A monovalent cation acts as structural and catalytic cofactor in translational GTPases

Abstract

Translational GTPases are universally conserved GTP hydrolyzing enzymes, critical for fidelity and speed of ribosomal protein biosynthesis. Despite their central roles, the mechanisms of GTP-dependent conformational switching and GTP hydrolysis that govern the function of trGTPases remain poorly understood. Here, we provide biochemical and high-resolution structural evidence that eIF5B and aEF1A/EF-Tu bound to GTP or GTPγS coordinate a monovalent cation (M(+)) in their active site. Our data reveal that M(+) ions form constitutive components of the catalytic machinery in trGTPases acting as structural cofactor to stabilize the GTP-bound "on" state. Additionally, the M(+) ion provides a positive charge into the active site analogous to the arginine-finger in the Ras-RasGAP system indicating a similar role as catalytic element that stabilizes the transition state of the hydrolysis reaction. In sequence and structure, the coordination shell for the M(+) ion is, with exception of eIF2γ, highly conserved among trGTPases from bacteria to human. We therefore propose a universal mechanism of M(+)-dependent conformational switching and GTP hydrolysis among trGTPases with important consequences for the interpretation of available biochemical and structural data.

Keywords: GTPase; catalytic mechanism; crystal structure; monovalent cation; translation.

Figure 1. GTP-bound eIF5B coordinates an M⁺ ion in its GTPase center

1. Overview of G domain and domain II of GTP-bound eIF5B with a Na⁺ ion (purple sphere) in the active site. P-loop and switch regions are shown in cyan; GTP is shown as balls and sticks. The Na⁺ ion is bound by a pentameric coordination sphere (inset; indicated by purple lines) formed by GTP, Asp^MC and Gly^MC. Mg²⁺ and water molecules are shown as spheres in light brown and gray, respectively; the catalytic histidine (His480) and highly conserved residues that interact with GTP or the cations are indicated; hydrogen bonds are shown as dashed lines.

2. The active site of eIF5B·GTPγS with a K⁺ ion (yellow sphere) bound in a heptameric coordination sphere.

3. Superposition of eIF5B·GTPγS structures with either Na⁺ (purple sphere) or K⁺ (yellow sphere) bound in the active site. Due to the shorter coordination distances to the Na⁺ ion, Asp^MC and Gly^MC (purple sticks) are drawn closer to the GTP molecule than in the K⁺-structure (yellow sticks).

4. Superposition of eIF5B·GTPγS·K⁺ and MnmE (purple; PDB: 2GJ8) bound to GDP, AlF₄ and K⁺.

Figure 2. Biochemical characterization of nucleotide binding and GTP hydrolysis by eIF5B

A GDPNP disrupts the coordination sphere for the M⁺ ion (blue sphere) formed by Asp^MC, Gly^MC and three oxygens from GTP by replacing the β-γ-bridging oxygen (O) with an NH group. B Temperature dependency of binding enthalpy changes (ΔH) upon eIF5B(517–858) interaction with GDP (•), GDPNP (○) or GTPγS (▾) measured by ITC. C Intrinsic GTPase activity of eIF5B(517–858) [wild-type (wt) or the D533A mutant] determined at 35°C in the presence of 200 mM of the indicated salts, 25 μM protein and 300 μM GTP and subsequent analysis by HPLC. The order in which the combinations are given on the right corresponds to the relative rates of GTP hydrolysis. D Intrinsic GTPase activity of eIF5B(517–858) wild-type (black), D533A (red), D533N (green) and D533R (green) in the presence of NaCl under conditions as in (C). E, F Dependency of the intrinsic GTPase activity of eIF5B(517–858) wild-type (•), D533A (○) and D533R (▾) on the concentration of KCl (E) or NaCl (F). Data information: Experiments were repeated two to three times; standard deviations are given by error bars (in some cases not visible because they are smaller than the symbol size).

Figure 3. Structural elements required for M⁺-coordination in eIF5B are universally conserved among trGTPases

1. Excerpt of a multiple sequence alignment of different trGTPases (orthologs of RF3, eIF5B, eIF2γ, SelB, eRF3, EF-Tu, LepA, EF-G) showing P-loop and switch 1. The upper and lower numbering corresponds to C. thermophilum eIF5B (Cthe) and Escherichia coli EF-Tu (Ecoli), respectively. Highly conserved residues are highlighted in dark blue, conserved residues in light blue. Asp^MC and Gly^MC are highlighted in red; residues in eIF2γ that replace Asp^MC and Gly^MC are highlighted in yellow.

