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. 2009 Mar;29(6):1592-607.
doi: 10.1128/MCB.01446-08. Epub 2008 Dec 29.

A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2

Andrés Gárriz  1 , Hongfang Qiu, Madhusudan Dey, Eun-Joo Seo, Thomas E Dever, Alan G Hinnebusch
Affiliations

A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2

Andrés Gárriz et al. Mol Cell Biol. 2009 Mar.

Abstract

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic representation of distinct dimer conformations in the KD crystal structures of inactive Gcn2 and active PKR. The Gcn2 KD dimer (left) has protomers arranged in antiparallel orientation, whereas the PKR KD dimer (right) displays a back-to-back, parallel mode of dimerization. The KD N and C lobes are colored light blue and green, respectively. The conserved R262-D266 salt bridge is depicted in the PKR dimer, whereas the corresponding residues in Gcn2 cannot interact in the antiparallel dimer. (B) Schematic representation of the inactive Gcn2 KD. L856 obstructs reorientation of αC and blocks formation of the K628-E643 salt bridge; improper rotation of αC is stabilized by the inappropriate E643-R843 salt bridge. The N793 “molecular flap” partially occludes the ATP-binding pocket. The inset shows the relevant residues in the ribbon diagram of the Gcn2 KD crystal structure, with stick representations of side chains, displayed using PyMOL software (http://www.pymol.org) and Protein Data Bank file 1ZYC. (C) Schematic depiction of the predicted active conformation of the Gcn2 KD. Phosphorylated T882 neutralizes the positive charge of R834 and disrupts the inhibitory R834-E643 salt bridge, allowing axial rotation and translation of αC (as shown) and formation of the critical K628-E643 salt bridge.
FIG. 2.
FIG. 2.
Alanine substitution of L856 constitutively activates Gcn2 in vivo. (A) L856A impairs cell growth dependent on eIF2α phosphorylation. Transformants of gcn2Δ SUI2 strain H1149 or gcn2Δ SUI2-S51A strain H1817 containing empty vector or the indicated plasmid-borne GCN2 alleles were streaked on synthetic complete medium lacking uracil and incubated for 3 days at 30°C. (B) L856A confers constitutive eIF2α phosphorylation in vivo. Duplicate cultures of strains from panel A were grown in synthetic complete medium lacking uracil and histidine to saturation, diluted into fresh medium at an optical density at 600 nm of ∼0.2, and grown for 6 h at 30°C. 3-AT was added at 10 mM to one culture for 1 h before harvesting. Whole cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western analysis using the indicated specific antibodies. The Western signals were quantified, and the ratios of eIF2α-P to total eIF2α were calculated. The ratios for the nonstarved cultures were normalized to those measured for the WT strain and indicated as eIF2α-P/eIF2α values below the appropriate lanes for nonstarved cultures; the ratios for the starved cultures were normalized to the nonstarved cultures for each strain and listed as (+)3-AT/(−)3-AT values under the lanes for each strain. (C) Hyperphosphorylation of eIF2α by L856A does not reduce Gcn2 expression in GCD2-K627T cells. Transformants of gcn2Δ GCD2-K627T strain HQY346 carrying the indicated plasmid-borne GCN2 alleles or empty vector were analyzed as described in panel B.
FIG. 3.
FIG. 3.
All but bulky hydrophobic substitutions at L856 constitutively activate Gcn2 in vivo. (A) Transformants of H1149 bearing empty vector or the indicated GCN2 alleles were streaked on synthetic complete medium lacking uracil and incubated for 3 days at 30°C. (B) Duplicate transformants from panel A were replica plated to SD, SD plus 30 mM 3-AT (3-AT), or SD plus 0.5 mM 5-FT (5-FT) medium and incubated for 3 days at 30°C. (C) The indicated strains from panel A were subjected to Western analysis of eIF2α phosphorylation as described in Fig. 2B.
FIG. 4.
FIG. 4.
Leu-856 functions in a network of hydrophobic residues to inhibit Gcn2 activity. (A) Residues making hydrophobic interactions with L856 are displayed on the ribbon diagram of the relevant portion of the Gcn2 KD crystal structure, with stick representations of the side chains (constructed from Protein Data Bank file 1ZYC). (B) Duplicate transformants of H1149 bearing empty vector or the indicated GCN2 alleles were replica plated to SD, SD plus 30 mM 3-AT (3-AT), or SD plus 0.5 mM 5-FT (5-FT) and incubated for 3 days at 30°C. (C) The indicated strains from panel B were subjected to Western analysis of eIF2α phosphorylation as described in Fig. 2B. (D) The indicated strains from panel B were streaked on synthetic complete medium lacking uracil and incubated for 3 days at 30°C. (E) Transformants of H1149 bearing empty vector or the indicated GCN2 alleles were subjected to Western analysis of eIF2α phosphorylation as described in Fig. 2B.
