PDBsum entry 1u1o

Transport protein/DNA

PDB id

1u1o

Contents

			Protein chain

					183 a.a. *

			DNA/RNA

			Waters ×87

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Human up1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (rrm).

Authors

J.C.Myers, Y.Shamoo.

Ref.

J Mol Biol, 2004, 342, 743-756. [DOI no: 10.1016/j.jmb.2004.07.029]

PubMed id

15342234

Abstract

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 A1 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 K(d) from 88nM to 209nM and 316nM, respectively. The loss of these 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 K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the 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 makes 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 (K(106)-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.



	Figure 2. Figure 2. Guide to modified bases used in these studies. Adenine (ade), guanine (gua), 7-deaza-guanine (7deazaG), 7-deaza-adenine (7deazaA), nebularine (neb), inosine (ino), 2-aminopurine (2AP). Arrows indicate positions that are good hydrogen bond donors or acceptors. The nomenclature of each modified oligonucleotide starts with the base to be substituted, followed by its position from the 5' end of the sequence 5'-d(TTAGGGTTAGGG)-3', and then by the base substitution.		Figure 3. Figure 3. Structures of UP1-oligonucleotide complexes for substitution of adenine 9. 2F[o] -F[c] composite omit electron density maps contoured at 1.25 s. Chevrons indicate the hydrogen bonding network. (a) Wild-type structure with Ade9 shown making hydrogen bonding contacts to the Arg178 guanidinium group through its N7 (2.7 Å) and from the main-chain carbonyl of Lys179 (3.0 Å). Ade9 is stacked directly over the conserved Phe108 of the RNP2 consensus sequence. (b) The UP1-A(9)Neb structure showed no substantive changes in either protein or DNA structures. The absence of the N6 amino group allowed the base to move slightly closer to Arg178 (2.5 Å). (c) UP1-A(9)7deazaA structure shows a large conformational rearrangement of the Arg178 side-chain. The Arg178 guanidinium group shifted 9.1 Å away from its position in the wild-type structure where it makes contacts to the O2 of Thy8 and N7 of Ade9 to make a new set of contacts to Glu93. All electron density Figures were made using PYMOL (DeLano Scientific, CA).

PROCHECK

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