The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-β-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices.

Tumor suppressor Nore1, its acronym coming from novel Ras effector, is one of the 10 members of the Rassf (Ras association domain family) protein family that have been identified. It is expressed as two mRNA splice variants, Nore1A and a shorter isoform, Nore1B. It forms homo- and heterocomplexes through its C-terminal SARAH (Sav/Rassf/Hpo) domain. The oligomeric state of Nore1 and other SARAH domain-containing proteins is important for their cellular activities. However, there are few experimental data addressing the structural and biophysical characterization of these domains. In this study, we show that the recombinant SARAH domain of Nore1 crystallizes as an antiparallel homodimer with representative characteristics of coiled coils. As is typical for coiled coils, the SARAH domain shows a heptad register, yet the heptad register is interrupted by two stutters. The comparisons of the heptad register of Nore1-SARAH with the primary structure of Rassf1-4, Rassf6, MST1, MST2, and WW45 indicate that these proteins have a heptad register interrupted by two stutters, too. Moreover, on the basis of the structure of Nore1-SARAH, we also generate structural models for Rassf1 and Rassf3. These models indicate that Rassf1- and Rassf3-SARAH form structures very similar to that of Nore1-SARAH. In addition, we show that, as we have previously found for MST1, the SARAH domain of Nore1 undergoes association-dependent folding. Nevertheless, the Nore1 homodimer has a lower affinity and thermodynamic stability than the MST1 homodimer, while the monomer is slightly more stable. Our experimental results along with our theoretical considerations indicate that the SARAH domain is merely a dimerization domain and that the differences between the individual sequences lead to different stabilities and affinities that might have an important functional role.

Hepatitis B virus is a major human pathogen, which forms enveloped virus particles. During viral maturation, membrane-bound hepatitis B surface proteins package hepatitis B core protein capsids. This process is intercepted by certain peptides with an “LLGRMKG” motif that binds to the capsids at the tips of dimeric spikes. With microcalorimetry, electron cryo microscopy and peptide microarray-based screens, we have characterized the structural and thermodynamic properties of peptide binding to hepatitis B core protein capsids with different secretion phenotypes. The peptide “GSLLGRMKGA” binds weakly to hepatitis B core protein capsids and mutant capsids with a premature (F97L) or low-secretion phenotype (L60V and P5T). With electron cryo microscopy, we provide novel structures for L60V and P5T and demonstrate that binding occurs at the tips of the spikes at the dimer interface, splaying the helices apart independent of the secretion phenotype. Peptide array screening identifies “SLLGRM” as the core binding motif. This shortened motif binds only to one of the two spikes in the asymmetric unit of the capsid and induces a much smaller conformational change. Altogether, these comprehensive studies suggest that the tips of the spikes act as an autonomous binding platform that is unaffected by mutations that affect secretion phenotypes.

The serine/threonine mammalian sterile 20-like kinase (MST1) is involved in promotion of caspase-dependent and independent apoptosis. Phosphorylation and oligomerization are required for its activation. The oligomerization domain, denoted as SARAH domain, forms an antiparallel coiled coil dimer, and it is important for both MST1 autophosphorylation and interactions with other proteins like the Rassf proteins containing also a SARAH domain. Here we show that the monomeric state of SARAH is thermodynamically unstable and that homodimerization is coupled with folding. Moreover, the influence of the inhibitory domain on SARAH stability and affinity is addressed. By investigating the thermal denaturation using differential scanning calorimetry and circular dichroism, we have found that the SARAH domain dissociates and unfolds cooperatively, without a stable intermediate monomeric state. Combining the data with information from isothermal titration calorimetry, a low thermodynamic stability of the monomeric species is obtained. Thus, it is proposed that the transition from MST1 SARAH homodimer to some specific heterodimer implies a non-native monomer intermediate. The inhibitory domain is found to be highly flexible and intrinsically unfolded, not only in isolation but also in the dimeric state of the inhibitory-SARAH construct. The existence of two caspase recognition motifs within the inhibitory domain suggests that its structural flexibility might be important for activation of MST1 during apoptosis. Moreover, the inhibitory domain increases the thermodynamic stability of the SARAH dimer and the homodimer affinity, while having almost no effect on the SARAH domain in the monomeric state. These results emphasize the importance of flexibility and binding-induced folding for specificity, affinity, and the capacity to switch from one state to another.

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.