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Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry.

ABSTRACT: Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

SUBMITTER: Krummenacher C

PROVIDER: S-EPMC1356314 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry.

Krummenacher Claude C Supekar Vinit M VM Whitbeck J Charles JC Lazear Eric E Connolly Sarah A SA Eisenberg Roselyn J RJ Cohen Gary H GH Wiley Don C DC Carfí Andrea A

The EMBO journal 20051117 23

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the posi ...[more]

PMID: 16292345

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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

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Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry.

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Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry.

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