Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here, we report the crystal structure of the full-length glycoprotein B, the fusogen from Herpes Simplex Virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. Hyperfusogenic mutations within the CTD destabilize it, targeting trimeric interfaces, structural motifs, and membrane-interacting elements. Thus, we propose that the CTD trimer observed in the structure stabilizes gB in its prefusion state despite being appended to the postfusion ectodomain. Our data suggest a model for how this dynamic, membrane-dependent “clamp” controls the fusogenic refolding of gB.

Enveloped viruses employ a class of proteins known as fusogens to orchestrate the merger of their surrounding envelope and a target cell membrane. Most fusogens accomplish this task alone, by binding cellular receptors and subsequently catalyzing the membrane fusion process. Surprisingly, in herpesviruses, these functions are distributed among multiple proteins: the conserved fusogen gB, the conserved gH/gL heterodimer of poorly defined function, and various non-conserved receptor-binding proteins. We summarize what is currently known about gB from two closely related herpesviruses, HSV-1 and HSV-2, with emphasis on the structure of the largely uncharted membrane interacting regions of this fusogen. We propose that the unusual mechanism of herpesvirus fusion could be linked to the unique architecture of gB.

Background:In ATP-binding cassette proteins, ATP binding produces association of the two nucleotide-binding domains (NBDs), but the molecular mechanism is unknown. Results: The NBDs separate following ATP hydrolysis. Conclusion: NBD dimers dissociate during the hydrolysis cycle supporting monomer/dimer models of operation. Significance: Knowledge of the molecular mechanism of hydrolysis will help us understand how ATP-binding cassette proteins work.

ATP-binding cassette exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward-and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer spectroscopy to study the prototypical ATP-binding cassette exporter MsbA reconstituted in nanodiscs at 37°C while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions. ATP-binding cassette (ABC)2 transporters constitute one of the largest families of membrane proteins and are found in all domains of life (1-3). They can be either importers (most prokaryote ABC transporters) or exporters (most mammal ABC transporters) (1-3). The core structure of ABC proteins consists of two transmembrane domains that form the translocation pathway and two conserved nucleotide-binding domains (NBDs) that bind and hydrolyze ATP (1-3), a process essential for their function. Binding of ATP promotes the formation of an NBD dimer in a head to tail orientation (4, 5). In the resulting structure two ATP molecules are "sandwiched" at the dimer interface, and residues from both NBDs form each of the two nucleotide-binding sites (1, 4, 5). NBD dimerization is essential for ATP hydrolysis and requires ATP binding to both nucleotide-binding sites (6), whereas dissociation of the dimers occurs following hydrolysis at only one of the two sites (7). This ATP-dependent NBDs dimerization/dissociation process is coupled to rearrangements of the transmembrane helices, switching the transporters from an inward-facing conformation (dissociated NBDs) to an outward-facing conformation (dimeric NBDs), with the concomitant translocation of substrate (1, 3).Two of the most studied ABC exporters are the multidrug resistance protein P-glycoprotein (MDR1 and ABCB1) that plays a role in the resistance to chemotherapy of some forms of cancer (3),and its bacterial homolog, the lipid flippase MsbA (8). MsbA is located in the inner membrane of Gram-negative bacteria, where it transports lipid A from the inner to the outer leaflet (9, 10). MsbA has been crystallized in inward-and outward-facing conformations (11) and has also been extensively studied by different spectroscopic techniques (12-18). Several studies point to large motions between the nucleotide-free "open" inward-facing conformation (NBDs separated by tens of Angstroms) and the ATP-bound "closed" outw...