PDBsum entry 1ask

Transport

PDB id: 1ask

Contents

Protein chains

125 a.a. *

Waters ×55

* Residue conservation analysis

PDB id:

1ask

Name:

Transport

Title:

Nuclear transport factor 2 (ntf2) h66a mutant

Structure:

Nuclear transport factor 2. Chain: a, b. Synonym: pp15, b2. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Organ: liver. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.30Å R-factor: 0.200 R-free: 0.243

Authors:

A.J.Mccoy,M.J.Stewart

Key ref:

W.D.Clarkson et al. (1997). Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran. J Mol Biol, 272, 716-730. PubMed id: 9368653 DOI: 10.1006/jmbi.1997.1255

Date:

11-Aug-97 Release date: 28-Jan-98

Protein chains

P61972  (NTF2_RAT) -  Nuclear transport factor 2 from Rattus norvegicus

Seq: 127 a.a.

Struc: 125 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1006/jmbi.1997.1255

J Mol Biol 272:716-730 (1997)

PubMed id: 9368653

Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran.

W.D.Clarkson, A.H.Corbett, B.M.Paschal, H.M.Kent, A.J.McCoy, L.Gerace, P.A.Silver, M.Stewart.

ABSTRACT

Nuclear transport factor 2 (NTF2) is associated with the translocation stage of nuclear protein import and binds both to nuclear pore proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in nuclear protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule's hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell nuclear protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient nuclear protein import.

Selected figure(s)

Figure 2.
Figure 2. 1D, 2D and 3D models of mutants generated. (a) Alignment of human and yeast NTF2 protein sequences showing the mutations introduced into the human protein above the sequences. The locations of two temperature-sensitive yeast NTF2 mutants M83T and D91G (Corbett & Silver, 1996) are indi- cated underneath the sequence. (b) A CPK space-filling model of a single polypeptide chain of NTF2 (Bullock et al., 1996) showing the positions of the residues of NTF2 that were mutated. (Note that the mutations are localised predomi- nantly within or near the central hydrophobic cavity.) Aspartate 23 (the D23N mutation) is not visible in this view, the residue being located towards the back of the protein. Residues 126 and 127 were not identifiable in the crystal struc- ture of this NTF2 chain, and so the F126delta truncation is not shown. (c) Schematic model of the NTF2 cavity and surrounding surface showing the key residues facing from the cavity, and the mutations introduced.

Figure 8.
Figure 8. Interaction of NTF2 with Ran and nucleoporins detected using a two-hybrid interaction trap assay. Interactions were quan- tified using a b-galactosidase activity assay. Each column rep- resents the average of three independent assays and error bars represent standard deviations. (a) Interactions between wild-type and mutant NTF2 and either Ran (black) or Nsp1 (white). Although all NTF2 constructs interact with Nsp1, mutant NTF2 constructs such as E42K and D92/94N do not interact with Ran effectively. Others, such as D117N, interact with Ran to the same extent as wild-type. (b) Gsp1 and Ran show the same pattern of binding to NTF2 mutants when tested for growth on selectable media, whereas neither wild-type nor mutant NTF2 bound to Ras. (c) and (d) Interactions between wild-type NTF2 and a range of nucleoporins as indicated. In (c) b- galactosidase assays show NTF2 interacts with FxFG nucleoporins such as Nsp1, Nup1 and Nup2, but not with nucleoporins contain- ing FG repeats such as Nup100, Nup116, Rip1 or Yrb2 (called Nup36 by Nehrbass & Blobel, 1996). The same spectrum of inter- actions was seen with E42K and D92/94N mutants on selective media (d).

The above figures are reprinted by permission from Elsevier: J Mol Biol (1997, 272, 716-730) copyright 1997.

Figures were selected by an automated process.