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    • We were able to obtain several crystalline

    2022-05-16

    We were able to obtain several crystalline forms of CHAV Gth. The first one corresponded to the post-fusion conformation [14]. The comparison of this structure with that of VSV-Gth post-fusion conformation revealed that the PHD is the most divergent domain, with the largest differences confined to the secondary structure of the major antigenic site of rhabdoviruses glycoproteins. Local differences also indicated that CHAV has evolved alternative structural solutions in hinge regions between PHD and FD as well as distinct pH-sensitive switches [14]. More interestingly, the asymmetric unit of the second crystalline form contains four CHAV Gth molecules exhibiting two conformations distinct from those of VSV Gth pre-fusion and CHAV/VSV Gth post-fusion conformations [31] (Figs. 1b and 2). Those crystals were grown at pH 7.5, a pH at which VSV Gth was shown to assume a range of intermediate monomeric conformations in solution [37]. The first protomer conformation found in the crystal asymmetric unit corresponded to an early intermediate (CHAV Gth EI) on the transition pathway (Fig. 1b). This protomer looks like the protomer of VSV G pre-fusion state with a single, functionally major difference: the R5 segment has already left the amg express llc groove it occupies in VSV G pre-fusion conformation [25], [31]. The second protomer conformation corresponded to a late intermediate (CHAV Gth LI) on the transition pathway [24], [31]. It is already in an elongated hairpin conformation. However, it is not superimposable with the protomer of CHAV Gth post-fusion state (Fig. 1b) due to incomplete refolding of R1, R4 and R5 [24], [31]. Those two structures offer a plausible pathway for the conformational change (Fig. 1b) with three major features. The first is that R5 can leave the TrD groove it occupies in the pre-fusion conformation before any other structural change. This movement is likely triggered by protonation of a cluster of histidines (H60, H162, and H407) (Fig. 1b), destabilizing the FD-R5 interaction in the pre-fusion state of the protomer [25]. The second feature is that the central helix elongates in two stages; in the LI, the helix is longer than in the pre-fusion protomer but shorter than in the post-fusion one. The third feature is that most of the structural transition occurs in a monomeric form and goes to an elongated hairpin-like conformation before trimerization. Therefore, in the case of rhabdovirus G, there is no need to postulate the existence of a trimeric pre-hairpin conformation resembling those proposed for class I and class II viral fusion glycoproteins.
    An antiparallel interaction between FDs required for fusion The orientational mobility of the CHAV Gth EI conformation, conferred by the release of the R5 segment from the groove it occupies in the pre-fusion conformation, allows EI to orientate its fusion loops toward the target membrane. However, in the crystal asymmetric unit, CHAV Gth molecules assemble in a compact dimer of EI–LI heterodimers [31] (Fig. 2a and b). The tetramer thus formed is a flat structure, from which protrude the fusion loops of the two EI, with a thickness of about 50Å (Fig. 2b). The EI/LI dimers are stabilized by extensive contacts between hydrophobic residues of the central helix (Fig. 2a), and by positioning the R5 segment of one molecule in the hydrophobic groove of the TrD of the other. In turn, the EI/LI dimers assemble by associating their four FDs in an antiparallel arrangement (Fig. 2a and c) in which the two EI fusion loops are projected outside the tetramer (Fig. 2b). This arrangement is stabilized by the formation of an intermolecular β-sheet between two EI and LI FDs, involving residues 75 to 80 in the EI strand (located downstream of the first G fusion loop) and 120 to 125 in the LI strand (located downstream of the second fusion loop) (Fig. 2c). In this region, two residues (D121, E123) were previously reported as sites of mutations affecting the fusion properties of both VSV [40], [41] and viral hemorrhagic septicemia virus (VHSV, a fish rhabdovirus) [42], and a mutation in position 76 was shown to compensate for a deleterious mutation in the fusion loops [43]. The crystal structures of the pre- and post-fusion conformations did not provide a molecular explanation of those phenotypes (Fig. 2d). However, the fact that in the intermolecular β-sheet found in the new crystal form, residues in positions 121 and 123 on the LI side are facing the conserved H80 on the EI side, provides an explanation (Fig. 2c).