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Biophysical Principles of Chaperone Function

Molecular chaperones play a key role in cellular processes, including protein homeostasis, but also in membrane protein transport and biogenesis. We employ high-resolution NMR studies of large chaperone-client complexes to provide an atomic resolution description of their structure and conformation and understand molecular mechanisms underlying their function.

Along these lines, we have used solution NMR spectroscopy to elucidate the mechanism underlying client recognition by the ATP-independent chaperones Spy, SurA and Skp at the atomic level (Burmann et al., Nat. Struct. Mol. Biol. (2013), Thoma et al., Nat. Struct. Mol. Biol. (2015), Morgado et al., Nat. Comm. (2017)). In these systems, we find that chaperones hold clients in a dynamic state that rapidly interconverts conformations while bound on the chaperone surface. Several chaperones were found to interact with the partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion (He et al., Sci. Adv. (2016), He et al., Angew. Chem. (2018)). 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 (Hiller, Trends Biochem. Sci. (2019)).

Recently, we have systematically investigated how molecular chaperones interact with the intrinsically disordered protein α-synuclein. α-synuclein plays a key role in Parkinson’s disease. We could show that molecular chaperones have a specific interaction signature and use this signature to elucidate how chaperones regulate the state of α-synuclein in living cells (Burmann et al., Nature (2020)). Our ongoing projects in the field of chaperone biophysics address fundamental questions. We want to derive quantitative predictors for chaperone function, unravel atomic-level details of how clients are recognized, understand how the interplay works between chaperone and client dynamics and study how basic chaperone function is embedded into complex functional cycles.



Mechanisms of Outer Membrane Protein Biogenesis

The chaperone machinery that facilitates outer membrane protein biogenesis in bacteria is most fascinating, both from the view of fundamental biophysics, as well as from the perspective of microbiology. While the process is catalyzed in vivo by an assembly line of more than eight chaperones, most β-barrel outer membrane proteins (Omps) can also autonomously fold and insert into a target membrane or membrane mimic. We aim at unraveling the underlying mechanisms at the atomic level, following our initial hypothesis of a hybrid barrel mechanism (Gruss et al., Nat. Struct. Mol. Biol. (2013)).

Towards elucidating the in vitro folding mechanism, 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 (Raschle et al., Angew. Chem. (2016), Rath et al., Angew. Chem. (2019)). We have resolved the folding mechanism of the b-barrel protein OmpX into lipid bilayers at the atomic level. The protein folds via a two-state mechanism, in contrast to published mechanisms for related proteins. OmpX folding into lipid bilayers is a rare, not a slow process, rate-limited by the membrane insertion probability. The data provide a rationale for the catalytic effect of BamA.

Towards understanding the functional mechanism of the β-barrel insertase BamA at the atomic level and to explore its role as a target for novel antibiotics, we have established NMR spectroscopy of the BamA barrel in detergent micelles (Hartmann et al., J. Am. Chem. Soc. (2018)). Our NMR assignments have allowed to monitor the conformational ensemble at the atomic level and to map its interactions with ligands and binding partners. We could then map the binding interface of a novel class of antibiotics, so-called OMPTAs, on BamA and studied their effect on the BamA conformational equilibrium. The OMPTAs interfere with the conformational equilibrium of BamA, providing an explanation for their mechanism of action (Luther et al., Nature (2019)). Subsequently, we have shown that the novel natural antibiotic darobactin directly binds and inhibits BamA (Imai et al., Nature (2019)). We are in the progress of unravelling the mechanism of darobactin function.



Mechanisms of bacterial cell signaling

Microorganisms can survive in challenging environments by rapidly adapting to the external conditions. Sensing external stimuli and transducing this information to the interior is in many cases mediated by phosphate signaling systems. Typical systems are composed of at least one sensor histidine kinase and one response regulator protein that communicate with each other via a phosphorylation cascade. In addition, Caulobacter and other bacteria use these systems to regulate their cell cycle. In such cases, not an external stimulus, but the fluctuating internal second messenger cyclic di-GMP is sensed.

In close collaboration with the Jenal and the Schirmer labs, we use a combination of methods, in particular X-ray crystallography and NMR spectroscopy, to elucidate the underlying molecular mechanisms at the atomic level. We have shown that the activity of the histidine kinase CckA from the CckA-CtrA pathway involved in initiation of chromosome replication is regulated by cyclic di-GMP, we identified a novel cyclic di-GMP binding pocket in CckA and demonstrated that cyclic di-GMP reversibly locks two kinase core domains in place, hindering the hinge motions required for autophosphorylation (Lori et al., Nature (2015), Dubey et al., Sci. Adv., (2016)).

Recently, we focused on the ShkA-TacA pathway involved in the G1/S phase transition and stalk formation (Kaczmarczyk et al., Nat. Comm., (2020)). We demonstrated that cyclic di-GMP binds to the pseudo-receiver domain of the ShkA hybrid histidine kinase, thereby discovering a novel binding mode for cyclic di-GMP. Specific methyl-labeling NMR strategies on the full-length protein proved that the interaction of ShkA with cyclic di-GMP leads to a large conformational change in which the C-terminal domain is liberated from its obstructing position (Dubey et al., PNAS, (2019)).

We are currently interested in understanding how the proteins in more complex signaling transduction systems communicate with each other. Due to their large size, these proteins have not yet been much studied by NMR spectroscopy, therefore the functional dynamics are largely unknown. With modern NMR technology solutions are however well within technical reach. In addition, we aim at dissecting the mechanisms of response regulator-histidine kinase hybrids, which have shown to be crucial for virulence and motility in pathogenic bacterial species.

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