Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM)
Myers, Jeffrey C.
Doctor of Philosophy
Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1--195 of hnRNP Al is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)n. We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in Kd from 70 nM to 160 and 280 nM, respectively. The loss of orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the Kd to 250 nM, a result suggesting that it is not simply the loss of affinity that gives rise to base mobility but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main chain to base hydrogen bonding make up an important aspect of ligand recognition; (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated; and (3) a conserved lysine position proximal to RNP-2 (K106-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.