Abstract
Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2) leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2) causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome.
Highlights
The functional structures of ADP-ribosylating toxins such as diphtheria toxin [1,2] and pseudomonas exotoxin [3] contain two interlinked moieties
While a growing body of knowledge has accumulated on the role of elongation factor 2 (EF2) and EF-G in the translocation of tRNAs on the ribosome [4,5,6,7,8,9,10], the specific events abolished by ADPRNEF2 that culminate to the inhibition of protein synthesis remain elusive
The second slowest step occurs at the EF2-mediated tRNA translocation (U~ k2 = 4.85 s21, k2 taken from [8,9]), followed by the POST release of EF2NGDP (k3 = 5 s21)
Summary
The functional structures of ADP-ribosylating toxins such as diphtheria toxin [1,2] and pseudomonas exotoxin [3] contain two interlinked moieties. A number of works reported that ADPRNEF2 reduced GTPase activity [13,14], reduced binding affinities to specific/non-specific rRNAs [15,16], to the 80S ribosome [14,17,18,19,20], and/or to GTP [20,21]. These findings suggest that inhibition of nascent protein syntheses may result from ADPRNEF2 abolishing the upstream event(s) before translocation. Countering evidence, advocated that ADPRNEF2 bound competitively against native EF2 for the PRE ribosome [11,22,23,24,25] or GTP [14,18,25,26,27], implying that ADPRNEF2 does not compromise the upstream processes of translocation but directly inhibits a late-phase tRNA translocation [7,25] to disrupt protein synthesis
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