The nuclear localization sequence of the epigenetic factor RYBP binds to human importin α3

Human Enzyme PADI4 Binds to the Nuclear Carrier Importin α3

“…Both regions are solvent-exposed in the folded structure of PADI4 and, therefore, available to bind Impα3 with no impediment ( Figure S7 ). Thus, we characterized the conformational propensities of the peptides corresponding to the isolated fragments of the two regions, and we measured their binding affinity for a specific importin, Impα3, for which we had already measured the affinity toward other cargos [ 41 , 42 , 43 ]. According to our in vitro measurements, both isolated regions were responsible for PADI4 binding to Impα3, as further confirmed by the molecular docking results ( Figure 10 and Figure 11 ).…”

“…PADI4, Impα3, and ΔImpα3 were purified as previously described [ 24 , 41 , 42 , 43 ]. The concentrations of the proteins were calculated by UV absorbance, using an extinction coefficient at 280 nm; this parameter was estimated from the number of tyrosines and tryptophans in each of these proteins [ 44 ].…”

“…Two-dimensional spectra of NLS2-PADI4 (at 1.2 mM) were acquired in each dimension in phase-sensitive mode by using the time-proportional phase incrementation technique [ 55 ] and a spectral width of 5500 Hz. Standard TOCSY (mixing time of 80 ms) [ 56 ] and NOESY experiments (a mixing time of 250 ms) [ 56 , 57 , 58 ], with the WATERGATE sequence [ 53 ], as well as experimental, processing, and assigning details, were the same used in acquiring, processing, and analyzing the spectra of other NLSs [ 41 , 42 , 43 ]. The chemical shift values of H α protons in random-coil regions were obtained from tabulated data, corrected by neighboring residue effects [ 59 , 60 , 61 ].…”

“…Molecular simulations were performed using the docking software AutoDock Vina (version 1.1.2) [ 64 ], following a methodology we already employed for the virtual screening of other NLS peptides binding to Impα3 [ 41 , 42 , 43 ]. The protein was modeled in the IBB-depleted form on the basis of the Protein Data Bank (PDB) entry 5XZX [ 65 ], which reports the crystallographic complex with the NLS of the Ran-binding protein 3 anchored within the major binding site of Impα3.…”

“…From an experimental point of view, Impα3 can also be easily expressed and purified for in vitro structural and binding studies [ 40 , 41 , 42 ]. In addition, Impα3 can be considered a model protein to investigate how the NLS sequence of the cargo can affect the thermodynamic parameters in the binding process, and we have already carried out several studies of the binding of Impα3 with other NLSs which can be used as a comparison [ 41 , 42 , 43 ]. Lastly, by studying both importin species (with and without the IBB), we could explore whether the absence of the IBB domain affects the binding of the peptide encompassing the NLS region, as studied in the case of other NLSs of several proteins (see [ 41 , 42 , 43 ] and references therein).…”