Synergy of Silent and Hot Spot Mutations in
Importin 
Reveals a Dynamic Mechanism for Recognition of a
Nuclear Localization Signal*
Carolin
Koerner
,
Tinglu
Guan,
Larry
Gerace§, and
Gino
Cingolani¶
From the Department of Cell and Molecular Biology, The Scripps
Research Institute, La Jolla, California 92037
Molecular recognition of the importin 
-binding
(IBB) domain of importin 
by importin is critical for the
nuclear import of protein cargoes containing a classical nuclear
localization signal. We have studied the function of four conserved
tryptophans of importin (Trp-342, Trp-430, Trp-472, and Trp-864)
located at the binding interface with the IBB domain by systematic
alanine substitution mutagenesis. We found that Trp-864 is a mutational hot spot that significantly affects IBB-binding and import activity, whereas residues Trp-342, Trp-430, and Trp-472 are mutationally silent
when analyzed individually. Interestingly, the combination of the hot
spot at residue Trp-864 with mutations in the other three tryptophans
gives rise to a striking synergy that diminishes IBB domain binding by
up to ~1000-fold and, in turn, abolishes import activity. We propose
that importin uses the tryptophans to select and stabilize a
helical conformation of the IBB domain, which, in turn, conveys
specific, high affinity binding.
*
The work was supported by National Institutes of Health
Grant GM41955 (to L. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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