NMR spectra of PB2 627, the RNA-binding domain in influenza A virus RNA polymerase that contains the pathogenicity factor lysine 627, and improvement of the spectra by small osmolytes

Yusuke S Kato; Masaru Tanokura; Takashi Kuzuhara

doi:10.1016/j.bbrep.2017.09.003

NMR spectra of PB2 627, the RNA-binding domain in influenza A virus RNA polymerase that contains the pathogenicity factor lysine 627, and improvement of the spectra by small osmolytes

Biochem Biophys Rep. 2017 Sep 20:12:129-134. doi: 10.1016/j.bbrep.2017.09.003. eCollection 2017 Dec.

Authors

Yusuke S Kato^{1

2

3}, Masaru Tanokura², Takashi Kuzuhara⁴

Affiliations

¹ Institute for Health Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
² Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
³ Institute for Enzyme Research, Tokushima University, Tokushima 770-8503, Japan.
⁴ Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.

Abstract

The influenza A virus, which has an RNA genome, requires RNA-dependent RNA polymerase for transcription and replication. The polymerase is comprised of the subunits PA, PB1, and PB2. The C-terminal RNA-binding domain in PB2 contains lysine 627 (PB2 627), which is associated with pathogenicity and host range. However, the structure and molecular mechanism of PB2 627 in solution remain obscure. Here, we investigated PB2 627 in solution by nuclear magnetic resonance (NMR) and detected inhomogeneity in the intensities of backbone amide proton signals due to local fluctuations in structure. To characterize the effects of chemical chaperones on spectral data and improve the data quality, we tested 20 different additives, including L-arginine L-glutamate salt, (L-arginine)₂SO₄, glycerol, β-octylglucoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Na₂SO₄, 1,5-diaminopentane, 1,4-diaminobutane, trehalose, sucrose, glycine, trimethylamine N-oxide, β-alanine, L-α-alanine, hydroxyectoine, betaine, L-proline, and non-detergent sulfobetaine 195, 201, and 256. We evaluated the quality of the resulting spectra by calculating the standard deviation and average of the ratio of signal intensities to noise level of amide peaks, as well as the ratio of the standard deviation to the average. NMR-profile analysis revealed diverse effects of additives on the dynamic properties of PB2 627. Based on such criteria, we found that small osmolytes such as glycine and L-α-alanine reduced structural fluctuations and improved the quality of spectral data, which is likely to facilitate a detailed NMR-based structural analysis. The methodology developed here may also be more generally useful for evaluating the effects of chemical chaperones on the structural integrity of proteins.

Keywords: CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; HSQC, heteronuclear single quantum coherence; Influenza A virus; Irel, ratio of signal intensity to noise level; NDSB, non-detergent sulfobetaine; NMR, additive; NMR, nuclear magnetic resonance; PB2 627; PB2 627, C-terminal RNA-binding domain of PB2 containing lysine 627; RNA polymerase; S/N, signal-to-noise ratio; TMAO, trimethylamine N-oxide.