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• W1997358511 abstract "•19F NMR is applied to 5-F-Trp-labeled Ud NS1A and its isolated RBD and ED domains•19F NMR reveals conformational exchange at ED dimer interface monitored by Trp187•ED dimer conformational exchange is >103 times faster than kinetics of dimerization•19F NMR shows Trp187 is exposed in full-length NS1A at subaggregate concentrations Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain 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, and we 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 more than 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 NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection. Nonstructural protein 1 of influenza A virus (NS1A) is a conserved virulence factor comprised of an N-terminal double-stranded RNA (dsRNA)-binding domain 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, and we 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 more than 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 NS1A at concentrations below aggregation. These results have important implications for the diverse roles of this NS1A epitope during influenza virus infection. Influenza A viruses constitute a genus of enveloped viruses in the Orthomyxoviridae family characterized by a segmented negative-stranded RNA genome encoding up to 14 proteins (Wise et al., 2012Wise H.M. Hutchinson E.C. Jagger B.W. Stuart A.D. Kang Z.H. Robb N. Schwartzman L.M. Kash J.C. Fodor E. Firth A.E. et al.Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.PLoS Pathog. 2012; 8: e1002998Crossref PubMed Scopus (161) Google Scholar) that are either incorporated into the virion particle or expressed in the infected host cell, so-called “structural” and “nonstructural” proteins, respectively (Medina and García-Sastre, 2011Medina R.A. García-Sastre A. Influenza A viruses: new research developments.Nat. Rev. Microbiol. 2011; 9: 590-603Crossref PubMed Scopus (438) Google Scholar). The multifunctional nonstructural protein 1 of influenza A virus (NS1A) plays an integral role in subverting the innate antiviral response of the host and also in regulating several virus functions (Hale et al., 2008bHale B.G. Randall R.E. Ortín J. Jackson D. The multifunctional NS1 protein of influenza A viruses.J. Gen. Virol. 2008; 89: 2359-2376Crossref PubMed Scopus (831) Google Scholar, Krug and Garcia-Sastre, 2013Krug R.M. Garcia-Sastre A. The NS1 protein: a master regulator of host and viral functions.in: Webster R.G. Monto A.S. Braciale T.J. Lamb R.A. Textbook of Influenza. John Wiley & Sons, Oxford, UK2013: 114-132Crossref Google Scholar). This highly conserved hub protein in influenza infection consists of a 73-residue N-terminal double-stranded RNA (dsRNA)-binding domain (RBD) tethered by a flexible linker to an effector domain (ED) that binds to a plethora of host cellular proteins, followed by an unstructured C-terminal polypeptide segment. Several structural studies on NS1A RBD (Cheng et al., 2009Cheng A. Wong S.M. Yuan Y.A. Structural basis for dsRNA recognition by NS1 protein of influenza A virus.Cell Res. 2009; 19: 187-195Crossref PubMed Scopus (100) Google Scholar, Chien et al., 1997Chien C.Y. Tejero R. Huang Y. Zimmerman D.E. Ríos C.B. Krug R.M. Montelione G.T. A novel RNA-binding motif in influenza A virus non-structural protein 1.Nat. Struct. Biol. 1997; 4: 891-895Crossref PubMed Scopus (99) Google Scholar, Liu et al., 1997Liu J. Lynch P.A. Chien C.Y. Montelione G.T. Krug R.M. Berman H.M. Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein.Nat. Struct. Biol. 1997; 4: 896-899Crossref PubMed Scopus (116) Google Scholar) and ED (Bornholdt and Prasad, 2006Bornholdt Z.A. Prasad B.V. X-ray structure of influenza virus NS1 effector domain.Nat. Struct. Mol. Biol. 2006; 13: 559-560Crossref PubMed Scopus (90) Google Scholar, Hale et al., 2008aHale B.G. Barclay W.S. Randall R.E. Russell R.J. Structure of an avian influenza A virus NS1 protein effector domain.Virology. 