Geissler, E. P. | bioRxiv Science

bioRxiv2025

Elp3 uses a conserved molecular tunnel to transport acetate between distant active sites and catalyze tRNA wobble base modification

The radical SAM enzyme Elp3 and eukaryotic Elongator complex catalyze formation of a key intermediate transfer RNA (tRNA) modification, 5-carboxymethyluridine (cm5U), in the anticodons of tRNAs across all domains of life. cm5U-derived modifications are important for fine tuning codon-anticodon interactions and efficient protein translation, and defects in this modification are linked to development of neurodegenerative disease in humans. Here we reconstitute tRNA modification activity with a model Elp3 enzyme and combine structural analyses, enzymology, and isotope incorporation experiments to show Elp3 harbors a conserved molecular tunnel that shuttles free acetate molecules from the acetyl-CoA binding domain to the radical SAM active site over 20 [A] away, where acetate undergoes radical-mediated reaction and addition to tRNA U34. Our model explains how Elp3 and Elongator bridge a large distance between active sites to catalyze tRNA carboxymethylation and illustrate a unique mechanism for intermediate transport in radical SAM enzymes. Graphical AbstractThe radical SAM enzyme Elp3 installs a critical tRNA wobble base modification in organisms across all domains of life. Here, the authors show how Elp3 uses a conserved molecular tunnel to transport acetate between distant Elp3 active sites to catalyze tRNA carboxymethylation, revealing a new mechanism for Elp3 and Elongator-mediated tRNA modification and the first example of acetate transport through an enzyme tunnel. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/652618v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@b3c25eorg.highwire.dtl.DTLVardef@19dd2b6org.highwire.dtl.DTLVardef@74d07corg.highwire.dtl.DTLVardef@123896f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease

The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis. Significance StatementViral proteases can strategically target human proteins to manipulate host biochemistry during infection. Here, we show that the SARS-CoV-2 main protease (Mpro) can specifically recognize and cleave the human tRNA methyltransferase enzyme TRMT1, and that cleavage of TRMT1 cripples its ability to install a key modification on human tRNAs that is critical for protein translation. Our structural and functional analysis of the Mpro-TRMT1 interaction shows how the flexible Mpro active site engages a conserved sequence in TRMT1 in an uncommon binding mode to catalyze its cleavage and inactivation. These studies provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.

biochemistry↗