Altering the RNA-binding mode of the U1A RBD1 protein.
The N-terminal RNA-binding domain (RBD1) of the human U1A protein is evolutionarily designed to bind its RNA targets with great affinity and specificity. The physical mechanisms that modulate the coupling (local cooperativity) among amino acid residues on the extensive binding surface of RBD1 are investigated here, using mutants that replace a highly conserved glycine residue. This glycine residue, at the strand/loop junction of beta3/loop3, is found in U1A RBD1, and in most RBD domains, suggesting it has a specific role in modulation of RNA binding. Here, two RBD1 proteins are constructed in which that residue (Gly53) is replaced by either alanine or valine. These new proteins are shown by NMR methods and molecular dynamics simulations to be very similar to the wild-type RBD1, both in structure and in their backbone dynamics. However, RNA-binding assays show that affinity for the U1 snRNA stem-loop II RNA target is reduced by nearly 200-fold for the RBD1-G53A protein, and by 1.6 x 10(4)-fold for RBD1-G53V. The mode of RNA binding by RBD1-G53A is similar to that of RBD1-WT, displaying its characteristic non-additive free energies of base recognition and its salt-dependence. The binding mode of RBD1-G53V is altered, having lost its salt-dependence and displaying site-independence of base recognition. The molecular basis for this alteration in RNA-binding properties is proposed to result from the inability of the RNA to induce a change in the structure of the free protein to produce a high-affinity complex.
Showalter
Scott A
SA
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110, USA.
Hall
Kathleen B
KB
D001665:Binding Sites; D003198:Computer Simulation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein