19F NMR Reveals Multiple Conformations at the Dimer Interface of the Nonstructural Protein 1 Effector Domain from Influenza A Virus

Abstract

SUMMARY Non-structural protein 1 of influenza A virus, NS1A, is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain (RBD) and a multifunctional C-terminal effector domain (ED), each of which can independently form symmetric homodimers. Here we apply 19F NMR to NS1A from influenza A/Udorn/307/1972 virus (H3N2) labeled with 5-fluorotryptophan (5-F-Trp), and demonstrate that the 19F signal of Trp187 is a sensitive, direct monitor of the ED helix-helix dimer interface. 19F relaxation dispersion data reveal the presence of conformational dynamics within this functionally important protein-protein interface, whose rate is over three orders of magnitude faster than the kinetics of ED dimerization. 19F NMR also affords direct spectroscopic evidence that Trp187, which mediates intermolecular ED:ED interactions required for cooperative dsRNA binding, is solvent exposed in full-length NS1Aat concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection.

GTP-Dependent K-Ras Dimerization

“…The range of crystal dimer interfaces observed in different crystal forms capture snapshots of heterogeneous dimer conformations. Interfaces, including those of Ras dimers, are dynamic, interconverting between substates (Aramini et al, 2014). Along these lines, a large-amplitude rocking motion has been detected by 19 F NMR in the dimer interface of influenza A virus nonstructural protein 1 (NS1A) (Aramini et al, 2014).…”

“…Interfaces, including those of Ras dimers, are dynamic, interconverting between substates (Aramini et al, 2014). Along these lines, a large-amplitude rocking motion has been detected by 19 F NMR in the dimer interface of influenza A virus nonstructural protein 1 (NS1A) (Aramini et al, 2014). We propose that both α-helical and β-sheet dimers are populated in membrane nanoclusters; a single interface type is unlikely to support nanocluster avidity.…”

The RAS-Binding Domain of Human BRAF Protein Serine/Threonine Kinase Exhibits Allosteric Conformational Changes upon Binding HRAS

“…The historic perception of protein structure as being static has been supplanted in recent years by a more dynamic view centered on the concept of conformational selection (Boehr et al, 2009), whereby proteins fluctuate between a range of structures and are thereby predisposed for interaction with binding partners (Huang and Montelione, 2005). To this end, NMR relaxation techniques have taken center stage in establishing the link between the intrinsic internal conformational dynamics of proteins, even for buried methyl-bearing residues, and macromolecular recognition and allosteric regulation (Aramini et al, 2014; Sekhar and Kay, 2013; Tzeng and Kalodimos, 2011, 2012; Wand, 2013). Moreover, β-sheet structures are particularly well suited for mediating allostery and signal transduction via correlated backbone motions causing global sheet bending and twisting, resulting in “channels of communication” running perpendicular to the strands (Fenwick et al, 2014).…”

19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

“…Resonance broadening or resonance overlap can further compound this impediment. Traditionally, resonance assignments of amino acid type-labeled proteins involves systematic, iterative substitution of all residues of the specific amino acid type in the protein with a structurally similar amino acid by site-directed mutagenesis (Aramini et al, 2014;Hyean-Woo et al, 2000;Li et al, 2009;Salopek-Sondi & Luck, 2002). Full spectral assignments can, in principle, be obtained by recording spectra of the complete set of single mutants and comparison of the remaining resonances in the mutants with those in the fully labeled native protein.…”

“…The approaches that have been used to incorporate 19 F-modified aromatic amino acids into proteins using biosynthetic microbial expression vary in detail but share several key steps (Aramini et al, 2014;Kim et al, 1990;Li et al, 2009). First, since addition of the 19 F-modifed amino acid can cause toxicity and interfere with protein synthesis, as a precaution, bacteria usually are not grown from an inoculum that was grown in 19 F-modified amino acidcontaining media.…”