Order and disorder control the functional rearrangement of influenza hemagglutinin

Abstract

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 HA 2 , the membranefusion 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 HA 2 . 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 "springloaded" model proposed for the HA 2 conformational transition.protein folding | structure-based model H emagglutinin (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 (HA 1 ), and a coiled-coil membrane-fusion domain anchored to the viral membrane (HA 2 ). 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 HA 1 from HA 2 (1) and an irreversible conformational transition of HA 2 . 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 HA 1 and HA 2 , and was purified from influenza virions (4). A postfusion conformation of HA 1 and HA 2 were obtained from prefusion viral HA th...

Lowered pH Leads to Fusion Peptide Release and a Highly Dynamic Intermediate of Influenza Hemagglutinin

“…The SBS 10 have indicated that an order-disorder transition of Loop3-4 (Fig. 1A,B) is integral to initiating the HA 2 rearrangement.…”

“…7 The post-fusion structure of HA 2 was obtained under low pH conditions and trimmed to remove the fusion peptides (FPs). 8,9 Comparison with the pre-fusion structure reveals dramatic changes in secondary, tertiary, and quaternary structure, 8,10 which opens the possibility of long-lived intermediate ensembles. Therefore, any search for structure-based therapeutics is incomplete without an understanding of the paths HA 2 follows during its conformational rearrangement.…”

“…To facilitate discussion of the HA 2 rearrangement, the trimer is partitioned into 7 sections identified from crystal structures as regions with distinct structural changes during the transition: 8,10 the fusion peptides (FPs), two beta-strands (TBS) and S1 to S5 (see Fig. 1, and details can be found in ref.…”

“…12,13 Importantly, there are titratable residues proximal to the burial pocket of the FPs in HA 2 that may encourage their removal. 14 Beyond the pH trigger, the paths that HA follows during the conformational rearrangement were recently studied for the first time 10 with dual-basin structure-based simulations (SBS). 15,16 These simplified protein folding models were parameterized using the pre- and post-fusion crystal structures of HA 2 .…”

“…This long-lived intermediate ensemble was characterized by a symmetry-breaking of the trimeric structure, which we called a “symmetry-broken intermediate” (SBI). 10 …”