Structural and Calorimetric Studies Demonstrate that Xeroderma Pigmentosum Type G (XPG) Can Be Imported to the Nucleus by a Classical Nuclear Import Pathway via a Monopartite NLS Sequence

Abstract

Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin-α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG.

Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits

“…A similar enthalpy release was measured in a control experiment where the classical SV40-NLS was injected against ⌬IBB-importin ␣ under identical experimental conditions (Fig. 4C), which gave equilibrium dissociation constants K d1 ϭ 1.5 Ϯ 0.3 M and K d2 ϭ 9.7 Ϯ 1.2 M. It should be pointed out that the equilibrium binding constants reported here are significantly lower than those measured using fluorescence depolarization (79) or surface-immobilized NLSs (80) but nonetheless consistent with published ITC studies that employed short NLS peptides (47,75,76,81). This is explained by the high solubility and charge of NLS peptides and their tendency to remain in solution as solvated ions.…”

Variations in nuclear localization strategies among pol X family enzymes

“…Inclusion of the N‐terminal Met1 residue in the NLS sequence results in increased binding affinity that can be attributed to an interaction of the Met1 backbone at the P‐1 position of the major motif. The NLS binding affinity required to support nuclear import has been subject to controversy, and studies of shorter NLS peptides that lack large extensions potentially capable of additional, non‐specific binding have generally yielded K d values in the range of 0.1 of 10 μM . As discussed recently, cooperative Impα binding of the major‐ and minor‐site NLS motifs—and potentially other motifs contained in the cargo protein, provides a strategy that is well suited to produce both high affinity binding and intra‐nuclear dissociation involving minor site‐directed dissociation factors …”

Structural characterisation of TNRC6A nuclear localisation signal in complex with importin-alpha

“…Lys1166 binding at the P2 pocket instead of Lys1167), as previously observed in other cNLS peptides [44, 56, 65]. Indeed, it has previously been suggested that the lower affinity measured for the XPG2 peptide compared to the XPG1 peptide may be related to peptide staggering or multiple interaction modes of the cNLS, evidenced through ambiguous electron density for XPG2 peptide when bound to mImpα1ΔIBB [65]. While no indication for register staggering was observed in the crystal structure of wild type TNRC6A cNLS in complex with mImpα1ΔIBB, further studies are required to establish whether mutation of the basic cluster motif, such as that of the P3 and/or P5 mutants, may cause the TNRC6A cNLS to adopt alternative binding register modes in order to maximise favourable interactions at the major binding site pockets.…”

“…Lys1166 binding at the P2 pocket instead of Lys1167), as previously observed in other cNLS peptides [44, 56, 65]. Indeed, it has previously been suggested that the lower affinity measured for the XPG2 peptide compared to the XPG1 peptide may be related to peptide staggering or multiple interaction modes of the cNLS, evidenced through ambiguous electron density for XPG2 peptide when bound to mImpα1ΔIBB [65].…”

“…(A) Comparison of cNLS binding at the mImpα P4 cavity. The mImpα1ΔIBB: XPG cNLS complex (PDB ID 5EKF [65]) is shown in blue. The mainchains of Arg106 and Glu107 (residue indicated) move to accommodate Arg binding at the P4 pocket in the case of TNC6A cNLS.…”