Solution NMR study of the interaction between NTF2 and nucleoporin FxFG repeats.
Interactions with nucleoporins containing FxFG repeat cores are crucial for the nuclear import of RanGDP mediated by nuclear transport factor 2 (NTF2). We describe here a solution NMR-based study that identifies primary and secondary FxFG-binding sites on NTF2 and accounts for a range of observations on the rate of NTF2 nuclear trafficking. We used three complementary NMR methods, namely amide group chemical shift titrations, NOE and cross-saturation measurements, to show that the major FxFG-binding site on the dimeric rat NTF2 (rNTF2) molecule is centred on Trp7 and is formed by residues from both NTF2 chains. A secondary FxFG-binding site is located at the rNTF2 hydrophobic cavity and these two sites, together with a surface hydrophobic cluster centred on Trp112, merge into an elongated hydrophobic stripe on the rNTF2 surface. The primary site centred on Trp7 is lost in the rNTF2-W7A mutant that has been shown to bind FxFG nucleoporins with greatly reduced affinity, whereas the secondary site at the rNTF2 hydrophobic cavity is retained. The interface between NTF2 and FxFG nucleoporins detected by NMR is more extensive than that detected by X-ray crystallography, and the presence of a secondary site at the NTF2 hydrophobic cavity accounts for the unexpectedly rapid nuclear import of rNTF2-W7R recently observed by others. The structure of the binding interfaces on these transport factors provides a rationale for the specificity of their interactions with nucleoporins that, combined with their weak binding constants, facilitates rapid translocation through NPCs during nuclear trafficking.
Morrison
Jonathan
J
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Yang
Ji-Chun
JC
Stewart
Murray
M
Neuhaus
David
D
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D020449:Repetitive Sequences, Amino Acid; D012996:Solutions / Q000737:chemistry; D013329:Structure-Activity Relationship; D017186:Titrimetry
D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503612:Nutf2 protein, rat; D012996:Solutions