2008; 378: 1-5Crossref PubMed Scopus (71) Google Scholar, Kerry et al., 2011Kerry P.S. Ayllon J. Taylor M.A. Hass C. Lewis A. García-Sastre A. Randall R.E. Hale B.G. Russell R.J. A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.PLoS ONE. 2011; 6: e17946Crossref PubMed Scopus (41) Google Scholar, Xia et al., 2009Xia S. Monzingo A.F. Robertus J.D. Structure of NS1A effector domain from the influenza A/Udorn/72 virus.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 11-17Crossref PubMed Scopus (39) Google Scholar) domains, as well as full-length NS1A (Bornholdt and Prasad, 2008Bornholdt Z.A. Prasad B.V. X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus.Nature. 2008; 456: 985-988Crossref PubMed Scopus (123) Google Scholar), have established that both domains adopt independent, unique homodimer structures. The isolated RBD forms a unique six-helical head-to-tail symmetric homodimer featuring an A-form RNA-binding epitope conserved across influenza A and B viruses (Cheng et al., 2009Cheng A. Wong S.M. Yuan Y.A. Structural basis for dsRNA recognition by NS1 protein of influenza A virus.Cell Res. 2009; 19: 187-195Crossref PubMed Scopus (100) Google Scholar, Yin et al., 2007Yin C. Khan J.A. Swapna G.V. Ertekin A. Krug R.M. Tong L. Montelione G.T. Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses.J. Biol. Chem. 2007; 282: 20584-20592Crossref PubMed Scopus (80) Google Scholar). The isolated ED adopts a novel α helix β-crescent fold and also forms a homodimeric structure (Bornholdt and Prasad, 2006Bornholdt Z.A. Prasad B.V. X-ray structure of influenza virus NS1 effector domain.Nat. Struct. Mol. Biol. 2006; 13: 559-560Crossref PubMed Scopus (90) Google Scholar, Hale et al., 2008aHale B.G. Barclay W.S. Randall R.E. Russell R.J. Structure of an avian influenza A virus NS1 protein effector domain.Virology. 2008; 378: 1-5Crossref PubMed Scopus (71) Google Scholar, Kerry et al., 2011Kerry P.S. Ayllon J. Taylor M.A. Hass C. Lewis A. García-Sastre A. Randall R.E. Hale B.G. Russell R.J. A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.PLoS ONE. 2011; 6: e17946Crossref PubMed Scopus (41) Google Scholar, Xia et al., 2009Xia S. Monzingo A.F. Robertus J.D. Structure of NS1A effector domain from the influenza A/Udorn/72 virus.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 11-17Crossref PubMed Scopus (39) Google Scholar). Some controversy concerning the exact nature of the biologically relevant dimer interface of NS1A ED was dispelled by recent biophysical studies conclusively establishing that the ED dimer interface in solution encompasses the C-terminal portion of the long α helix in each subunit (Aramini et al., 2011Aramini J.M. Ma L.C. Zhou L. Schauder C.M. Hamilton K. Amer B.R. Mack T.R. Lee H.W. Ciccosanti C.T. Zhao L. et al.Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.J. Biol. Chem. 2011; 286: 26050-26060Crossref PubMed Scopus (52) Google Scholar, Kerry et al., 2011Kerry P.S. Ayllon J. Taylor M.A. Hass C. Lewis A. García-Sastre A. Randall R.E. Hale B.G. Russell R.J. A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.PLoS ONE. 2011; 6: e17946Crossref PubMed Scopus (41) Google Scholar). The focal point of this “helix:helix” dimer interface is a highly conserved tryptophan residue, Trp187. This ED dimerization epitope interacts with host targets, such as the 30 kDa subunit of cleavage and polyadenylation specificity factor (CPSF30) (Das et al., 2008Das K. Ma L.C. Xiao R. Radvansky B. Aramini J. Zhao L. Marklund J. Kuo R.L. Twu K.Y. Arnold E. et al.Structural basis for suppression of a host antiviral response by influenza A virus.Proc. Natl. Acad. Sci. USA. 2008; 105: 13093-13098Crossref PubMed Scopus (180) Google Scholar). Structural studies of NS1A ED in complex with domains from CPSF30 (Das et al., 2008Das K. Ma L.C. Xiao R. Radvansky B. Aramini J. Zhao L. Marklund J. Kuo R.L. Twu K.Y. Arnold E. et al.Structural basis for suppression of a host antiviral response by influenza A virus.Proc. Natl. Acad. Sci. USA. 2008; 105: 13093-13098Crossref PubMed Scopus (180) Google Scholar) and the p85β subunit of phosphoinositide 3-kinase (Hale et al., 2010Hale B.G. Kerry P.S. Jackson D. Precious B.L. Gray A. Killip M.J. Randall R.E. Russell R.J. Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein.Proc. Natl. Acad. Sci. USA. 2010; 107: 1954-1959Crossref PubMed Scopus (90) Google Scholar) have also revealed that ED dimer dissociation is a prerequisite for complex formation. Independent of these host protein interactions, this same surface epitope of the ED plays an important role in the mechanism of cooperative dsRNA binding by full-length NS1A in vitro (Aramini et al., 2011Aramini J.M. Ma L.C. Zhou L. Schauder C.M. Hamilton K. Amer B.R. Mack T.R. Lee H.W. Ciccosanti C.T. Zhao L. et al.Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.J. Biol. Chem. 2011; 286: 26050-26060Crossref PubMed Scopus (52) Google Scholar) and in vivo (Ayllon et al., 2012Ayllon J. Russell R.J. García-Sastre A. Hale B.G. Contribution of NS1 effector domain dimerization to influenza A virus replication and virulence.J. Virol. 2012; 86: 13095-13098Crossref PubMed Scopus (25) Google Scholar). This promiscuous behavior of ED facilitates the multiple functions of NS1A in a spatial and temporal manner within the infected host cell (Kerry et al., 2011Kerry P.S. Ayllon J. Taylor M.A. Hass C. Lewis A. García-Sastre A. Randall R.E. Hale B.G. Russell R.J. A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.PLoS ONE. 2011; 6: e17946Crossref PubMed Scopus (41) Google Scholar). Moreover, the ability of NS1A to bind multiple partner proteins suggests underlying conformational plasticity (Nobeli et al., 2009Nobeli I. Favia A.D. Thornton J.M. Protein promiscuity and its implications for biotechnology.Nat. Biotechnol. 2009; 27: 157-167Crossref PubMed Scopus (377) Google Scholar). All of these studies, coupled with recent progress in the design of NS1A-based inhibitors (Jablonski et al., 2012Jablonski J.J. Basu D. Engel D.A. Geysen H.M. Design, synthesis, and evaluation of novel small molecule inhibitors of the influenza virus protein NS1.Bioorg. Med. Chem. 2012; 20: 487-497Crossref PubMed Scopus (32) Google Scholar) and attenuated viruses (Richt and García-Sastre, 2009Richt J.A. García-Sastre A. Attenuated influenza virus vaccines with modified NS1 proteins.Curr. Top. Microbiol. Immunol. 2009; 333: 177-195PubMed Google Scholar), underscore the growing interest in the NS1A protein as a target for the development of novel therapeutics to combat future outbreaks of potentially deadly forms of influenza A virus (Imai et al., 2012Imai M. Watanabe T. Hatta M. Das S.C. Ozawa M. Shinya K. Zhong G. Hanson A. Katsura H. Watanabe S. et al.Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets.Nature. 2012; 486: 420-428Crossref PubMed Scopus (1077) Google Scholar). Since the 1960s, 19F has been recognized as a valuable NMR probe for biological systems due to its numerous favorable properties, including its nuclear spin (I = ½), high natural abundance (100%), extremely high resonance frequency and sensitivity (83% that of 1H), minimal inherent 19F background signals, and the exquisite sensitivity of its chemical shift to changes in local environment (Danielson and Falke, 1996Danielson M.A. Falke J.J. Use of 19F NMR to probe protein structure and conformational changes.Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 163-195Crossref PubMed Scopus (274) Google Scholar, Gerig, 1994Gerig J.T. Fluorine NMR of proteins.Prog. Nucl. Magn. Reson. Spectrosc. 1994; 26: 293-370Abstract Full Text PDF Scopus (239) Google Scholar, Kitevski-LeBlanc and Prosser, 2012Kitevski-LeBlanc J.L. Prosser R.S. Current applications of 19F NMR to studies of protein structure and dynamics.Prog. Nucl. Magn. Reson. Spectrosc. 