Biological Projects

Structure and binding mechanism of RIP2 CARD

Activation of the innate immune pattern recognition receptor NOD2 by the bacterial muramyl-dipeptide peptidoglycan fragment triggers recruitment of the downstream adaptor kinase RIP2, eventually leading to NF-kB activation and proinflammatory cytokine production. Full-length RIP2 can form long filaments mediated by its caspase recruitment domain (CARD), in common with other innate immune adaptor proteins. We showed that the NOD2 tandem CARDs bind to one end of the RIP2 CARD filament, suggesting a mechanism for polar filament nucleation by activated NOD2. By combining X-ray, solid-state NMR and cryo-EM in a collaborative project with the group of S. Cusack, Grenoble, the structure of the helical RIP2 CARD filament was determined, revealing the intermolecular interactions that stabilize the assembly. Using structure-guided mutagenesis, the importance of RIP2 polymerization for the activation of NF-kB signalling by NOD2 was demonstrated. Furthermore, we would like to describe the binding interface of the RIP2 CARD and NOD2 CARD domains. For this study we use a dynamic polarization (DNP)-enhanced solid-state NMR approach. The 15N-13C TEDOR DNP correlation spectra (see figure below) represent a first step into this direction.



Pellegrini E, Desfosses A, Wallmann A, Schulze WM, Rehbein K, Mas P, Signor L, Gaudon S, Zenkeviciute G, Hons M, Malet H, Gutsche I, Sachse C, Schoehn G, Oschkinat H, H. & Cusack S(2018) RIP2 filament formation is required for NOD2 dependent NF-κB signalling.Nature Communications 9:4043. DOI: 10.1038/s41467-018-06451-3

15N13C RIP2 CARD TEDOR spectrum

(a) 13C’ region (b) 13Ca region. The spectrum was recorded at 280K at 700 MHz. The 15N,12C RIP2 CARD-14N,13C NOD2 CARD TEDOR spectrum was recorded at 100K on a 400 DNP spectrometer. RIP2 CARD- NOD2 CARD complex TEDOR DNP spectrum (red) overlayed with the RIP2 CARD controlled spectrum (blue) (c) 13C’ region (d) 13Ca region.

(click on image to enlarge)

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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