X-ray structure of a cysteine-less mutant galectin-1

Abstract

Influenza A viruses are important human pathogens resulting in periodic pandemic threaten, while nonstructural protein 1 of influenza A virus (NS1A) shields the virus against host defense as an immunosuppressor. Mutational inactivation of the dsRNA binding activity of NS1A highly attenuates virus replication. This study investigated the structural principles of dsRNA recognition by NS1A protein. The complex crystal of NS1A RNA binding domain (NS1A RBD) with dsRNA diffracted X-rays to 1.7 A and was in space group C2 with unit cell dimensions of a=60.707 A, b=57.218 A, and c=83.709 A. The crystal structure revealed that NS1A RBD forms a dimeric six-helical fold and used a dimeric anti-parallel helices α 2/α2’ to recognize the major groove of the dsRNA as a sequenceindependent mode. The RNA helix adopted 40°bending towards the NS1A RBD at both ends of the helix to facilitate the RNA-protein interactions. The highly conserved residues within a positive patch, including R35, R37, R38, and K41 played the primary roles for dsRNA binding by hydrogen bonds and electrostatic interactions. Outside this positive patch, conserved residues, such as T5, D29, D34, S42 and T49, also contributed for dsRNA binding through hydrogen bonds directly or via water bridges. The significant conformational change of invariable residue R38 before and after NS1A RBD binding to dsRNA indicated that R38 played a key role for dsRNA binding by penetrating its side chain into dsRNA helix. The protein-RNA interactions observed from the crystal structure were further supported by the isothermal titration calorimetry assay of NS1A RBD and its mutants binding to dsRNA. Moreover, Agrobacterium co-infiltration assay suggested that arginine 38 may also play important roles for dsRNA binding in vivo.