp186 (183TKVD186/G4), and Ser231/Asn232 (G5) are shown in the GDP- and GppNHp-bound conformation of IIGP1wt. Switch I residues Gly103-Thr108 of the IIGP1wt-GDP complex (cyan) and the IIGP1wt-GppNHp complex (transparent gray) are shown together with residues Thr102, Gly103, and Val105-Thr108 in the switch I region of the M173A-GppNHp complex (black). Other nucleotide binding residues (Figure 6) and the Mg2+ ions of the GppNHp complexes are omitted for clarity. The sulfate ion bound to molecule 2 of the nucleotide-free enzyme is shown in dark green. (C) 2σ composite omit electron density map in the IIGP1wt-GDP active site (stereoview). GDP and surrounding regions (P loop, switch I, Asp126) are shown as atomic stick figures. Mg2+ and three water ligands are shown as yellow and red spheres. (D and E) Van der Waals surface representation of the nucleotide binding pocket in the IIGP1wt-GDP- (D) and M173A-GppNHp (E) complexes. The surface is colored according to its electrostatic potential as calculated by GRASP (Nicholls et al., 1991). GDP, GppNHp, and Mg2+ are shown as atomic stick figure and yellow sphere. Molecular Cell 2004 15, 727-739DOI: (10.1016/j.molcel.2004.07.017)

Figure 6 Schematic Drawing of GDP and GppNHp Interactions of IIGP1 (A) M173A-GppNHp, (B) IIGP1wt-GDP, (C) IIGP1wt-GppNHp. Nucleotide-interacting IIGP1 residues (bold) and the analogous residues in Ras (italics) are labeled. Dashed lines indicate hydrogen bonds. The respective interatomic distances (Å) are annotated. Molecular Cell 2004 15, 727-739DOI: (10.1016/j.molcel.2004.07.017)