The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

Abstract

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a 'lure and lock' model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the 'lure' step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on 'top' of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein-RNA complexes.

Figure 1

Representation of the protein, protein–RNA complex and RNA target. (A) The amino acid sequence of the N-terminal RRM of U1A showing structural features in blue, conserved RNP domains in green, and positively charged amino acids mutated in this study in red. (B) Space filling model of the U1A/U1hpII complex as taken from the X-ray crystal structure (22) (pdb ID 1URN). Highlighted in blue are the positively charged residues mutated in this study; the RNA target is indicated in red. For orientation purposes, Lys20 is pointing into the plane away from the viewer, while Lys22 is pointing out of the plane towards the viewer. (C) U1hpII RNA used in our kinetic analyses. Nucleotides U-5 to G15 are identical to the wild-type RNA target. The numbering scheme is based on the loop residues numbered 1 through 10, with backward and forward numbering used for the 5′ and 3′ ends of the stem, respectively. The loop residues essential for high affinity U1A binding are seen in a red box; the 5′ A carries the biotin linker.

Figure 2

Sensorgrams showing kinetic analyses of wild-type U1A and mutant proteins with U1hpII RNA. Black lines represent triplicate injections which were performed in random order over a U1hpII surface at the indicated concentrations. Association was monitored for 1 min followed by a 5 min dissociation phase. Red lines represent the global fit of datasets using CLAMP (32). Kinetic parameters for the experiments are shown in Table 1.

Figure 3

Effects of electrostatic mutations on k_a, k_d and K_D. To visualize the differences between mutants and wild type protein, we plotted the logarithm of wild type/mutant values for k_a (open bars) and mutant/wild-type values for k_d (gray bars) and K_D (closed bars). Error bars indicate the SEM while stars represent values that are statistically significantly different from wild type.

Figure 4

The effect of salt concentration on the behavior of U1A and its mutants. Upper panel: bars represent the loss in association rate for each protein at 220 (open), 330 (gray) and 500 (closed) mM NaCl, compared with 150 mM NaCl. Note that for the triple Glu mutant there is actually a relative gain in the association rate at 220 and 550 mM NaCl (Table 2). Lower panel: bars indicate the increase in dissociation rate for each protein at 220, 330 and 500 mM NaCl, compared with 150 mM NaCl. The SEM is indicated by error bars.

Figure 5

Interactions of Lys20, Lys22 and Lys23 with RNA phosphates during a 2 ns molecular dynamics simulation of the U1A/U1hpII complex. Each plot shows the distance between the N-zeta group of the indicated amino acid and the phosphorous atom of the indicated base over time. Note that the Lys22 interactions are much more consistent than those of Lys20, and that Lys23 interacts with the C10 phosphate but not with that of C9.