2012; 62: 1-33Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Indeed, 19F chemical shift ranges for a given residue type incorporated into a folded protein can span 5 to 20 ppm (Gerig, 1994Gerig J.T. Fluorine NMR of proteins.Prog. Nucl. Magn. Reson. Spectrosc. 1994; 26: 293-370Abstract Full Text PDF Scopus (239) Google Scholar). Moreover, because of the comparable atomic radii of hydrogen and fluorine, incorporation of fluorinated amino acids into proteins generally results in relatively minor structural perturbations, although this is highly probe and protein dependent (Danielson and Falke, 1996Danielson M.A. Falke J.J. Use of 19F NMR to probe protein structure and conformational changes.Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 163-195Crossref PubMed Scopus (274) Google Scholar, Kitevski-LeBlanc and Prosser, 2012Kitevski-LeBlanc J.L. Prosser R.S. Current applications of 19F NMR to studies of protein structure and dynamics.Prog. Nucl. Magn. Reson. Spectrosc. 2012; 62: 1-33Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). As a result of these traits, 19F NMR has been successfully applied to a wide variety of protein structural and dynamics studies, including probing site-specific conformational changes in proteins, protein complexes, and membrane proteins, ligand binding studies, and in-cell NMR studies (Didenko et al., 2013Didenko T. Liu J.J. Horst R. Stevens R.C. Wüthrich K. Fluorine-19 NMR of integral membrane proteins illustrated with studies of GPCRs.Curr. Opin. Struct. Biol. 2013; 23: 740-747Crossref PubMed Scopus (68) Google Scholar, Kitevski-LeBlanc and Prosser, 2012Kitevski-LeBlanc J.L. Prosser R.S. Current applications of 19F NMR to studies of protein structure and dynamics.Prog. Nucl. Magn. Reson. Spectrosc. 2012; 62: 1-33Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). In particular, the general importance of tryptophan at protein:protein interfaces combined with the relative paucity of this amino acid in proteins makes the combination of 19F NMR and incorporation of fluorinated tryptophan analogs an ideal strategy for directly probing protein interaction surfaces in complexes, including dimers (Liu et al., 2012Liu L. Byeon I.J.L. Bahar I. Gronenborn A.M. Domain swapping proceeds via complete unfolding: a 19F- and 1H-NMR study of the Cyanovirin-N protein.J. Am. Chem. Soc. 2012; 134: 4229-4235Crossref PubMed Scopus (44) Google Scholar). Here we have applied 19F NMR to the study of NS1A from influenza A/Udorn (Ud)/307/1972 virus (H3N2), Ud NS1A. Full-length Ud NS1A contains a total of four tryptophan residues, with one residue in the RBD (Trp16) and three residues in the ED (Trp102, Trp187, and Trp203) (Figure 1A). We incorporated 5-fluorotryptophan (5-F-Trp), containing a fluorine substitution at the C5 (International Union of Pure and Applied Chemistry, Cζ3) position of the indole ring, into the RBD, ED, and a truncated form of full-length Ud NS1A lacking the C-terminal 22 residues (NS1A′) and assigned the 19F signals corresponding to the four 5-F-Trp residues by site-directed mutagenesis. We demonstrate that the 19F signal for Trp187 can be used to directly monitor the oligomerization state (i.e., monomer and dimer) of the ED. Moreover, the concentration dependence of 19F line shapes and relaxation properties of the 19F signal for Trp187 demonstrate conformational heterogeneity and microsecond-to-millisecond motional dynamics within the ED dimer at this important protein interaction interface. Such interfacial dynamics provide a biophysical mechanism by which the energetics of molecular recognition can be modulated by attenuating the entropy changes associated with dimerization (Huang and Montelione, 2005Huang Y.J. Montelione G.T. Proteins flex to function.Nature. 