Order and disorder control the functional rearrangement of influenza hemagglutinin

Influenza hemagglutinin (HA), a homotrimeric glycoprotein crucial for membrane fusion, undergoes a large-scale structural rearrangement during viral invasion. X-ray crystallography has shown that the pre- and postfusion configurations of HA₂, the membrane-fusion subunit of HA, have disparate secondary, tertiary, and quaternary structures, where some regions are displaced by more than 100 Å. To explore structural dynamics during the conformational transition, we studied simulations of a minimally frustrated model based on energy landscape theory. The model combines structural information from both the pre- and postfusion crystallographic configurations of HA₂. Rather than a downhill drive toward formation of the central coiled-coil, we discovered an order-disorder transition early in the conformational change as the mechanism for the release of the fusion peptides from their burial sites in the prefusion crystal structure. This disorder quickly leads to a metastable intermediate with a broken threefold symmetry. Finally, kinetic competition between the formation of the extended coiled-coil and C-terminal melting results in two routes from this intermediate to the postfusion structure. Our study reiterates the roles that cracking and disorder can play in functional molecular motions, in contrast to the downhill mechanical interpretations of the “spring-loaded” model proposed for the HA₂ conformational transition.Hemagglutinin (HA) is a viral receptor-binding and membrane-fusion glycoprotein involved in the invasion of influenza virions into host cells (1). Structural rearrangements of HA during membrane fusion are crucial for the delivery of the viral genome. The postfusion conformation of HA shows considerable similarity to other viral fusion proteins and eukaryotic membrane receptors involved in intracellular vesicle trafficking (2), suggesting there may be common mechanisms in the function of these proteins. Therefore, HA may serve as a model system, allowing characterization of the molecular and energetic details that underlie its conformational transition to provide insights into general principles of membrane fusion (3).HA is a homotrimer consisting of two domains connected by disulfide bonds (4): a globular receptor binding domain (HA₁), and a coiled-coil membrane-fusion domain anchored to the viral membrane (HA₂). Recognized by the sialic acid receptor of a host cell, the intact virus enters the cell via endocytosis. Low pH in a late endosome then induces the dissociation of HA₁ from HA₂ (1) and an irreversible conformational transition of HA₂. Experimentally, this conformational change can be triggered by either low pH, high temperature, or urea denaturation (5).Structures of HA in pre- and postfusion pH conformations have been solved by X-ray crystallography. The structure of the prefusion ectodomain contains both HA₁ and HA₂, and was purified from influenza virions (4). A postfusion conformation of HA₁ and HA₂ were obtained from prefusion viral HA that was sequentially treated with low pH and trypsin (6, 7). Comparison of these two structures shows no structural changes in HA₁, but a major rearrangement in HA₂, including secondary, tertiary, and quaternary structural changes. The N-terminal domain of HA₂, initially adjacent to the transmembrane region in the prefusion configuration, undergoes a large movement (over 100 Å) during the transition. The C-terminal domain of HA₂ changes from a globular structure to three extended loops packed against the central coiled-coil.Although experiments have probed the fusion mechanism through mutation (8) and provided measures of fusion kinetics (9), there is a lack of structural information about how HA₂ transitions from the prefusion to postfusion conformations. Theoretical models have been suggested to describe the fusion mechanism based on the available experimental kinetic data (10–12). However, because of the large scale of the HA₂ rearrangement, only limited computational techniques, such as targeted molecular dynamics (13), have been applied to study the molecular details of the transition.In this study we applied the principles of the energy landscape theory as developed in the context of protein folding (14–16) to examine structural details of the HA₂ conformational transition. We used a structure-based model (SBM) (17, 18) built with a dual-funneled landscape (19, 20) that has both the pre- and postfusion structures as explicit minima. The HA₂ landscape has at least two competing basins of attraction, corresponding to the pre- and postfusion structures of HA₂, respectively. HA₁ dissociation sterically enables HA₂ to explore beyond the prefusion local free-energy minimum and to diffuse toward the postfusion configuration. The long-length scale and extensive shuffling of secondary and tertiary structures is reminiscent of protein folding, but distinct in that both ends of the HA₂ transition can be described by ensembles of structurally similar configurations. Just as in protein folding, there may be free-energy barriers and structural intermediates along the HA₂ transition caused by the imperfect cancellation of energy and entropy. These intermediate ensembles may be interesting candidates for drug design to inhibit HA function.Previously, a “spring-loaded” model has been applied to describe the mechanism for the HA₂ transition (21). This model suggested a downhill mechanical transition of the N-terminal region of HA₂ into an ordered helical structure that orients the fusion peptides away from the virus and toward the host membrane. Our simulations expand this view by showing that the conformational change of the N-terminal domains is associated with an entropic barrier and the unfolding of the C-terminal region is associated with the major energetic barrier during the HA₂ conformational transition. Kinetic competition between these two events creates a long-lived metastable intermediate that allows for two dominant routes. The first route (the “sequential route”) resembles the spring-loaded model, and the second route (the “cooperative route”) involves cooperative interactions between the N-terminal and C-terminal domains in forming the central coiled-coil. The presence of these distinct routes suggests multiple mechanisms for HA₂ rearrangement and membrane fusion.