Large‐scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl‐tRNA synthetase

Abstract

a b s t r a c tStructural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a ''closed" form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl-tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.

Crystal Structure of Human Mitochondrial PheRS Complexed with tRNAPhe in the Active “Open” State

“…Experimental data suggest that the conformational mobility of ABD is highly relevant to hmPheRS operation. 19 First, solution studies suggest the availability of two forms of the enzyme, with predominance of the open form. Secondly, aminoacylation assays performed after introduction of two cysteines (K33C/T351C, separated by ∼ 5 Å), allowing disulfide bond formation under nonreducing conditions, exhibit negligible activity for hmPheRS, as compared to the wild-type (WT) enzyme when it is restrained in the closed conformation.…”

“…Biochemical and small-angle X-ray scattering experiments suggest that formation of the catalytically active PheRS-tRNA Phe complex in human mitochondria per se requires substantial ABD reorientation. 18,19 Starting from these data, we suggest that two operating conformations, closed and open, should exist for hmPheRS. However, the distinctive sites in tRNA that govern interactions with the monomeric PheRS remain unknown, and this information could not be deduced from the crystal structure of heterodimeric enzyme complexed with tRNA Phe .…”

Exclusive Use of trans-Editing Domains Prevents Proline Mistranslation

“…Some ARSs such as human mitochondrial LeuRS (48), yeast and human mitochondrial PheRSs (49,50), mitochondrial and cytosolic eukaryotic ProRSs, archaeal ProRSs, and some bacterial ProRSs such as C. crescentus ProRS investigated here (51,52), have lost their editing function and are in many cases more selective at the initial amino acid activation step (48,(52)(53)(54). Nevertheless, transediting factors such as ProXp-ala are encoded in some genomes.…”

Translation in Mammalian Mitochondria: Order and Disorder Linked to tRNAs and Aminoacyl-tRNA Synthetases

“…Instead of forming complex heterodimeric assemblies as bacterial, archaeal, and cytosolic enzymes, it forms a two-domain monomer, which only maintains the catalytic domain characteristic of class II. This human mitochondrial version is the smallest known aaRS (Yadavalli et al 2009). …”