N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil - PubMed (original) (raw)

N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil

J Chen et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The structure of a stable recombinant ectodomain of influenza hemagglutinin HA(2) subunit, EHA(2) (23-185), defined by proteolysis studies of the intact bacterial-expressed ectodomain, was determined to 1.9-A resolution by using x-ray crystallography. The structure reveals a domain composed of N- and C-terminal residues that form an N cap terminating both the N-terminal alpha-helix and the central coiled coil. The N cap is formed by a conserved sequence, and part of it is found in the neutral pH conformation of HA. The C-terminal 23 residues of the ectodomain form a 72-A long nonhelical structure ordered to within 7 residues of the transmembrane anchor. The structure implies that continuous alpha helices are not required for membrane fusion at either the N or C termini. The difference in stability between recombinant molecules with and without the N cap sequences suggests that additional free energy for membrane fusion may become available after the formation of the central triple-stranded coiled coil and insertion of the fusion peptide into the target membrane.

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Figures

Figure 1

Schematic of influenza virus HA and HA2 sequences. EHA2 (_E. coli_-expressed HA2 residues 23–185) is the protein structure determined here. TM, transmembrane anchor; S—S, interchain disulfide bond from HA1 Cys-14 to HA2 Cys-137; Flag octapeptide (in F185), bold.

Figure 2

Proteolysis of the F185 ectodomain of HA2 defined a stable EHA2 (23–185) ectodomain. (A) SDS/PAGE of digestion products of F185 (Fig. 1) with thermolysin, trypsin, α-chymotrypsin, elastase, and V8-protease. The first two lanes are controls without protease. Lanes labeled E are the proteases alone. (B) SDS/PAGE analysis of degradation products of F185 stored at 4°C for 60 days. The full length protein (22 kDa) was completely degraded to ≈18 kDa and ≈10 kDa bands. Fig. prepared with

molscript

(42).

Figure 3

Ribbon diagrams of monomeric and trimeric HA2 ectodomains. (A) Stereodiagram of the EHA2 (23–185) monomer (blue) superimposed on HA2 (red) from neutral pH BHA (5). Residues 1–33 not visible in EHA2 (23–185) are colored white in the HA2. (B) Trimer of HA2 (blue, red, yellow monomers) from neutral pH BHA (5). Residues 1–33 not visible in EHA2 (23–185) are colored white in HA2. (C) Trimer of the EHA2 (23–185) structure (colored as in B). Figure prepared with

ribbons

(43).

Figure 4

N cap domain of EHA2(23–185). (A) Stereo ribbon diagram of the N-terminal residues (dark blue, dark red, dark yellow) and the C-terminal residues (light colors) of EHA2 (23–185), viewed down the molecular threefold-symmetry axis. (B) Stereoatomic diagram of the N-terminal residues (colors and view as in A) and the C-terminal residues of EHA2 (23–185). Potential hydrogen bonds are green dashed lines. (C) Stereoribbon diagram as in A but viewed perpendicular to the molecular threefold-symmetry axis. (D) Stereoatomic diagram as in B but viewed as in C. Figure prepared with

ribbons

(43).

Figure 5

Comparison of TBHA2 from low pH-treated HA and EBHA2(23–185). (A) TBHA2 (residues 1–27 of HA1 and 38–175 of HA2) from thermolytic digestion of low pH-treated viral BHA. The white molecular surface is the central triple-stranded coiled coil. The atomic models are the C-terminal residues beyond residue 106 colored differently for each monomer. The last residues visible in the electron density at the termini are labeled. (B) EBHA2 (23–185) with the N-terminal residues from 34 to 40 colored yellow. The rendering is as in A with the visible terminal residues labeled. Figure prepared with

grasp

(44).

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N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil - PubMed (original) (raw)