Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

Abstract

While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that ␤-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.

Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus–cell fusion

“…Out of 26 residues, the PIV5 TMD contains 10 β-branched residues Val and Ile (SI Appendix, Table S5), which are known to destabilize α-helices. High Val and Ile contents in synthetic LV peptides and other viral TMDs have been shown to cause higher fusogenicity, possibly by increasing conformational flexibility and reducing the α-helical content (12,13,53,54). Indeed, β-branched residues constitute 30-75% of the TMD sequences of 10 paramyxoviruses and HIV, suggesting that transition to the β-strand conformation in PE-rich membranes may be general for this class of fusion proteins.…”

“…13 C and 15 N chemical shifts indicate that the two termini of the peptide have high β-strand propensities, whereas the center is more helical, suggesting that this strand-helix-strand motif may endow the TMD with the ability to perturb the two membrane leaflets differently to generate NGC. The 26-residue peptide is sufficient to span the membrane: as a pure α-helix, it would be ∼39 Å long, and the β-strand residues further increase the peptide length, making it possible for the TMD to tilt, bend, and acquire local disorder while still spanning the membrane.…”

“…Because the PIV5 FP structure depends on the membrane environment, including the membrane surface charge, lipid chain unsaturation, and lipid intrinsic curvature (11,26), we measured the TMD chemical shifts in six lipid membranes in the gel-and liquid-crystalline phases, to probe both the rigid-limit structure and the dynamics of the peptide. Two-dimensional 13 C-13 C correlation spectra of the TMD ( Fig. 1 and SI Appendix, Fig.…”

“…To investigate if negatively charged lipids and cholesterol affect the TMD conformation, we measured 2D 13 C-13 C and 15 N- 13 C correlation spectra of TMD in the POPC/1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylglycerol (POPG) and POPC/cholesterol membranes (Figs. 1 and 2 and SI Appendix, Fig.…”

“…In comparison, little is known about the structures of the C-terminal TMD of viral fusion proteins. Cysteine-scanning and sedimentation equilibrium data showed that various paramyxovirus TMDs form three-helix bundles in lipid membranes (12,13). Circular dichroism (CD) and Fourier-transform infrared (FT-IR) data of influenza HA TMD indicate an α-helical structure parallel to the membrane normal (14).…”