PDBsum entry 1h2v

Nuclear protein

PDB id: 1h2v

Contents

Protein chains

722 a.a. *

93 a.a. *

Waters ×294

* Residue conservation analysis

PDB id:

1h2v

Name:

Nuclear protein

Title:

Structure of the human nuclear cap-binding-complex (cbc)

Structure:

80 kda nuclear cap binding protein. Chain: c. Fragment: mif4g domain, residues 20-790. Synonym: ncbp 80 kda subunit, cbp80. Engineered: yes. Mutation: yes. Other_details: deletion of the first 19 residues in n-terminal, engineered mutation ala 479 ser. 20 kda nuclear cap binding protein.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: trichoplusia ni. Expression_system_taxid: 7111. Expression_system_cell_line: high five. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.00Å R-factor: 0.212 R-free: 0.247

Authors:

C.Mazza,A.Segref,I.W.Mattaj,S.Cusack

Key ref:

C.Mazza et al. (2002). Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex. EMBO J, 21, 5548-5557. PubMed id: 12374755 DOI: 10.1093/emboj/cdf538

Date:

16-Aug-02 Release date: 17-Oct-02

Protein chain

Q09161  (NCBP1_HUMAN) -  Nuclear cap-binding protein subunit 1 from Homo sapiens

Seq: 790 a.a.

Struc: 722 a.a.*

Protein chain

P52298  (NCBP2_HUMAN) -  Nuclear cap-binding protein subunit 2 from Homo sapiens

Seq: 156 a.a.

Struc: 93 a.a.

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

DOI no: 10.1093/emboj/cdf538

EMBO J 21:5548-5557 (2002)

PubMed id: 12374755

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.

C.Mazza, A.Segref, I.W.Mattaj, S.Cusack.

ABSTRACT

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.

Selected figure(s)

Figure 1.
Figure 1 (A) Time course of limited proteolysis. A 42 g aliquot of CBC was incubated at room temperature with 210 ng of trypsin in the absence or presence of 10 mM cap analogue m^7GpppG in a total volume of 60 l. Aliquots of 10 l were taken off every 0, 5, 10, 20, 40 and 60 min, denatured by 3 l of denaturing buffer (125 mM Tris pH 6.8, 260 mM DTT, 30% glycerol, 10% SDS and 0.025% Coomassie Blue) and loaded onto a 13.5% Tricine SDS−polyacrylamide gel. (B) Cap-binding activity of CBP80 653−701. [^35S]Methionine-labelled wild-type or mutant CBP80 was incubated for 30 min at room temperature in the absence (−) or presence of either 1.7 M (high) or 53 nM (low) m^7GpppG-capped (m7) or ApppG-capped (A) unlabelled U1 Sm RNAs. The samples were fractionated by native 6% PAGE followed by fluorography. Free CBP80, CBC and the CBC−RNA complex are indicated. In the lanes indicated by an asterisk, the corresponding CBP80 proteins were loaded without CBP20. (C) Shuttling activity of CBP80 NLS 653−701 in Xenopus oocytes. [^35S]methionine-labelled CBP80 NLS2 or CBP80 NLS2 653−701 were injected together with [^35S]methionine-labelled GST−M10 into Xenopus oocyte nuclei either (1) alone (lanes 1 and 2, and 7 and 8), (2) together with m7GpppG-capped unlabelled U1 Sm RNAs (lanes 3 and 4, and 9 and 10) or (3) together with ApppG-capped U1 Sm RNAs (lanes 5 and 6, and 11 and12). Oocytes were dissected either immediately (lanes 1 and 2, and 7 and 8) or 5 h after injection (lanes 3−6 and 9−12) and the proteins analysed by SDS−PAGE followed by fluorography. GST−M10 is a mutant of HIV Rev with a non-functional nuclear export signal used as a negative control. See Ohno et al. (2000) or Segref et al. (2001) for more details.

Figure 2.
Figure 2 (A) Structure of CBP20 in the CBC NLS complex. Ribbon representation of the cap-free conformation of CBP20 showing residues 30−125 (grey). Residues involved in cap binding are shown in yellow (already in their cap-bound conformation) and blue (undergo a conformational change to interact with the cap) (compare with B). Phe49 (light green) changes conformation to help stabilize the C-terminal domain (compare with B). Salt bridges and hydrogen bonds are indicated by dashed lines. (B) Stabilization of the N- and C-terminal extensions of CBP20 upon cap binding. Ribbon representation of cap-bound CBP20 showing residues 32−125 (already ordered in the cap-free form) in grey and the N- and C-terminal extensions that fold upon cap binding in green and orange, respectively. Yellow residues stabilize the folded conformation of these two extensions through interactions with the cap. This stabilization is reinforced by protein−protein interactions involving the light green residues. D116, R123 and R127 take part to both kinds of contacts. CBP80 residues interacting with residues 5−13 from CBP20 are depicted in pink. Hydrogen bonds and salt bridges are represented as dashed lines, and the hydrophobic contacts as dashed bars. (C and D) Two views of CBC bound to the cap analogue m^7GpppG. The three MIF4G domains of CBP80 are represented in pink, yellow and green for domains 1, 2 and 3, respectively. CBP20 is depicted in red, and the cap analogue m^7GpppG in cyan. (A), (B) and (C) were generated with Molscript (Kraulis, 1991) and Render (Merritt and Murphy, 1994).

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2002, 21, 5548-5557) copyright 2002.

Figures were selected by an automated process.