A presumed homologue of the regulatory subunits of eIF2B functions as ribose-1,5-bisphosphate isomerase in Pyrococcus horikoshii OT3

Prerana Gogoi,Shankar Prasad Kanaujia

doi:10.1038/s41598-018-20418-w

Prerana Gogoi, Shankar Prasad Kanaujia

Open Access

https://doi.org/10.1038/s41598-018-20418-w

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Journal: Scientific Reports	Publication Date: Jan 30, 2018
Citations: 5	License type: open-access

Affiliation: Indian Institute of Technology Guwahati

Abstract

The homologues of the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B) are assumed to be present in archaea. Likewise, an ORF, PH0208 in Pyrococcus horikoshii OT3 have been proposed to encode one of the homologues of regulatory subunits of eIF2B. However, PH0208 protein also shares sequence similarity with a functionally non-related enzyme, ribose-1,5-bisphosphate isomerase (R15Pi), involved in conversion of ribose-1,5-bisphosphate (R15P) to ribulose-1,5-bisphosphate (RuBP) in an AMP-dependent manner. Herein, we have determined the crystal structure of PH0208 protein in order to decipher its true function. Although structurally similar to the regulatory subunits of eIF2B, the ability to bind R15P and RuBP suggests that PH0208 would function as R15Pi. Additionally, this study for the first time reports the binding sites of AMP and GMP in R15Pi. The AMP binding site in PH0208 protein clarified the role of AMP in providing structural stability to R15Pi. The binding of GMP to the ‘AMP binding site’ in addition to its own binding site indicates that GMP might also execute a similar function, though with less specificity. Furthermore, we have utilized the resemblance between PH0208 and the regulatory subunits of eIF2B to propose a model for the regulatory mechanism of eIF2B in eukaryotes.

Highlights

The resemblance of the protein translation initiation process between eukaryotes and archaea has long been postulated[1]
Later, based on an in-depth in silico analysis, we have demonstrated that the protein PH0208 encode an enzyme, ribose-1,5-bisphosphate isomerase (R15Pi) (EC 5.3.1.29) with all the essential amino acid residues conserved[20]
The crystal structure of PH0208 protein was solved by molecular replacement method using the atomic coordinates of Tk-R15Pi (PDB id: 3VM6), which has a sequence identity of 86% with PH0208

Summary

Introduction

The resemblance of the protein translation initiation process between eukaryotes and archaea has long been postulated[1]. OT3, a hyperthermophillic archaeon, three open reading frames (ORFs) (PH0440, PH0702 and PH0208) were postulated to encode aIF2Bα, aIF2Bβ and aIF2Bδ, respectively[19] This assumption was merely based on sequence similarity to the α, β, and δ subunits of eIF2B and lacked convincing evidence. We attempt to abolish the uncertainty regarding the (un)availability of a homologue of the regulatory subunits of eIF2B in archaea by performing a meticulous characterization of PH0208 protein. We have determined the three-dimensional structure of PH0208 protein in complex with the cognate substrate as well as product of the enzyme, R15Pi. To understand the role of purine nucleotides in structure and activity, crystal structures of PH0208 protein (wild type and mutants) in complex with only AMP, only GMP and both AMP & GMP were deciphered. A model depicting the regulatory mechanism of eIF2B in eukaryotes has been proposed based on the similitude perceived between PH0208 protein and the regulatory subunits of eIF2B

Methods

Results

Conclusion