Structural and Hydrodynamic Characterization of Dimeric Human Oligoadenylate Synthetase 2

Amit Koul,Darren Gemmill,Nikhat Lubna,Markus Meier,Natalie Krahn,Evan P Booy,Jörg Stetefeld,Trushar R Patel,Sean A Mckenna

doi:10.1016/j.bpj.2020.04.025

Abstract

Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.

Highlights

The 20-50-oligoadenylate synthetases (OASs) are interferoninduced proteins that catalyze the synthesis of 20-50-linked oligomers (2-5A) upon interaction with double-stranded RNA and are often triggered by viral infection [1]. 2-5A can activate latent ribonuclease L by causing its dimerization [2], which leads to the degradation of viral and cellular RNAs [3,4]
Extrapolation of the plot of rH measured at each concentration to infinite dilution provided a value of 7.5 5 0.26 nm (Fig. 1 B), which is significantly larger than the reported value obtained for OAS1 (3.0 5 0.3 nm) [34]
In the absence of any structural and hydrodynamic information on OAS2, we used a combination of different biophysical techniques, including analytical ultracentrifugation (AUC), dynamic light scattering (DLS), circular dichroism (CD), and SAXS, to investigate the solution properties and hydrodynamic parameters of OAS2

Summary

Introduction

The 20-50-oligoadenylate synthetases (OASs) are interferoninduced proteins that catalyze the synthesis of 20-50-linked oligomers (2-5A) upon interaction with double-stranded RNA (dsRNA) and are often triggered by viral infection [1]. 2-5A can activate latent ribonuclease L by causing its dimerization [2], which leads to the degradation of viral and cellular RNAs [3,4]. 2-5A can activate latent ribonuclease L by causing its dimerization [2], which leads to the degradation of viral and cellular RNAs [3,4] This degradation limits protein synthesis, thereby impairing viral replication [5,6]. Three aspartic acid residues in the active site form the core catalytic triad responsible for the coordination of two magnesium ions and are required for both the binding of ATP and 2-5A chain formation [11]. Mutation of these residues to alanine abrogates OAS activity [12]. OAS3 has three OAS1-like domains (domains I, II, and III), with domain III harboring the catalytic triad at positions D816, D818, and D888 [15]

Methods

Results

Conclusion