2. Superposition of eIF5B·GTPγS·K⁺ (colored as in Fig 1) with ribosome-bound EF-G·GDPCP (brown; PDB: 4JUW). Ribosome-bound EF-G provides all structural elements to bind the M⁺ ion; however, its coordination is prevented by the CH₂ group of GDPCP in lieu of the β-γ-bridging oxygen (arrow). A water molecule (red sphere) is bound next to the M⁺-binding site instead.

3. Similarly, EF-Tu·GDPNP (purple; PDB: 2C78) provides all structural elements to bind the M⁺ ion; however, its coordination is prevented by the NH group of GDPNP (arrow). A water molecule (red sphere) is bound next to the M⁺ binding site instead.

Figure 4. The GTP-bound EF-Tu ortholog aEF1A coordinates an M⁺ ion in its GTPase center

1. Overview of domains I (G) to III of GTP-bound aEF1A with a Na⁺ ion (purple sphere) in the active site. P-loop and switch regions are shown in yellow. The inset shows a detailed view on the active site with the coordination sphere of the Na⁺ ion (indicated by purple lines) formed by GTP, Asp^MC and Gly^MC in the MC-loop.

2. Superposition of GTP-bound aEF1A with either a Na⁺ (purple sphere) or NH₄⁺ ion (blue sphere) in the active site. Both ions are coordinated by the identical sphere, however, with different coordination distances.

3. Superposition of EF-Tu·GDPNP (switch 1 in red; PDB: 1EXM) with EF-Tu·GDPNP in the ternary complex (TC) with aa-tRNA (blue; PDBs: 1TTT, 1OB2) and aEF1A·GTP·M⁺ (M⁺ in blue; switch 1 in yellow). The M⁺ ion stabilizes a conformation of helix A″ that is required for stable TC formation.

4. Dependency of the intrinsic GTPase activity of E. coli EF-Tu wild-type (•) or the D21A mutant (○) on the concentration of K⁺ ions, determined in the presence of 0–1 M KCl under single turnover conditions (20 μM GTP-bound EF-Tu) at 30°C and subsequent analysis by HPLC.

5. Intrinsic GTPase activity of E. coli EF-Tu determined in the presence of 200 mM of the indicated salts under single turnover conditions. The order in which the combinations are given on the right corresponds to the relative rates of GTP hydrolysis.

Data information: Experiments were repeated two to three times; standard deviations are given by error bars (in some cases not visible because they are smaller than the symbol size).

Figure 5. The mechanism of M⁺-dependent conformational switching in trGTPases

1. The nucleotide-dependent conformational switch in the G domain of a/eIF5B. In eIF5B·GDP, switch 1 and 2 are oriented away from the nucleotide binding pocket. Upon exchange of GDP for GTP, the switch regions undergo a large conformational rearrangement that results in direct contacts with the γ-phosphate. Here, the M⁺ ion (blue sphere) provides a direct contribution to the stabilization of switch 1 (inset). This contribution is missing in aIF5B·GDPNP (PDB: 1G7T), allowing it to crystallize in the GDP-like “off” state conformation.

2. Schematic presentation of the M⁺-dependent conformational switch mechanism in trGTPases.

Figure 6. The M⁺ ion as catalytic element in the GTP hydrolysis reaction

A Model of aEF1A·GTP·M⁺ on the ribosome [the sarcin-ricin loop (SRL) is shown as green sticks], based on a superposition with ribosome-bound EF-Tu (gray; PDB: 2XQD). Upon productive interactions with the SRL, the imidazole moiety of His^cat is reoriented from its inactive ground state (purple) to the active position (yellow) in which it forms a hydrogen bond to W^cat (Voorhees et al, 2010). The invariant P-loop lysine, the Mg²⁺ ion (light brown sphere) and the M⁺ ion (blue sphere) form a triangle of positively charged moieties around the β-γ-bridging oxygen. The M⁺ ion is thus suitably positioned to stabilize negative charges that develop in the TS of the hydrolysis reaction. B Schematic presentation of the active site of a ribosome-bound trGTPase with GTP in the transition state of the hydrolysis reaction stabilized by the M⁺ ion (blue). TrGTPase and ribosome are colored in purple and green, respectively; negative charges, the P-loop lysine and the Mg²⁺ ion are omitted for clarity. For simplicity, His^cat is shown in its neutral form, although it might be double protonated in its activated state (Adamczyk & Warshel, ; Liljas et al, ; Aleksandrov & Field, ; Wallin et al, 2013) (modified from Bos et al, and Rodnina, 2009). C, D The M⁺ ion in eIF5B and aEF1A (purple) is coordinated in a position analogous to the arginine-finger in the complex of Ras (gray) and RasGAP (green) (modified from Bos et al, and Rodnina, 2009).