The mechanism for activation of GTP hydrolysis on the ribosome

Abstract

Protein synthesis requires several guanosine triphosphatase (GTPase) factors, including elongation factor Tu (EF-Tu), which delivers aminoacyl-transfer RNAs (tRNAs) to the ribosome. To understand how the ribosome triggers GTP hydrolysis in translational GTPases, we have determined the crystal structure of EF-Tu and aminoacyl-tRNA bound to the ribosome with a GTP analog, to 3.2 angstrom resolution. EF-Tu is in its active conformation, the switch I loop is ordered, and the catalytic histidine is coordinating the nucleophilic water in position for inline attack on the γ-phosphate of GTP. This activated conformation is due to a critical and conserved interaction of the histidine with A2662 of the sarcin-ricin loop of the 23S ribosomal RNA. The structure suggests a universal mechanism for GTPase activation and hydrolysis in translational GTPases on the ribosome.

Figure 1

The ternary complex bound to the ribosome in the activated state. A) EF-Tu (red) and aminoacyl-tRNA (green) bound to the 70S ribosome, stabilized by GDPCP (blue). B) Unbiased F_o-F_c electron density for GDPCP, the water molecule (orange), and His84 positioned in a catalytically active conformation. E.coli number is used throughout the text. C) Unbiased F_o-F_c electron density displayed for the Switch I loop.

Figure 2

The active site of EF-Tu during decoding. A) Superposition of the GTPase centers from the isolated TC (grey)(19) and the TC-GDPCP-ribosome (red). Small movements are observed for hydrophobic gate residues Val20 and Ile60. B) GTPase activation allows the phosphate of A2662 of the SRL (orange) to position His84 into the active site. After GTP hydrolysis (15) and Pi release Switch I becomes disordered (dashed-line) and His84 rotates away from GTP.

Figure 3

Chemical mechanism of GTP hydrolysis. A) Highly conserved His84 acts as a general base to activate the catalytic water molecule, which is positioned by interactions with Thr61, Gly83 and His84. B) Chemical drawing depicting the interactions between EF-Tu and GTP that stabilize the β and γ phosphates, allowing GTP hydrolysis to occur (see also fig s3).