Biochemical and Biophysical Research Communications
Structural basis for the regulation of nuclear import of Epstein-Barr virus nuclear antigen 1 (EBNA1) by phosphorylation of the nuclear localization signal
Graphical abstract
Introduction
Epstein-Barr virus (EBV) is a ubiquitous human herpes virus associated with a diverse range of tumors of both lymphoid and epithelial origin [1]. EBV nuclear antigen 1 (EBNA1) is a DNA-binding protein expressed in every EBV-positive tumor. EBNA1 plays an essential role in the maintenance and replication of the episomal EBV genome through its direct interaction with sequences in the EBV latent origin of replication (oriP) [2], [3], [4], [5], and also acts as a transcriptional regulator [6], [7]. It has been shown that EBNA1 induces B cell lymphomas in transgenic mice [8], enhances cell survival [9], and induces genetic instability [10], indicating that EBNA1 might contribute directly to oncogenesis. EBNA1 is phosphorylated at multiple serine residues when expressed in human and insect cells [11], [12], [13], [14]. Although the physiological significance of EBNA1 phosphorylation remains incompletely understood, it has been suggested that phosphorylation of EBNA1 serine residues contributes to segregation and maintenance of the EBV genome, transcriptional activation, and nuclear import of EBNA1 [15], [16], [17], [18].
Transport of macromolecules between the cytoplasm and nucleus occurs through nuclear pore complexes (NPCs) [19]. Eukaryotic cells control and finely tune many biological processes by regulating nuclear transport [20]. Phosphorylation of cargoes has emerged as one of the important mechanisms to regulate a multitude of nuclear transport pathways [20], including the importin (Imp) α:β-dependent nuclear import pathway [21], [22]. The Impα adaptor proteins bind cargo proteins possessing the nuclear localization signal (NLS), and heterodimerize with Impβ through the N-terminal Impβ-binding (IBB) domain, forming the heterotrimeric Impα:β:NLS-cargo complexes that permeate through NPCs and deliver NLS-cargoes into the nucleus [19], [20], [21], [22]. Mammalian cells have at least seven Impα isoforms, whose expression is tightly regulated depending on cell type and developmental stage [23], [24]. Each isoform of Impα has different substrate specificity, and many cellular and viral cargoes have been shown to associate preferentially with specific isoforms of Impα [23], [24].
Previous cell biological and biochemical studies identified the NLS of EBNA1 (379KRPRSPSS386) [25] and demonstrated that EBNA1 binds to the nuclear import adaptor Impα1 [26], [27], [28] as well as Impα5 [28]. The amino-terminal K379 and R380 of the EBNA1 NLS are essential for nuclear translocation [18]. Although the serine residues (S383, S385, and S386) are not essential for the EBNA1 NLS, both S383 and S385 are important for nuclear translocation [18]. Phosphorylation of S385 increases nuclear import efficiency [18] and also increases the binding affinity of the EBNA1 NLS to Impα1 as well as Impα5 [18], [29]. In this study, we used X-ray crystallography to elucidate how phosphorylation of EBNA1 NLS can regulate its interaction with Impα.
Section snippets
Preparation of protein-peptide complexes for crystallization
N-terminally His6-and S-tagged ΔIBB Impα1 (mouse, residues 70–529) was expressed from pET30a (Novagen) [30] in the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene), and was purified over Ni-NTA (Novagen) and gel filtration over Superdex200 (GE Healthcare). S385-phosphorylated EBNA1 NLS peptide 378EKRPRPRSP-pS-S386 (pS stands for phosphoserine) and non-phosphorylated EBNA1 NLS peptide 378EKRPRPRSPSS386 were synthesized by GenScript. Prior to crystallization, ΔIBB Impα1 and the NLS peptide
Crystal structure of Impα1 bound to S385-phosphorylated EBNA1 NLS peptide
We obtained crystals of the NLS-binding armadillo (ARM) repeat domain of Impα1 bound to the S385-phosphorylated NLS peptide of EBNA1, and determined the structure at 2.0 Å resolution by molecular replacement (Table 1). The structure was refined to free and working R-factor values of 21.2% and 18.6%, respectively. Residues 378–385 and 378–383 of the NLS peptide bound to the minor and major NLS-binding sites, respectively, were identified unambiguously in the electron density map (Fig. 1A and B).
Acknowledgements
We thank the staff of Photon Factory for assistance during X-ray diffraction data collection.
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