Abstract
Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan, Trypanosoma brucei, are transcribed individually. Also unlike most mitochondrial mRNAs, the 5′ end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTP-bound and RNA-bound T. brucei MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2-like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.
Highlights
Kinetoplastids, a group of flagellated protists that include the parasitic protozoa, Trypanosoma brucei, T. cruzi, and Leishmania spp., are named after their unusual mitochondrial DNA called the kinetoplast (Vickerman 1985; Stevens 2008)
Data analyses suggest that the amino-terminal region likely functions as a signal sequence; MERS1 is chromosomally encoded and the protein transported into the mitochondria
Crystals of the apo protein, MERS1–GTP complex and MERS1 bound to the RNA site 5′-GAGAGGG GGGUU-3′ were produced (Materials and Methods)
Summary
Kinetoplastids, a group of flagellated protists that include the parasitic protozoa, Trypanosoma brucei, T. cruzi, and Leishmania spp., are named after their unusual mitochondrial DNA called the kinetoplast (Vickerman 1985; Stevens 2008). Reveals that there are insertions within its Nudix motifs, which are interaction motifs that may be involved fold compared to the other hydrolases, resi- in binding MERS1 and/or RNA. This helix encompasses residues 278–283 forms part of a shield over docks near the MERS1 Nudix motif and is proximal to the the guanine nucleotide-binding site and interacts with acidic residues of this motif.
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