Backbone resonance assignments and secondary structure of Ebola nucleoprotein 600-739 construct

Abstract

Ebola viral infections have resulted in several deadly epidemics in recent years in West and Central Africa. Because only one of the seven proteins encoded by the viral genome possesses enzymatic activity, disruption of protein-protein interactions is a promising route for antiviral drug development. We carried out a screening campaign to identify small, drug-like compounds that bind to the C-terminal region of the multifunctional Ebola nucleoprotein (eNP) with the objective of discovering ones that disrupt its binding to other Ebola proteins or to the single-stranded RNA genome. In the course of this effort we assigned the backbone ¹H, ¹⁵N, and ¹³C resonances of residues 600‒739, the region that contains the critical eVP30 binding region 600‒615 targeted by host factors, and used the assigned chemical shifts to predict secondary structural features and peptide dynamics. This work supports and extends the previous X-ray crystal structures and NMR studies of residues 641‒739. We found that the 600‒739 domain consists of separate regions that are largely disordered and ordered.

Keywords: Ebola virus; Non-segmented negative-stranded RNA virus; Nucleoprotein; Virus replication.

Fig.1

2D ¹H, ¹⁵N HSQC-TROSY spectrum of 1.3 mM eNP-CTD_600‒739 in 10 mM HEPES buffer, pH 7.0, containing 150 mM sodium chloride, 2 mM TCEP, and 5% D₂O, acquired at 298 K on a Varian spectrometer operating at 800 MHz (¹H). Assignments are annotated on the spectrum.

Fig. 2

Structural analysis results of eNP-CTD_600‒739 by TALOS-N and PECAN. a Secondary structure propensities predicted by TALOS-N (green, helix; blue, strand; gray box, sequence-based prediction for unassigned residue). Structural regions in agreement with PECAN (Fig.2. b) are denoted as α1–5 and β1–4. b Secondary structure probabilities predicted by PECAN (green, helix; blue, strand). c Random coil index order parameters (RCI-S²) predicted by TALOS-N (green to red, ordered to flexible).