Electrostatics Drive the Molecular Chaperone BiP to Preferentially Bind Oligomerized States of a Client Protein.

Hsp70 chaperones bind short monomeric peptides with a weak characteristic affinity in the low micromolar range, but can also bind some aggregates, fibrils, and amyloids, with low nanomolar affinity. While this differential affinity enables Hsp70 to preferentially target potentially toxic aggregates, it is unknown how a chaperone can differentiate between monomeric and aggregated states of a client protein and why preferential binding is only observed for some aggregated clients but not others. Here we examine the interaction of BiP (the Hsp70 paralog in the endoplasmic reticulum) with the client proIGF2, the pro-protein form of IGF2 that includes a long and mostly disordered E-peptide region that promotes proIGF2 oligomerization. By dissecting the mechanism by which BiP targets proIGF2 and E-peptide oligomers we discover that electrostatic attraction is a powerful driving force for oligomer recognition. We identify the specific BiP binding sites on proIGF2 and as monomers they bind BiP with characteristically weak affinity in the low micromolar range, but electrostatic attraction to E-peptide oligomers boosts the affinity to the low nanomolar level. The dominant role of electrostatics is manifested kinetically as a steering force that accelerates the binding of BiP to E-peptide oligomers by approximately two orders of magnitude as compared against monomeric peptides. Electrostatic targeting of Hsp70 provides an explanation for why preferential binding has been observed for some aggregated clients but not others, as all the currently-documented cases in which Hsp70 binds aggregates with high-affinity involve clients that have an opposite charge to Hsp70.

The shape of pleomorphic virions determines resistance to cell-entry pressure.

Many enveloped animal viruses produce a variety of particle shapes, ranging from small spherical to long filamentous types. Characterization of how the shape of the virion affects infectivity has been difficult because the shape is only partially genetically encoded, and most pleomorphic virus structures have no selective advantage in vitro. Here, we apply virus fractionation using low-force sedimentation, as well as antibody neutralization coupled with RNAScope, single-particle membrane fusion experiments and stochastic simulations to evaluate the effects of differently shaped influenza A viruses and influenza viruses pseudotyped with Ebola glycoprotein on the infection of cells. Our results reveal that the shape of the virus particles determines the probability of both virus attachment and membrane fusion when viral glycoprotein activity is compromised. The larger contact interface between a cell and a larger particle offers a greater probability that several active glycoproteins are adjacent to each other and can cooperate to induce membrane merger. Particles with a length of tens of micrometres can fuse even when 95% of the glycoproteins are inactivated. We hypothesize that non-genetically encoded variable particle shapes enable pleomorphic viruses to overcome selective pressure and may enable adaptation to infection of cells by emerging viruses such as Ebola. Our results suggest that therapeutics targeting filamentous virus particles could overcome antiviral drug resistance and immune evasion in pleomorphic viruses.

Structural cavities are critical to balancing stability and activity of a membrane-integral enzyme.

Packing interaction is a critical driving force in the folding of helical membrane proteins. Despite the importance, packing defects (i.e., cavities including voids, pockets, and pores) are prevalent in membrane-integral enzymes, channels, transporters, and receptors, playing essential roles in function. Then, a question arises regarding how the two competing requirements, packing for stability vs. cavities for function, are reconciled in membrane protein structures. Here, using the intramembrane protease GlpG of Escherichiacoli as a model and cavity-filling mutation as a probe, we tested the impacts of native cavities on the thermodynamic stability and function of a membrane protein. We find several stabilizing mutations which induce substantial activity reduction without distorting the active site. Notably, these mutations are all mapped onto the regions of conformational flexibility and functional importance, indicating that the cavities facilitate functional movement of GlpG while compromising the stability. Experiment and molecular dynamics simulation suggest that the stabilization is induced by the coupling between enhanced protein packing and weakly unfavorable lipid desolvation, or solely by favorable lipid solvation on the cavities. Our result suggests that, stabilized by the relatively weak interactions with lipids, cavities are accommodated in membrane proteins without severe energetic cost, which, in turn, serve as a platform to fine-tune the balance between stability and flexibility for optimal activity.