PDBsum entry 2jdq

Protein transport

PDB id: 2jdq

Contents

Protein chains

424 a.a.

63 a.a.

65 a.a.

Waters ×265

References listed in PDB file

Key reference

Title

Structure and nuclear import function of the c-Terminal domain of influenza virus polymerase pb2 subunit.

Authors

F.Tarendeau, J.Boudet, D.Guilligay, P.J.Mas, C.M.Bougault, S.Boulo, F.Baudin, R.W.Ruigrok, N.Daigle, J.Ellenberg, S.Cusack, J.P.Simorre, D.J.Hart.

Ref.

Nat Struct Mol Biol, 2007, 14, 229-233. [DOI no: 10.1038/nsmb1212]

PubMed id

17310249

Abstract

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Figure 1.
Figure 1. Identification and solution NMR structure of PB2 C-terminal domain. (a) Protein expression screen of 26,880 random deletion constructs of the pb2 gene. Stable expression of soluble protein results in efficient in vivo labeling of a C-terminal biotin acceptor peptide. Detection is by fluorescent streptavidin and fluorimaging. (b) Ribbon diagram of the ten lowest-energy NMR structures superimposed using backbone heavy atoms (r.m.s. deviation 0.94 Å for 224 atoms). Indicated are Asp701, Arg702 and Ser714, which are implicated in cross-species transmission, and basic regions corresponding to the minor (purple) and major (gold) sites of the bipartite NLS. (c) Primary sequence alignment comparing influenza (FLU) A, B and C strains. Indicated are residues implicated in cross-species transmission (blue triangles), the minor site (purple triangles) and major site (gold triangles) of the bipartite NLS, conserved buried hydrophobic residues (black ovals) and secondary structure elements.

Figure 3.
Figure 3. X-ray structure of the PB2 C-terminal domain complexed with importin 5. (a) Ribbon diagram showing DPDE (red) bound to human importin 5 (blue), comprising ten armadillo repeats. The C-terminal helix of the importin is unpacked and mediates domain-swap dimer formation in the crystal. The bipartite NLS at the C terminus of DPDE binds classically within the superhelical groove of importin 5. Basic residues Arg737, Lys738 and Arg739 from the minor site (purple) interact with the C-terminal armadillo repeats; Lys752, Arg753 and Arg755 from the major site (gold) interact with the N-terminal armadillo repeats. Lys736 does not interact with importin 5 but makes intramolecular hydrogen bonds in DPDE, perhaps preventing further unfolding of the C terminus. Lys718 makes three hydrogen bonds with importin 5. (b) Comparison of the PB2 domain structure in complexed (red) and free solution state (cyan) demonstrates unfolding of residues 736–759 (purple) upon binding to importin 5. Residue Asp701, important in host specificity and virulence, forms a salt bridge with Arg753 of the major NLS motif and tethers the C terminus to the core of the domain in the unbound state. Residues Arg702 and Ser714 are also implicated in interspecies transmission. Note different orientations of the N-terminal helix of DPDE in the two structures.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2007, 14, 229-233) copyright 2007.