Omp Biogenesis, Chaperones, and Inflammasome Signalling
We are interested in describing structural and functional details of integral outer membrane proteins and their biogenesis at the atomic level. The outer membrane proteins of Gram-negative bacteria and mitochondria are responsible for a wide range of essential cellular functions, including signal transduction, catalysis, respiration, and transport. At the same time, some of them are good targets for novel antibiotics. Our main technique of expertise is nuclear magnetic resonance (NMR) spectroscopy, which we combine with complementary techniques. Further interests are structural determinants for chaperone-client interactions and structural and functional aspects of the innate immune response.
Molecular chaperones play a key role in outer membrane protein biogenesis, forming a natural assembly line for transport of the unfolded proteins to their target membrane. We employ high-resolution NMR studies of large 70-100 kDa membrane protein-chaperone complexes to provide an atomic resolution description of the underlying molecular mechanisms, such as Skp (Fig. 1, Burmann et al., Nat. Struct. Mol. Biol. (2013)). Subsequent projects address the other chaperones. We want to know the details how the polypeptide transport is accomplished, how the substrates are recognized and how the final folding and insertion step is catalyzed.
Along these lines, we have used NMR spectroscopy to elucidate the mechanism underlying client recognition by the ATP-independent chaperones Spy, SurA and Skp at the atomic level (Fig. 2, He et al., Sci. Adv. (2016)). The chaperones interact with the partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. Spy destabilizes a partially folded client, but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. The similarity of the interactions suggests that the underlying principle of recognizing frustrated segments is of fundamental nature.
Many β-barrel outer membrane proteins (Omps) can autonomously fold and insert into a target membrane or membrane mimic. The Omp folding mechanism is biophysically intriguing but only poorly understood. We have developed an experimental setup that allowed for the first time the observation of hydrogen bond formation during Omp folding by combining H/D-exchange with NMR spectroscopy and mass spectrometry (Fig. 3, Raschle et al., Angew. Chem. Int. Ed. (2016)). OmpX folding into detergent micelles is rate-limited by circular barrel closure from a rapidly exchanging confomational equilibrium. Folding is thus a rare, not a slow process.
A combination of single-molecule force spectroscopy (SMFS) and NMR spectroscopy was employed to characterize how the periplasmic holdase chaperones SurA and Skp shape the folding trajectory of the large β-barrel Omp FhuA from E. coli (Fig. 4, Thoma et al., Nat. Struct. Mol. Biol. (2015)). The unfolded FhuA polypeptide is prone to misfolding and cannot insert back into the membrane. The chaperones SurA and Skp prevent unfolded FhuA polypeptide from misfolding by stabilizing a dynamic state, allowing a search for structural intermediates. The SurA-chaperoned FhuA polypeptide refolds by stepwise inserting individual β-hairpins into the lipid membrane. Thereby the lipid membrane acts as a free energy sink and physically separates transient folds from the chaperones. This trapping of intermediates funnels the unfolded FhuA polypeptide towards its native structure.
As the final step of outer membrane biogenesis, Omp substrates are folded and inserted into the membrane by members of the Omp85 family of proteins. This family comprises the proteins BamA and TamA, but also two-partner secretion systems such as FhaC in Gram-negative bacteria, and Sam50 in mitochondria. We want to determine the molecular mechanisms of folding and insertion by NMR spectroscopy, X-ray crystallography and complementary techniques. Our hypothesis for the functional mechanism is the formation of a hybrid barrel as the folding intermediate, which would allow the translocation of a passenger domain for autotransporter proteins (Fig. 5, Gruss et al., Nat. Struct. Mol. Biol. (2013)).
The innate immune response reacts to pathogens, danger- and damage-related intracellular signals by assembling large inflammasome complexes. We have determined the structure of the mouse ASC inflammasome filament (Fig. 6, Sborgi et al., Proc. Natl. Acad. Sci. USA (2015)) and characterized its polymerization process by employing a combination of NMR spectroscopy and cryo-electron microscopy. In the future, we are interested in a quantitative, atomic-resolution description of inflammasome signaling and its regulation by biomacromolecules and environmental parameters, as well as the molecular mechanisms of gasdermin function.