A pH-dependent cluster of charges in a conserved cryptic pocket on flaviviral envelopes

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Flaviviruses are enveloped viruses which include numerous human pathogens of escalating global health concern that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making traditional vaccine-based routes of fighting viral infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it also has the potential to diminish the effects of ADE. Here, we systematically examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of a ∼400,000 atom raft system that represents a substantial portion of the curved flaviviral envelope. We utilised a benzene-mapping approach over a total of 14 μs of sampling time, leading to discovery of shared hotspots and elucidation of the dynamic behaviour of conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited significant strain-specific characteristics. An alternative conserved cryptic site located at the E protein domain interfaces showed a more consistent dynamic behaviour across flaviviruses and contains a buried, conserved cluster of ionisable residues that includes His144, previously implicated in the pH-dependent conformational switch required for fusion. The dynamics of the cluster were further explored in extensive constant-pH simulations and revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer.


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  1. eLife assessment

    The study represents an impressive effort to use atomistic simulations to probe cryptic binding sites in the envelope of six flaviviruses. Moreover, using constant pH simulations, the authors suggest that a cluster of ionizable residues contribute to the pH dependent conformational rearrangements required in the infection process. Therefore, the study provides new mechanistic insights that can be helpful in future efforts to develop drugs that target flaviviruses.

  2. **Reviewer #1 (Public Review):
    The pH-dependent conformational change of the envelope protein in flaviviruses is required for the infection process, thus it represents an attractive target for drug development. In this study, the authors conducted extensive atomistic simulations for models for the envelope in six flaviviruses. Using a benzene-mapping approach, they were able to identify several cryptic binding sites that can be targeted for drug development. One of the cryptic binding site was observed in a previous study to be occupied by a detergent molecule, while the other cryptic binding site is located at domain interface. The second binding site involves a cluster of ionizable residues. Using constant pH simulations, the authors suggested that the cluster of ionizable residues contribute to the pH dependent conformational rearrangements. This cluster model helps to explain the inconsistencies reported in the literature regarding the role of several key histidine residues as pH sensors. Overall, the study has provided new mechanistic insights that can be taken advantage of in future drug developments that target flaviviruses. The work also highlights the importance of constant pH simulations to the analysis of pH sensitive biological processes.

  3. Reviewer #2 (Public Review):

    The authors made an applaudable attempt to identify druggable cryptic pockets and address a controversy regarding a pH switch of a very large system of significant biological and Pharmaceutical interest. Due to the size of the system and uncertainty in the membrane interactions/curvature the draft produces etc, it is a nontrivial task. By using a previously validated mixed solvent (i.e., benzene mapping) protocol, the authors were able to analyze the potential pockets in the entire system. This is big technical advance and the protocol can be used by other works in the field for studying cryptic pockets.

  4. Reviewer #3 (Public Review):

    This work dives into the inner molecular workings of viruses such as yellow fever, Zika, and tick borne encephalitis. Due to their pathogenic nature, these are active targets for drug development, and motivated by this, the authors set out to search for so-called "cryptic" binding pockets, concealed from the protein surface and therefore often missed. Using atomistic computer simulations of viral rafts embedded in lipid membranes, the authors present new methodology to detect and characterise structural and electrostatic features of viral envelope proteins. By mixing in a small organic co-solvent (benzene) that acts as a drug proxy, structural fluctuations are enhanced, which reveal hitherto hidden binding pockets. The authors convincingly show that this perturbation has only a minute effect on protein secondary structure. The technique revealed a new cryptic binding pocket that is well conserved across multiple flaviviruses.

    The cryptic site involves four potentially charged residues and to understand their interplay, constant pH molecular dynamics simulations are combined with a detailed structural and electrostatic analysis of the binding pocket.
    Due it's multi-dimensional nature, the response to a possible pH change is a complex process and the authors present a compelling analysis involving charge states, inter-residue distances (reduced using PCA), and structural features of the pocket. An important conclusion is that the role of histidine is less important than previously thought: the pH dependent behaviour is a collective property of the pocket.

    This study is an important contribution to computer aided drug-design. In particular, using co-solutes to induce structural fluctuations seems very helpful for uncovering new binding sites. Of equal importance are methodology to analyse complex trajectories. This work is a good example of how multiple dimensions can be reduced and rationalised using e.g. solvent accessibly surface area (SASA), radius of gyration, net-charge, and principal component analysis. There are likely several other properties that could aid in this rationalising and the present work is a solid platform for exploring these.