2005; 438: 36-37Crossref PubMed Scopus (64) Google Scholar). Finally, in full-length NS1A, where the N-terminal RBD forms a tight dimer, 19F NMR provides direct spectroscopic evidence that intermolecular ED:ED interactions within the ∼50 kDa full-length NS1A protein dimer do not occur in solution at subaggregate protein concentrations (≤50 μM). These data support a mechanism of cooperative dsRNA binding in which the surface epitope including Trp187 becomes buried only upon formation of the functionally important protein:protein interface between dimeric NS1A molecules (Aramini et al., 2011Aramini J.M. Ma L.C. Zhou L. Schauder C.M. Hamilton K. Amer B.R. Mack T.R. Lee H.W. Ciccosanti C.T. Zhao L. et al.Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.J. Biol. Chem. 2011; 286: 26050-26060Crossref PubMed Scopus (52) Google Scholar). Using the protocols described below, we consistently obtained high levels (≥90%) of biosynthetic 5-F-Trp incorporation into Ud NS1A constructs (Figure S1 available online). To examine the structural effects of 5-F-Trp incorporation, we obtained 1H-15N TROSY-HQSC spectra of 5-F-Trp-labeled and unfluorinated Ud NS1A ED (Figure S2A). Aside from the expected absence of the backbone and indole 15N-1H resonances in fluorinated ED, consistent with the high percentage of 5-F-Trp incorporation, the similar spectral patterns are indicative of relatively minor structural differences between the proteins. In fact, when mapped onto the crystal structure of Ud NS1A ED (Xia et al., 2009Xia S. Monzingo A.F. Robertus J.D. Structure of NS1A effector domain from the influenza A/Udorn/72 virus.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 11-17Crossref PubMed Scopus (39) Google Scholar), backbone amide chemical shift perturbations (CSPs) are localized to residues in proximity to the fluorinated tryptophans (Figure S2B). Given the established critical role of a tryptophan (Trp187) within the helix:helix dimer interface of NS1A ED (Aramini et al., 2011Aramini J.M. Ma L.C. Zhou L. Schauder C.M. Hamilton K. Amer B.R. Mack T.R. Lee H.W. Ciccosanti C.T. Zhao L. et al.Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.J. Biol. Chem. 2011; 286: 26050-26060Crossref PubMed Scopus (52) Google Scholar, Kerry et al., 2011Kerry P.S. Ayllon J. Taylor M.A. Hass C. Lewis A. García-Sastre A. Randall R.E. Hale B.G. Russell R.J. A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.PLoS ONE. 2011; 6: e17946Crossref PubMed Scopus (41) Google Scholar), we next examined the effect of 5-F-Trp labeling on the dimerization dissociation constant (KD) of Ud NS1A ED by sedimentation equilibrium (SEQ) (Figure S3). The wild-type (WT) and 5-F-Trp-labeled NS1A ED exhibit comparable KD values in low salt pH 8 buffer (KD = 12 ± 6 and 7 ± 5 μM for WT and 5-F-Trp NS1A ED, respectively). Taken together with the CSP data above, we conclude that incorporation of 5-F-Trp in Ud NS1A results in only minor biophysical perturbations, making this fluorinated tryptophan analog an appropriate spectroscopic reporter for this protein, particularly its biologically important dimer interface. The 19F NMR spectrum of 5-F-Trp-labeled Ud NS1A ED exhibits three distinct resonances in a chemical shift window characteristic of 5-F-Trp (δ of ∼−120 to −127 ppm), and conservative W→F single-residue mutants facilitate their assignment (Figure 1B). We observe that the 19F resonance corresponding to Trp187 located in the helix:helix dimer interface of NS1A ED (Aramini et al., 2011Aramini J.M. Ma L.C. Zhou L. Schauder C.M. Hamilton K. Amer B.R. Mack T.R. Lee H.W. Ciccosanti C.T. Zhao L. et al.Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.J. Biol. Chem. 2011; 286: 26050-26060Crossref PubMed Scopus (52) Google Scholar, Hale et al., 2008aHale B.G. Barclay W.S. Randall R.E. Russell R.J. Structure of an avian influenza A virus NS1 protein effector domain.Virology. 2008; 378: 1-5Crossref PubMed Scopus (71) Google Scholar, Xia et al., 2009Xia S. Monzingo A.F. Robertus J.D. Structure of NS1A effector domain from the influenza A/Udorn/72 virus.