FIG. 5.
FIG. 5.
Disrupting the L856 hydrophobic network reduces the requirement for tRNA binding and autophosphorylation at T882 in Gcn2 activation. (A) H1149 transformants containing the indicated GCN2 alleles or empty vector were replica plated to SD, SD plus 30 mM 3-AT (3-AT), or SD plus 0.5 mM 5-FT (5-FT) and incubated for 3 days at 30°C. (B and C) The indicated strains from panel A were subjected to Western analysis of eIF2α phosphorylation as described in Fig. 2B. (D) Strains from panel A were streaked on synthetic complete medium lacking uracil and incubated for 3 days at 30°C.
FIG. 6.
FIG. 6.
Enhancing the parallel dimer interface reduces the requirement for T882 autophosphorylation and tRNA binding in Gcn2 activation. (A) H1149 transformants containing the indicated GCN2 alleles or empty vector were replica plated to SD, SD plus 30 mM 3-AT (3-AT), or SD plus 0.5 mM 5-FT (5-FT) and incubated for 3 days at 30°C. (B and C) Strains indicated in panel A were subjected to Western analysis of eIF2α phosphorylation as described in Fig. 2B.
FIG. 7.
FIG. 7.
The L856A and Hyper activating mutations reduce the requirement for the parallel dimer interface in Gcn2 activation. (A) H1149 transformants containing the indicated GCN2 alleles or empty vector were replica plated to SD, SD plus 30 mM 3-AT (3-AT), or SD plus 0.5 mM 5-FT (5-FT) and incubated for 3 days at 30°C. (B) The indicated strains from panel A were subjected to Western analysis of eIF2α phosphorylation as described in Fig. 2B. (C) Strains from panel A were streaked on synthetic complete medium lacking uracil and incubated for 3 days at 30°C.
FIG. 8.
FIG. 8.
Different orientations of the DFG + 1 Leu residue correlate with distinct positions of the DFG Asp in inactive versus active forms of eIF2α kinases and CDK2. In the structures of the inactive forms of Gcn2 (A) and CDK2 (C), interaction of the DFG + 1 Leu residue (L856 or L148, respectively, shown in purple) with hydrophobic residues in β3, β5, and αC (all in orange) correlates with an incorrect position of the Asp (in cyan) of the DFG motif, wherein its ∼9-Å separation from the DFG Gly residue (shown in yellow) precludes H bonding. The proposed disruption of the hydrophobic network during kinase activation of PKR (B) (considered a model for activated Gcn2) and CDK2 (D) might help to achieve the correct position of the DFG Asp and its H bonding to the DFG Gly residue.
FIG. 9.
FIG. 9.
Hypothetical model for multistep activation of Gcn2 by uncharged tRNA. The mode of KD dimerization and disposition of αC and key residues controlling ATP binding and catalysis are indicated schematically for different states in the proposed activation pathway for Gcn2. In all states, we envision that Gcn2 is dimerized through the self-interaction of the C-term (not shown), and the model depicts only the disposition of the KDs within the full-length dimer. (A and B) At the low levels of uncharged tRNA in nonstarved cells, Gcn2 exists in equilibrium between two inactive conformations, with that shown in panel A being highly favored; it contains nondimerized KDs in which ATP binding is hindered by a closed conformation of the N and C lobes and by N793, which forms a flap over the ATP-binding pocket, and catalysis is blocked by hinge rigidity, the incorrect rotation of αC, and the attendant E643-R834 inhibitory salt bridge. (B) The KDs adopt the parallel dimer conformation in which αC is properly oriented, but catalysis and ATP binding are still hindered by hinge rigidity, the closed conformation of N and C lobes, and the N793 flap. (C to E) Binding of tRNA to the HisRS domain occurs in starved cells and shifts the equilibrium to the three different states, of which the fully activated form (E) is highly favored. In all three of these states, tRNA binding to the HisRS domain has derigidified the hinge, removing the flap over the ATP-binding site and increasing interlobe flexibility to expand the separation between the N and C lobes. (C) This state is inactive, owing to the absence of parallel dimerization of the KDs and incorrect rotation of αC. (D) This state is active, containing the proper conformation of αC and the crucial K628-E643 salt bridge, and is capable of autophosphorylation to produce the fully functional kinase locked into the active conformation (E).
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