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Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z.

ABSTRACT: The enzyme tRNase Z, a member of the metallo-?-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: NdomTprox, located at the carboxy end of the amino domain proximal to the linker, and Tflex, a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F329 and L332) at the carboxy end of NdomTprox show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the Ndom- linker boundary. The Ala substitution for N378 in the Tflex region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R382 reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.

SUBMITTER: Saoura M

PROVIDER: S-EPMC5646807 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z.

Saoura Makenzie M Pinnock Kyla K Pujantell-Graell Maria M Levinger Louis L

PloS one 20171018 10

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3' trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase ZS, functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase ZL, related to tRNase ZS through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase ZS. tRNase ZL consists of related but ...[more]

PMID: 29045449

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Project description:Calmodulin (CaM) is a highly-expressed Ca2+ binding protein known to bind hundreds of protein targets. Its binding selectivity to many of these targets is partially attributed to the protein's flexible alpha helical linker that connects its N- and C-domains. It is not well established how its linker mediates CaM's binding to regulatory targets yet. Insights into this would be invaluable to understanding its regulation of diverse cellular signaling pathways. Therefore, we utilized Martini coarse-grained (CG) molecular dynamics simulations to probe CaM/target assembly for a model system: CaM binding to the calcineurin (CaN) regulatory domain. The simulations were conducted assuming a 'wild-type' calmodulin with normal flexibility of its linker, as well as a labile, highly-flexible linker variant to emulate structural changes that could be induced, for instance, by post-translational modifications. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 μs of simulation time; of these, 1.7% sampled the fully-bound state observed in the experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be ka= 8.7 × 108 M-1 s-1, which is similar to the diffusion-limited, experimentally-determined rate of 2.2 × 108 M-1 s-1. Furthermore, our simulations recapitulated its well-known inverse relationship between the association rate and the solution ionic strength. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states which are controlled by the linker flexibility. Therefore, our simulations suggest that variations in the CaM linker's propensity for alpha helical secondary structure can modulate the kinetics of target binding.

Dataset Information

Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z.

Publications

Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase ZL, the long form of tRNase Z.

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