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 11-17Crossref PubMed Scopus (39) Google Scholar) is quite broad, a sign of conformational exchange at this site (see below). A broad 1H linewidth is also observed for the Hε1 resonance of Trp187 in native unfluorinated NS1A ED (Figure 1C). In addition, the resonance assigned to Trp203 is split into two resonances under high salt conditions, indicative of two slightly different local environments for the Trp203 side chain (Xia et al., 2009Xia S. Monzingo A.F. Robertus J.D. Structure of NS1A effector domain from the influenza A/Udorn/72 virus.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 11-17Crossref PubMed Scopus (39) Google Scholar). Interestingly, alanine substitution of individual tryptophans results in small chemical shift changes to the remaining tryptophan resonances, particularly Trp187, indicative of allosteric structural perturbations (Figure S4). The 19F resonance of Trp187 is highly sensitive to the dimer↔monomer solution equilibrium of Ud NS1A ED. Dilution of 5-F-Trp-labeled Ud NS1A ED results in a progressive decrease in the broad resonance for Trp187 and concomitant increase in a second sharper upfield resonance (Figure 2A). This upfield-shifted resonance for Trp187 is also observed for the K110A mutant of Ud NS1A ED (Figure 2B), a mutant of this protein domain that is monomeric (Aramini et al., 2011Aramini J.M. Ma L.C. Zhou L. Schauder C.M. Hamilton K. Amer B.R. Mack T.R. Lee H.W. Ciccosanti C.T. Zhao L. et al.Dimer interface of the effector domain of non-structural protein 1 from influenza A virus: an interface with multiple functions.J. Biol. Chem. 2011; 286: 26050-26060Crossref PubMed Scopus (52) Google Scholar). Hence, this sharp upfield-shifted resonance corresponds to Trp187 in the exposed monomeric state. Note that the line width of the 19F resonance for Trp187 in the dimer is also significantly broader than those for Trp102 and Trp203 over the entire dilution series (see below). Fitting the concentration dependence of the dimer and monomer states of NS1A ED in slow exchange on the 19F time scale yields a dimer KD value of 35 ± 6 μM (Figure 2C). This value is comparable to the SEQ results, albeit slightly weaker. This disparity may be attributed to the vastly different protein concentrations required by the two techniques and the poor solubility of NS1A ED at concentrations above ∼600 μM, which may complicate the estimate of KD. On the basis of one-dimensional (1D) 19F saturation transfer experiments (Figure S5), we estimate exchange rates (kex) for ED monomer→dimer and dimer→monomer interconversion under these conditions of kex = 1.2 ± 0.3 and 0.8 ± 0.2 s−1, respectively; that is, with a time constant of ∼1 s. These rates are, as expected, well below the frequency difference between the Trp187 dimer and monomer 19F resonances (Δν of ∼650 Hz at magnetic field strength [B0] = 11.7 T). Interestingly, the KD value for the ED homodimer is highly salt dependent, in that increasing salt significantly weakens the dimerization, resulting in intermediate/fast exchange behavior on the 19F time scale and weaker KD values by SEQ (Figure S6). This is consistent with the presence of intermolecular salt bridges at the dimer interface in the structure of Ud NS1A ED (e.g., Lys110:Asp189′) (Xia et al., 2009Xia S. Monzingo A.F. Robertus J.D. Structure of NS1A effector domain from the influenza A/Udorn/72 virus.Acta Crystallogr. D Biol. Crystallogr. 2009; 65: 11-17Crossref PubMed Scopus (39) Google Scholar). The slow exchange between the broad dimer and narrow monomer 19F resonances of Trp187 observed under low salt conditions is strongly indicative of conformational exchange within the ED dimer interface, rather than exchange between bound dimer and free monomer states. The line shapes for such a slow/intermediate exchange between monomer and dimer would feature an identical line width for both the bound- and free-state Trp187 19F resonances when the populations are equal, and different line widths throughout the dilution experiment correlated with the fraction of monomer and dimer (Palmer et al., 2001Palmer 3rd, A.G. Kroenke C.D. Loria J.P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules.Methods Enzymol. 