|Biochemistry (2004) 43(7):1950-62|
|Northeast Structural Genomics Consortium|
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The influenza virus nonstructural protein 1 encoded by influenza A virus (NS1A protein) is a multifunctional protein involved in both protein-protein and protein-RNA interactions. ...
NS1A binds nonspecifically to double-stranded RNA (dsRNA) and to specific protein targets, and regulates several post-transcriptional processes. The N-terminal structural domain corresponding to the first 73 amino acids of the NS1 protein from influenza A/Udorn/72 virus [NS1A(1-73)] possesses all of the dsRNA binding activities of the full-length protein. Both NMR and X-ray crystallography of this domain have demonstrated that it is a symmetric homodimer which forms a six-helix chain fold, a unique structure that differs from that of the predominant class of dsRNA-binding domains, termed dsRBDs, that are found in a large number of eukaryotic and prokaryotic proteins. Here we describe biophysical experiments on complexes containing NS1A(1-73) and a short 16 bp synthetic dsRNA duplex. From sedimentation equilibrium measurements, we determined that the dimeric NS1A(1-73) binds to the dsRNA duplex with a 1:1 stoichiometry, yielding a complex with an apparent dissociation constant (K(d)) of approximately 1 microM. Circular dichroism and nuclear magnetic resonance (NMR) data demonstrate that the conformations of both NS1A(1-73) and dsRNA in the complex are similar to their free forms, indicating little or no structural change in the protein or RNA upon complex formation. NMR chemical shift perturbation experiments show that the dsRNA-binding epitope of NS1A(1-73) is associated with helices 2 and 2'. Analytical gel filtration and gel shift studies of the interaction between NS1A(1-73) and different double-stranded nucleic acids indicate that NS1A(1-73) recognizes canonical A-form dsRNA, but does not bind to dsDNA or dsRNA-DNA hybrids, which feature B-type or A/B-type intermediate conformations, respectively. On the basis of these results, we propose a three-dimensional model of the complex in which NS1A(1-73) sits astride the minor groove of A-form RNA with a few amino acids in the helix 2-helix 2' face forming an electrostatically stabilized interaction with the phosphodiester backbone. This mode of dsRNA binding differs from that observed for any other dsRNA-binding protein.
|chemistry metabolism isolation & purification |
|Protein Structure, Tertiary RNA-Binding Proteins Ultracentrifugation Influenza A virus RNA, Viral DNA, Viral Nitrogen Isotopes Nuclear Magnetic Resonance, Biomolecular RNA, Double-Stranded Chromatography, Gel Thermodynamics Protons Circular Dichroism Nucleic Acid Heteroduplexes Epitope Mapping Viral Nonstructural Proteins Carbon Isotopes Protein Binding Peptide Fragments |
|86 (Last update: 07/15/2017 11:12:29am)|
|Biochemistry. 2004 Feb 24;43(7):1950-62.|