Structural Switch of the γ Subunit in an Archaeal aIF2αγ Heterodimer Laure Yatime, Yves Mechulam, Sylvain Blanquet, Emmanuelle Schmitt  Structure  Volume 14, Issue 1, Pages 119-128 (January 2006) DOI: 10.1016/j.str.2005.09.020 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 S. solfataricus aIF2αγ Heterodimer (A) Overall structure. The structure of the Ss-aIF2αγ heterodimer is represented as a cartoon. In the Ss-aIF2γ subunit, the three structural domains are colored as follows: G domain (residues 1–210) in green, domain II (residues 211–320) in yellow, and domain III (residues 321–415) in orange. The zinc ion is represented as a green sphere. The Gpp(NH)p molecule is drawn in yellow sticks, and the Mg ion is drawn as a yellow sphere. In Ss-aIF2α, the three structural domains are colored as follows: domain 1 (residues 1–85) in dark blue, domain 2 (residues 86–175) in marine, and domain 3 (residues 176–266) in cyan. Secondary structure elements are labeled according to the numbering used for the Pa-aIF2α and Pa-aIF2γ structures (Schmitt et al., 2002; Yatime et al., 2005). The γL1 loop involved in the binding of aIF2α is indicated by an arrow. (B) Close-up view of the interface region between aIF2α and aIF2γ. Domain 3 of aIF2α is drawn as a cyan cartoon. The molecular surface of domain 2 of aIF2γ is shown in yellow. Cartoons of this domain are visible by transparency. Residues of aIF2γ involved in the binding of aIF2α are shown as sticks. (C) Stereoview of the interface region. aIF2α is drawn as cyan sticks, and aIF2γ is drawn as yellow sticks. Residues of aIF2α and of aIF2γ involved in the interface are labeled. Electrostatic interactions are drawn with dotted lines. Figures 1, 3, and 4 were drawn with Pymol (http://pymol.sourceforge.net/). Structure 2006 14, 119-128DOI: (10.1016/j.str.2005.09.020) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 Sequences and Topologies of aIF2γ and aIF2α (A) aIF2γ: Sequence alignment of aIF2γ from M. jannashii, P. abyssi, and S. solfataricus with Thermus thermophilus EF1A (Tt-EF1A). The alignment shown is deduced from a multiple sequence alignment with 50 eukaryotic eIF2γ sequences, 22 archaeal aIF2γ sequences, and 97 EF1A sequences from the 3 life domains. Strictly conserved residues (defined as displaying no more than four exceptions) in e/aIF2γ and EF1A sequences are colored in cyan. Strictly conserved residues (no more than four exceptions) in e/aIF2γ are colored in red. Positions with conservative replacements (residues of the same family with no more than four exceptions) are in green. Conservation families are as follows: hydrophobic (A, V, I, L, M, F, and C), aromatic (W, Y, and F), basic (R and K), acidic (D and E), small (G and A), and hydroxyl groups (S and T). For the sake of clarity, the N-terminal sequence of Mj-aIF2γ has been truncated until residue 27. Methionine 1 of EF1A is omitted. The numberings and the secondary structure elements of Ss-aIF2γ and Thermus thermophilus EF1A are schematized below the sequences. Dots symbolize undefined regions in the electronic density. Red dots show residues involved in tRNA binding according to site-directed mutagenesis experiments (see text and Figure 4B). Green triangles designate residues involved in the binding of aIF2α. (B) aIF2α sequence from Sulfolobus solfataricus. Conserved residues are deduced from multiple sequence alignments of 66 eukaryotic and archaeal aIF2α sequences. Strictly conserved residues are in red (no more than 4 exceptions out of the 66 analyzed sequences). Positions with conservative replacements are in green. Conservation families are the same as those described above. The numbering and the secondary structure elements of Ss-aIF2α are schematized at the bottom of the sequence. Dots below the sequence indicate an undefined region in the electronic density. Green triangles correspond to residues involved in the binding of aIF2γ. Structure 2006 14, 119-128DOI: (10.1016/j.str.2005.09.020) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 Gpp(NH)p Binding Site (A) Superimposition of domain I of the Pa-aIF2γ:GDP structure on domain I of the Ss-aIF2αγ:Gpp(NH)p structure. The Ss-aIF2αγ:Gpp(NH)p structure is represented as a cartoon; domain I is in green, and domain II is in yellow. For the sake of clarity, only domain I of Pa-aIF2γ:GDP is shown as a slate-blue cartoon. The Gpp(NH)p molecule bound to Ss-aIF2αγ is drawn as a stick. The magnesium ion is represented as a yellow sphere. The water molecule bound to the magnesium ion is shown as a red sphere. The locations of the peptidic motifs involved in nucleotide binding are indicated. The positions of switch 1 and switch 2 are indicated. Residues T46 (switch 1) and D93 (switch 2) are drawn as sticks. The movement of the switch 1 loop from the GDP state (Pa-aIF2γ:GDP structure) to the Gpp(NH)p state (Ss-aIF2αγ:Gpp(NH)p structure) is underlined with an arrow. This arrow illustrates how the movement of the switch region opens the channel between domains I and II, therefore favoring tRNA binding. (B) Stereodiagram focusing on the Gpp(NH)p binding pocket on aIF2γ. Domain I of aIF2γ is shown as a green stick. Residues involved in Gpp(NH)p binding are colored with N atoms in blue and O atoms in red. Gpp(NH)p is drawn as a stick; the Mg ion is drawn as a yellow sphere, and the water molecule bound to the Mg ion is drawn as a red sphere. Hydrogen bonds are shown with gray, dotted lines. Penta coordination of the magnesium ion by the β and γ phosphates, by T23 and T46, and by a water molecule is indicated. (C) Positions of the switch regions. The views are deduced from superimpositions of domain I of Ss-aIF2αγ:Gpp(NH)p, of EF1A:Gpp(NH)p:tRNA (PDB ID: 1TTT), of Pa-aIF2γ:Gpp(NH)p (PDB ID: 1KK1), and of Pa-aIF2γ:GDP (PDB ID: 1KK3). Domain 1 of Ss-aIF2γ was used as the reference. In all four panels, switch 1 is colored in orange, switch 2 is colored in red, and the region that links switch 1 to switch 2 is colored in green. The bound nucleotides are drawn as sticks. The positions of the crucial residues corresponding to the conserved threonine of switch 1 and to the conserved aspartate of switch 2 are shown. Magnesium and zinc ions are drawn with green spheres. Left view: Switch regions of Ss-aIF2αγ:Gpp(NH)p. Middle-left view: Switch regions of EF1A:Gpp(NH)p:tRNA. Middle-right view: Switch regions of Pa-aIF2γ:Gpp(NH)p. Right view: Switch regions of Pa-aIF2γ:GDP. Note the limited movements of the switch regions observed in the case of Pa-aIF2γ crystals soaked either in Gpp(NH)p or GDP nucleotide solutions. The most affected region corresponds to the loop of switch 2, which, upon binding of Gpp(NH)p, is displaced in such a way that D90 no longer interacts with the magnesium ion (Schmitt et al., 2002). Structure 2006 14, 119-128DOI: (10.1016/j.str.2005.09.020) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 Docking of Phe-tRNAPhe onto the Ss-aIF2αγ Heterodimer The docking model was directly deduced from superimposition of the entire Ss-aIF2γ molecule on the entire EF1A:Phe-tRNAPhe complex (PDB ID: 1TTT). The docking did not require any adjustment. (A) Surface representation of the Ss-aIF2αγ heterodimer; basic residues are drawn in blue, and acidic residues are drawn in red. The tRNA molecule is drawn as a stick. (B) A transparent molecular surface of the Ss-aIF2αγ heterodimer is represented. Cartoons of the model are visible by transparency. The color code is the same as that used in Figure 1: γ domain I is in green, γ domain II is in yellow, γ domain III is in orange, α domain 3 is in cyan, and α domain 2 is in marine. The surface of the Gpp(NH)p molecule is in white. The position of γL1 is indicated. Residues important for tRNA recognition, as discussed in the text, are drawn as sticks and are labeled. Structure 2006 14, 119-128DOI: (10.1016/j.str.2005.09.020) Copyright © 2006 Elsevier Ltd Terms and Conditions