2001; 339: 204-238Crossref PubMed Scopus (837) Google Scholar). In contrast, the presence of both broad bound (due to exchange broadening within the dimer) and narrow free (characteristic of the environment in the monomer state) Trp187 19F resonances throughout the entire dilution series (Figure 2A) is attributed to conformational heterogeneity at the helix:helix interface of the dimer complex. To test this hypothesis and gain further insight into the conformational dynamics within the NS1A ED dimer interface, 1D 19F longitudinal (T1), transverse (T2), and Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments were performed on 5-F-Trp-labeled NS1A ED, at high (dimer only) and intermediate (dimer plus monomer) concentrations, and on its monomeric K110A mutant (Table 1; Figure 3A). Although T1 values for the various 5-F-Trp residues are relatively insensitive to ED oligomerization state, the 19F T2 of Trp187 is significantly shorter for the dimer resonance compared with the monomer resonance (Table 1) and is shorter than the 19F T2 of residues Trp102 and Trp203 in the dimer state. This is consistent with conformational exchange within the interface of the ED dimer, rather than exchange broadening due to interconversion between bound and free forms (see below), or a simple effect due to the slower overall rotational correlation time of the dimer. For the monomeric mutant, the 19F T2 of Trp187 is even longer, indicative of full solvent and rotameric accessibility. In addition, 19F CPMG relaxation dispersion results for WT and [K110A] NS1A ED dramatically demonstrate that the value of the transverse relaxation rate (R2) for Trp187 in the dimer interface is extremely sensitive to the delay time (τcp), in contrast to the transverse relaxation behavior of Trp187 in monomeric ED as well as the other two tryptophan residues, Trp102 and Trp203, in this domain (Figure 3A).Table 119F T1 and T2 Relaxation Data for 5-F-Trp Residues in Ud NS1A ED, [K110A] NS1A ED, and [C116S,G183C] NS1A EDSamplePeakT1 (s)T2 (ms)aValues for W187 are in bold.NS1A ED (600 μM)W1020.58 (0.06)4.51 (0.41)W187 dimer0.63 (0.06)3.34 (0.16)W2030.61 (0.10)5.44 (0.59)NS1A ED (250 μM)W1020.49 (0.07)5.67 (1.09)W187 dimer0.75 (0.26)4.0 (1.2)W187 monomer0.71 (0.11)11.76 (3.02)W2030.52 (0.06)5.86 (1.29)[K110A] NS1A ED (200 μM)W1020.35 (0.04)9.83 (2.23)W187 monomer0.65 (0.14)27.06 (5.89)W2030.42 (0.05)10.71 (2.14)[C116S,G183C] NS1A ED (200 μM)W1020.62 (0.05)3.16 (0.32)W1870.65 (0.06)4.42 (0.56)W2030.68 (0.07)3.84 (0.55)The 19F relaxation data shown were obtained at 470.18 MHz in low salt pH 8 buffer and at 20°C. All T2 data shown correspond to a νCPMG of 2,500 Hz. SEs representing 95% confidence bounds for the exponential fits are given in parentheses.a Values for W187 are in bold. Open table in a new tab The 19F relaxation data shown were obtained at 470.18 MHz in low salt pH 8 buffer and at 20°C. All T2 data shown correspond to a νCPMG of 2,500 Hz. SEs representing 95% confidence bounds for the exponential fits are given in parentheses. To further test this model, we generated and studied a disulfide-crosslinked ED dimer that locks the NS1A ED in a dimeric form. By mutating Gly183 to a cysteine, we generated a disulfide-bonded dimer that was confirmed by polyacrylamide gel electrophoresis (Figure S7). The locked dimer exhibits a significant reduction in Trp187 19F resonance line width, with concomitant downfield shift compared with WT NS1A ED (Figure 3B). Locking down the interfacial dynamics of the NS1A dimer also abolishes the 19F relaxation dispersion effect observed for Trp187 in the WT protein (Figure 3A). Taken together, these data conclusively demonstrate the presence of slow motion (microsecond-to-millisecond) conformational" @default.
