PDBsum entry 1rkj

Transcription/RNA

PDB id

1rkj

Contents

			Protein chain

					175 a.a. *

			DNA/RNA

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Solution structure of the complex formed by the two n-Terminal RNA-Binding domains of nucleolin and a pre-Rrna target.

Authors

C.Johansson, L.D.Finger, L.Trantirek, T.D.Mueller, S.Kim, I.A.Laird-Offringa, J.Feigon.

Ref.

J Mol Biol, 2004, 337, 799-816. [DOI no: 10.1016/j.jmb.2004.01.056]

PubMed id

15033352

Abstract

Nucleolin is a 70 kDa multidomain protein involved in several steps of eukaryotic ribosome biogenesis. In vitro selection in combination with mutagenesis and structural analysis identified binding sites in pre-rRNA with the consensus (U/G)CCCG(A/G) in the context of a hairpin structure, the nucleolin recognition element (NRE). The central region of the protein contains four tandem RNA-binding domains (RBDs), of which the first two are responsible for the RNA-binding specificity and affinity for NREs. Here, we present the solution structure of the 28 kDa complex formed by the two N-terminal RNA-binding domains of nucleolin (RBD12) and a natural pre-rRNA target, b2NRE. The structure demonstrates that the sequence-specific recognition of the pre-rRNA NRE is achieved by intermolecular hydrogen bonds and stacking interactions involving mainly the beta-sheet surfaces of the two RBDs and the linker residues. A comparison with our previously determined NMR structure of RBD12 in complex with an in vitro selected RNA target, sNRE, shows that although the sequence-specific recognition of the loop consensus nucleotides is the same in the two complexes, they differ in several aspects. While the protein makes numerous specific contacts to the non-consensus nucleotides in the loop E motif (S-turn) in the upper part of the sNRE stem, nucleolin RBD12 contacts only consensus nucleotides in b2NRE. The absence of these upper stem contacts from the RBD12/b2NRE complex results in a much less stable complex, as demonstrated by kinetic analyses. The role of the loop E motif in high-affinity binding is supported by gel-shift analyses with a series of sNRE mutants. The less stable interaction of RBD12 with the natural RNA target is consistent with the proposed role of nucleolin as a chaperone that interacts transiently with pre-rRNA to prevent misfolding.



	Figure 4. Figure 4. Superposition of the ensemble of the 14 lowest-energy structures of the nucleolin RBD12/b2NRE complex. a, The nucleolin RBD12/b2NRE complex showing the backbone superposition of RBD12 (11-170) and the lowest-energy structure of b2NRE. b, Heavy-atom superposition (nucleotides 2-15, 17-21) of b2NRE and the lowest-energy RBD12 structure shown in ribbon representation. c, Superposition of backbone atoms of RBD12 (T11-Y170) and all heavy atoms of the b2NRE loop (nucleotides 8-15) (Table 1). RBD1 is shown in blue, the linker in red, RBD2 in green, and b2NRE in orange.		Figure 5. Figure 5. Global view of the nucleolin RBD12/b2NRE complex structure. The lowest-energy structure is shown. a, Stereoview of the complex, showing b2NRE and nucleolin RBD12 in stick and ribbon representations, respectively. The b2NRE (orange) loop is sandwiched between the two RBDs, with RBD1 (blue) interacting with nucleotides at the 3' side of the RNA (C12, G13 and A14) and RBD2 (green) contacting nucleotides of the 5' side (U9 and C10). The linker (red) spans the major groove of the loop. Some of the amino acid side-chains (green) at the interface (Y58, F56, K94, R127, Y140) are shown in stick representation. b, Surface representation of the whole complex. The side-chains of linker residues K95 and R97 (red), pack closely against the RNA. The view and color scheme is the same as in a. c, Surface representation of the whole complex with the molecule turned vert, similar 180° relative to b, showing the two holes in the RNA loop. The linker residue K94 (red) inserts into the upper hole, whereas R97 packs against the lower hole, possibly involved in water-mediated interactions with the RNA.

PROCHECK

	Headers