Abstract
Pulsed electron–electron double resonance (PELDOR) is an electron paramagnetic resonance (EPR) spectroscopy technique for nanometer distance measurements between paramagnetic centers such as radicals. PELDOR has been recognized as a valuable tool to approach structural questions in biological systems. In this manuscript, we demonstrate the value of distance measurements for differentiating competing structural models on the dimerization of the effector domain (ED) of the non-structural protein 1 (NS1) of the influenza A virus. Our results show NS1 to be well amenable to nanometer distance measurements by EPR, yielding high quality data. In combination with mutants perturbing protein dimerization and in silico prediction based on crystal structures, we can exclude one of two potential dimerization interfaces. Furthermore, our results lead to a viable hypothesis of a NS1 ED:ED interface which is flexible through rotation around the vector interconnecting the two native cysteines. These results prove the high value of pulse EPR as a complementary method for structural biology.
Highlights
Influenza A viruses remain a continuing threat to public health.[1]
As spin-labeled dimers of full length non-structural protein 1 (NS1) can bear four spin labels for dimers and even more for oligomers leading to more challenging theoretical treatment,[38] we approached the dimerization of the individual domains by performing measurements between spin labels incorporated into the individual RNA binding domain (RBD) and effector domain (ED) dimers using Pulsed electron−electron double resonance (PELDOR)
The primary PELDOR data for this dimer reveals at least four full dipolar modulations (Figure 2, top), which is exceptional for proteins spin-labeled with MTSSL via cysteines and clearly indicates very constrained spin-label conformations giving rise to a very narrow distance distribution
Summary
Influenza A viruses remain a continuing threat to public health.[1]. In addition to seasonal epidemics, the virus has the potential to cause worldwide pandemics, such as the 2009 H1N1 “swine flu” outbreak. The multifunctional NS1 (non-structural 1) protein of influenza A has been proposed to interact with a wide range of cellular and viral factors.[2,3] Most notably, expression of NS1 suppresses stimulation of the innate immune response through interactions with cellular pathogen recognition receptors (e.g., RIG-I and TRIM25),[4,5] by suppression of the host mRNA maturation via binding to the cellular and splicing processivity factor 30 (CPSF30)[6] and by sequestration of double stranded RNA (dsRNA) produced during viral infection.[7] NS1 stimulates viral growth through interactions with phosphoinositide-3-kinase (PI3K)[8] and the transcription factor eIF4E.9. Both domains form homodimeric interactions, and the full-length protein can oligomerize at higher concentrations.[10−15] While the dimer interface of the RBD is unambiguous, several dimer interfaces have been proposed for the ED, based upon X-ray crystallography of the ED and full-length NS1 protein (see Figure 1).[10,13,16]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
