Vaccinia virus N1L protein resembles a B cell lymphoma-2 (Bcl-2) family protein

Abstract

Poxviruses encode immuno-modulatory proteins capable of subverting host defenses. The poxvirus vaccinia expresses a small 14-kDa protein, N1L, that is critical for virulence. We report the crystal structure of N1L, which reveals an unexpected but striking resemblance to host apoptotic regulators of the B cell lymphoma-2 (Bcl-2) family. Although N1L lacks detectable Bcl-2 homology (BH) motifs at the sequence level, we show that N1L binds with high affinity to the BH3 peptides of pro-apoptotic Bcl-2 family proteins in vitro, consistent with a role for N1L in modulating host antiviral defenses.

Figure 1.

Structure of vaccinia N1L and comparison with Bcl-2 family proteins. (A) Stereo view of the N1L monomer. Helices and termini are labeled. (B) Stereo superposition of N1L (navy) and Bcl-X_L (gray; 1MAZ; Muchmore et al. 1996). N1L helices are labeled. Functionally important BH regions of Bcl-X_L are colored in magenta (BH4), green (BH3), orange (BH1), and cyan (BH2). (C) Structure-based sequence alignment of N1L with Bcl-2 family members: Mcl-1, mouse myeloid cell leukemia-1 (PDB code 1WSX; Day et al. 2005); KSHV, Kaposi sarcoma virus Bcl-2 homolog (PDB code 1K3K; Huang et al. 2002); human Bcl-X_L (PDB code 1MAZ; Muchmore et al. 1996). Hydrophobic residues are highlighted in green, and acidic/basic residues are in red/blue. Secondary structures of N1L and Bcl-X_L are indicated below the sequence, and consensus BH motifs are indicated above, with the same color scheme as in B. The highly conserved Bcl-2 signature motif, NWGR, is boxed.

Figure 2.

N1L adopts a dimeric structure. (A) Stereo view of the N1L homodimer. The α1 and α6 helices from one N1L monomer (blue) interact in an antiparallel way with equivalent helices in another monomer (green). N and C termini, and helices of each subunit are labeled. (B) Specific α1 and α6 residues at the N1L dimer interface. In the antiparallel N1L dimer, Ile6, Leu10, Phe95, and Phe99 constitute a critical hydrophobic patch whereas Arg7/Asp14 and Arg90/Glu103 pairs form complementary electrostatic surfaces, not present in Bcl-X_L. The N1L monomer (blue) in this view is related to the blue monomer in A by 90° about a vertical axis. (C) The same view for Bcl-X_L showing analogous residues, which are either not hydrophobic or not complementary in charge. BH1–4 domains are colored in magenta (BH4), green (BH3), orange (BH1), and cyan (BH2), as in Figure 1, B and C.

Figure 3.

N1L binds to BH3 peptides. (A,B) Hydrophobic surfaces of N1L and Bcl-X_L. The solvent-accessible surface of N1L indicates the presence of a small hydrophobic groove on the same face of the molecule as the BH3 binding groove in Bcl-X_L. Phe, Trp, Tyr, Met, Ile, Leu, Val, and Ala are colored in yellow. Approximate positions of helices surrounding the groove are indicated on the surfaces. The orientation of each protein is similar to that in Figure 1B. (C) Fluorescence polarization plots (mP = 1000 × fluorescence polarization) of FITC-labeled BH3 domains (Bid, Bim, Bak, and Bad) in the presence of varying concentrations of N1L (▪) or Bcl-X_L (□). (D) Tabulated EC₅₀ values from these plots.