Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b-7 interacts with the lipid bilayer prior to recruiting C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

The membrane attack complex (MAC) is one of the immune system’s first responders. Complement proteins assemble on target membranes to form pores that lyse pathogens and impact tissue homeostasis of self-cells. How MAC disrupts the membrane barrier remains unclear. Here we use electron cryo-microscopy and flicker spectroscopy to show that MAC interacts with lipid bilayers in two distinct ways. Whereas C6 and C7 associate with the outer leaflet and reduce the energy for membrane bending, C8 and C9 traverse the bilayer increasing membrane rigidity. CryoEM reconstructions reveal plasticity of the MAC pore and demonstrate how C5b6 acts as a platform, directing assembly of a giant β-barrel whose structure is supported by a glycan scaffold. Our work provides a structural basis for understanding how β-pore forming proteins breach the membrane and reveals a mechanism for how MAC kills pathogens and regulates cell functions

Is Base of Support Greater in Unsteady Gait?

Abstract Background and Purpose. We investigated dynamic interfoot distance (IFD) throughout the gait cycle in people with unsteady gait caused by vestibulopathy and in people without known neuromuscular pathology. We expected that the subjects with unsteady gait would use a greater IFD than subjects without neuromuscular pathology and that this IFD would be correlated with other measures of locomotor stability. Subjects and Methods. Simultaneous whole-body (11-segment) dynamic kinematic data were collected from 22 subjects with vestibulopathy and 22 subjects without known neuromuscular pathology who were matched for age, height, weight, and body mass index. Two trials each of the participants' gait at preferred speed and paced gait at 120 steps/min were analyzed with a repeated-measures design with multiple dependent variables. Quantitative data were analyzed descriptively and with inferential statistics. Results. Interfoot distance at preferred gait speed did not differentiate unsteady subjects with vestibulopathy from the comparison subjects. Paced gait IFD total range and IFD in single-limb support differed between groups, but IFD at heel-strike did not. However, IFD at heel-strike, the traditional measure of “base-of-support width,” was correlated with measurements of whole-body center-of-gravity stability (r=.32–.55). Discussion and Conclusion. Gait at preferred speed permitted the unsteady subjects and the comparison subjects to select similar IFD values, but at the cost of slower gait in the unsteady subjects. When required to walk at a “normal” pace of 120 steps/min, subjects with vestibulopathy increased their IFD. These data suggest that wide-based gait alone cannot differentiate between subjects with and without balance impairments. Base of support and other whole-body kinematic variables are mechanical compensations of vestibulopathic instability. Further studies are needed to determine whether development of active control of these whole-body control variables can occur after vestibular rehabilitation.</jats:p