PDBsum entry 2ix0

Hydrolase

PDB id: 2ix0

Contents

Protein chain

637 a.a. *

Ligands

C5P

Metals

_CA

_MG

Waters ×145

* Residue conservation analysis

PDB id:

2ix0

Name:

Hydrolase

Title:

Rnase ii

Structure:

Exoribonuclease 2. Chain: a. Fragment: residues 6-644. Synonym: rnase ii, exoribonuclease ii, ribonuclease ii. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Strain: c600. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.44Å R-factor: 0.190 R-free: 0.236

Authors:

C.Frazao,C.E.Mcvey,M.Amblar,A.Barbas,C.Vonrhein,C.M.Arraiano, M.A.Carrondo

Key ref:

C.Frazão et al. (2006). Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex. Nature, 443, 110-114. PubMed id: 16957732 DOI: 10.1038/nature05080

Date:

05-Jul-06 Release date: 05-Oct-06

Protein chain

P30850  (RNB_ECOLI) -  Exoribonuclease 2 from Escherichia coli (strain K12)

Seq: 644 a.a.

Struc: 637 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.13.1 - exoribonuclease Ii.
• Reaction: Exonucleolytic cleavage in the 3'- to 5'-direction to yield nucleoside 5'-phosphates.

DOI no: 10.1038/nature05080

Nature 443:110-114 (2006)

PubMed id: 16957732

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex.

C.Frazão, C.E.McVey, M.Amblar, A.Barbas, C.Vonrhein, C.M.Arraiano, M.A.Carrondo.

ABSTRACT

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.

Selected figure(s)

Figure 2.
Figure 2: RNase II–ssRNA interactions.
Figure 2 : RNase II|[ndash]|ssRNA interactions. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com-
a, Atomic interactions scheme between RNA (enclosed atoms in black or in red for O2'-protein interactions) and protein residues (coloured by domains as in Fig. 1a). Hb indicates hydrogen bonds up to 3.2 Å, and vdW indicates van der Waals interactions up to 3.6 Å. b, c, RNA docking catalytic region (b) with residues 364–381 deleted for clarity, and anchor region (c) involving canonical oligonucleotide-binding motifs^16 L[23] of CSD2, and chain 3 and loop L[45] of S1. RNA (coloured as in Fig. 1) and protein hydrogen-bonding side-chain residues shown in sticks (carbon in yellow, nitrogen in cyan, oxygen in red), enzyme domains as cartoons (coloured as in Fig. 1). Hydrogen bonds shown as dashed green lines, Mg shown as green sphere with coordination in dashed yellow lines. Nucleotides (Nt) 9–13 are clamped between the aromatic side-chains of Tyr 253 and Phe 358 (with labels highlighted in green), nucleotides 3 and 4 are between Phe 588 and Pro 104, and nucleotide 5 is nestled between His 103, Pro 104, Asp 102 and Arg 167.

Figure 3.
Figure 3: RNA degradation by RNase II.
Figure 3 : RNA degradation by RNase II. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com-
a, Stereo view of RNase II D209N mutant active site; bonds are shown as sticks (oxygen in red, nitrogen in blue, phosphorus in orange), waters as red spheres, Mg as a green sphere and its coordination as dashed orange lines, and hydrogen bonds as dashed green lines, superposed with sigma-A corrected Fourier synthesis electron density map (brown mesh) contoured at 1 . Additionally, N1 and N6 of nucleotide 13 are in the vicinity of the carboxylate oxygens of Glu 542, at 3.2 Å (hydrogen bond as dashed green lines) and at 4.3 Å (distance as dotted orange line), respectively. b, Magnified view of the Mg ion and its coordinating environment (distances in Å) superposed with the positive 3 sigma-A corrected Fourier difference map (green mesh) calculated with the Mg and coordinating waters omitted from the model. c, Stereo view of the superposition of the RNase II D209N mutant (yellow) and RNase H (grey) with magnesium coordinating spheres (opposite view to Fig. 3a). RNase H displays two magnesium ions ligated by Glu 109, D132N, Glu 188 and Asp 192 that correspond in RNase II to Asp 201, Asp 210, D209N and Asp 207, respectively. d, Proposed catalytic mechanism for RNase II, showing the postulated second Mg (Mg II) and the attacking hydroxyl group (grey). e, Model for RNA degradation by RNase II. ssRNA (red) is threaded into the catalytic cavity and clamped between Tyr 253 and Phe 358. The additional stabilization of RNA inside the cavity drives the RNA translocation after each cleavage, up to a final four-nucleotide fragment.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2006, 443, 110-114) copyright 2006.

Figures were selected by an automated process.