Nanomechanical interactions of phenylalanine-glycine nucleoporins studied by single molecule force-volume spectroscopy.
Phenylalanine-glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-beta. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-beta, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-beta-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG-receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG-receptor interactions instead of FG-FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force-volume (FV) mode.
Lim
Roderick Y H
RY
M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel 4056, Switzerland. roderick.lim@unibas.ch
Köser
Joachim
J
Huang
Ning-Ping
NP
Schwarz-Herion
Kyrill
K
Aebi
Ueli
U
D005998:Glycine / Q000737:chemistry*; D006801:Humans; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry*; D013057:Spectrum Analysis; D028961:beta Karyopherins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D028861:Nuclear Pore Complex Proteins; D028961:beta Karyopherins; D010649:Phenylalanine; D005998:Glycine