PDBsum entry 1zzn

Structural protein/RNA

PDB id: 1zzn

Contents

Protein chain

95 a.a.

DNA/RNA

Metals

_MG ×5

__K

Waters ×54

References listed in PDB file

Key reference

Title

Structural evidence for a two-Metal-Ion mechanism of group i intron splicing.

Authors

M.R.Stahley, S.A.Strobel.

Ref.

Science, 2005, 309, 1587-1590. [DOI no: 10.1126/science.1114994]

PubMed id

16141079

Abstract

We report the 3.4 angstrom crystal structure of a catalytically active group I intron splicing intermediate containing the complete intron, both exons, the scissile phosphate, and all of the functional groups implicated in catalytic metal ion coordination, including the 2'-OH of the terminal guanosine. This structure suggests that, like protein phosphoryltransferases, an RNA phosphoryltransferase can use a two-metal-ion mechanism. Two Mg2+ ions are positioned 3.9 angstroms apart and are directly coordinated by all six of the biochemically predicted ligands. The evolutionary convergence of RNA and protein active sites on the same inorganic architecture highlights the intrinsic chemical capacity of the two-metal-ion catalytic mechanism for phosphoryl transfer.

Figure 1.
Fig. 1. The group I intron splicing reaction. (A) Secondary structure of the pre-2S crystallization construct. The residues discussed in the text are shown superimposed on the secondary structure. RNA connectivity is depicted with a dashed line with small arrows to show the 5' to 3' orientation. Exons are shown in red. The coloring of other residues corresponds to the structural element in which they are located: P4 to P6 (green), P3 to P9 (blue), and J8/7 (purple). (B) Summary of the biochemically defined ligands for active-site metal coordination. The six oxygens shown in orange have been implicated in metal-ion coordination on the basis of metal specificity switch experiments (10-15), including four in the substrates and two in the intron. Ligands biochemically shown to coordinate the same metal are depicted with double-ended arrows. The exon splicing reaction involving attack of the U-1 O3' on the scissile phosphate with loss of the G O3' is shown with curved arrows. (C) Proposed three-metal-ion mechanism based on differential Mn2+ affinity to sulfur/amino-substituted substrates (21, 22). The four substrate ligands in (B) are coordinated to three metal ions, M[A], M[B], and M[C].

Figure 3.
Fig. 3. A two-metal mechanism for group I intron splicing. (A) F[O]-F[C] omit map (active-site metals were not included in the model) used to assign M[1] and M[2] positions, superimposed on the refined structure. The native density (5 ) for each metal is depicted in blue. The other residues are as labeled. In (A), (B), and (D), the scissile bond, nucleophile, and leaving group are shown in yellow. (B) Active-site coordination to M[1] and M[2]. In this and (D), the active-site Mg2+ ions are shown as large orange spheres, the predicted inner and outer sphere ligands are shown as small orange spheres, and the metal-to-metal distance is labeled. Orange lines indicate inner sphere coordinations. Labels for the individual nucleotides are as in Fig. 2A. All the coordinations depicted in Fig. 1B are satisfied in this structure. (C) Model of the group I intron transition state stabilized by a two-metal mechanism. (D) Two-metal active-site coordination within the T7 DNA polymerase (1). The incoming deoxy-nucleotide triphosphate (dNTP), the primer oligonucleotide, and active-site aspartates are labeled. The nucleophile was not present in the crystal structure but is modeled here for comparison.

The above figures are reprinted by permission from the AAAs: Science (2005, 309, 1587-1590) copyright 2005.

Secondary reference #1

Title

Crystal structure of a self-Splicing group i intron with both exons.

Authors

P.L.Adams, M.R.Stahley, A.B.Kosek, J.Wang, S.A.Strobel.

Ref.

Nature, 2004, 430, 45-50. [DOI no: 10.1038/nature02642]

PubMed id

15175762

Figure 3.
Figure 3: Recognition of the P1 substrate helix by the active site. a, Experimental electron density contoured at 1 of the G U wobble pair docked into J5/4 -J4/5. The density for the active-site metal ions is also visible. b, Stereo view of minor-groove-mediated P1 -P2 helix docking. Enlarged atoms correspond to the functional groups identified by interference analysis as being important for ribozyme function21,22. c, Wobble -wobble receptor motif that specifies the 5'-splice site. The U-1 2'-OH (blue) is modelled into the structure with the predicted network of transition state stabilizing hydrogen bonds in red. The scissile phosphate is also shown.

Figure 4.
Figure 4: Active-site metal ions and their ligands. a, Identity of the metals based upon heavy-metal soaks. The two anomalous X-ray scattering electron-density maps for Yb^3+ (orange) and Tl+ (blue) are overlaid on the active site and contoured at 30 and 33 , respectively. The locations of the metal ions in the native structure are shown as solid spheres. b, Coordination of the active-site metal ions. The Mg2+ ion (M[1]) and its ligands are shown in orange. The K+ ion (M[2]) and its ligands are depicted in blue. The nucleophile, the scissile phosphorous, the leaving group and the labile bond are coloured yellow. All other residues are coloured grey, except for the phosphorus atoms, which are black. The residue labels are coloured according to the scheme in Fig. 1a. c, Proposed reaction mechanism for the second step of splicing by the bacterial intron. M[1] and M[2] are probably both Mg2+ ions.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #2

Title

Crystal structure of a group I intron splicing intermediate.

Authors

P.L.Adams, M.R.Stahley, M.L.Gill, A.B.Kosek, J.Wang, S.A.Strobel.

Ref.

Rna, 2004, 10, 1867-1887.

PubMed id

15547134

Secondary reference #3

Title

In vitro splicing of the ribosomal RNA precursor of tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence.

Authors

T.R.Cech, A.J.Zaug, P.J.Grabowski.

Ref.

Cell, 1981, 27, 487-496. [DOI no: 10.1016/0092-8674(81)90390-1]

PubMed id

6101203

Secondary reference #4

Title

Self-Splicing introns in tRNA genes of widely divergent bacteria.

Authors

B.Reinhold-Hurek, D.A.Shub.

Ref.

Nature, 1992, 357, 173-176.

PubMed id

1579169