• W1997358511 created "2016-06-24" @default.
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• W1997358511 date "2014-04-01" @default.
• W1997358511 modified "2023-10-15" @default.
• W1997358511 title "19F NMR Reveals Multiple Conformations at the Dimer Interface of the Nonstructural Protein 1 Effector Domain from Influenza A Virus" @default.
• W1997358511 cites W1552774315 @default.
• W1997358511 cites W1582836242 @default.
• W1997358511 cites W159622809 @default.
• W1997358511 cites W1969987257 @default.
• W1997358511 cites W1975123527 @default.
• W1997358511 cites W1975948012 @default.
• W1997358511 cites W1978078396 @default.
• W1997358511 cites W1983443072 @default.
• W1997358511 cites W1985477676 @default.
• W1997358511 cites W1989458326 @default.
• W1997358511 cites W1993371465 @default.
• W1997358511 cites W1995510727 @default.
• W1997358511 cites W1995550922 @default.
• W1997358511 cites W1997584810 @default.
• W1997358511 cites W2003412353 @default.
• W1997358511 cites W2003639258 @default.
• W1997358511 cites W2006934214 @default.
• W1997358511 cites W2008880130 @default.
• W1997358511 cites W2009239521 @default.
• W1997358511 cites W2011516085 @default.
• W1997358511 cites W2015094506 @default.
• W1997358511 cites W2015117663 @default.
• W1997358511 cites W2015337210 @default.
• W1997358511 cites W2017178787 @default.
• W1997358511 cites W2022925934 @default.
• W1997358511 cites W2032660941 @default.
• W1997358511 cites W2033843609 @default.
• W1997358511 cites W2039236902 @default.
• W1997358511 cites W2039241525 @default.
• W1997358511 cites W2044172420 @default.
• W1997358511 cites W2045590420 @default.
• W1997358511 cites W2047696147 @default.
• W1997358511 cites W2048822589 @default.
• W1997358511 cites W2055377264 @default.
• W1997358511 cites W2056080643 @default.
• W1997358511 cites W2061151008 @default.
• W1997358511 cites W2061486876 @default.
• W1997358511 cites W2062961793 @default.
• W1997358511 cites W2063227656 @default.
• W1997358511 cites W2066962978 @default.
• W1997358511 cites W2067847540 @default.
• W1997358511 cites W2083741754 @default.
• W1997358511 cites W2085419867 @default.
• W1997358511 cites W2088484786 @default.
• W1997358511 cites W2089911961 @default.
• W1997358511 cites W2093400164 @default.
• W1997358511 cites W2093482724 @default.
• W1997358511 cites W2097985410 @default.
• W1997358511 cites W2099458577 @default.
• W1997358511 cites W2109863561 @default.
• W1997358511 cites W2110214166 @default.
• W1997358511 cites W2112170460 @default.
• W1997358511 cites W2112901090 @default.
• W1997358511 cites W2115612309 @default.
• W1997358511 cites W2117815641 @default.
• W1997358511 cites W2136125021 @default.
• W1997358511 cites W2140343664 @default.
• W1997358511 cites W2147601655 @default.
• W1997358511 cites W2169821755 @default.
• W1997358511 cites W2257974587 @default.
• W1997358511 cites W30334872 @default.
• W1997358511 doi "https://doi.org/10.1016/j.str.2014.01.010" @default.
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