Struktur und Interaktion von Proteinen aus dem Reggie-like Operon von Prokaryoten / Structure and Interaction of Proteins of the Reggie-like Operon in Prokaryotes
Zusammenfassung der Projektergebnisse
Reggie/Flotillin proteins belong to the diverse class of SPFH proteins and play key roles during neuronal regeneration and cell signaling events. In eukaryotes, reggies/flotillins are involved in the formation of lipid microdomains within the plasma membrane of cells, and they are thought to serve as adapter proteins that recruit partner proteins into these lipid microdomains finally leading to a functional state. How these proteins act during formation of lipid microdomains and molecular details of their interactions with partner proteins is even today largely unclear. In this project, we have investigated the bacterial homologues of reggies/flotillins (reggie-like proteins, RLPs). Recently, it was shown that prokaryotic reggies/flotillins localize to raft-like structures in the plasma membrane, and according to this, the prokaryotic reggies/flotillins seem to be engaged in similar functions and interactions as their eukaryotic counterparts. We have determined high-resolution structures of three NfeD proteins. These proteins are encoded in the same operon structures as the RLPs (in this context summarized as reggieassociated proteins, RAPs), and are putative functional if not physical partner proteins of RLPs. Despite low conservation of their amino acid sequences, all reggie-associated NfeD proteins have surprisingly similar 3D structures. This, together with extensive sequence analysis of bacterial genomes, allowed us to derive evolutionary relationships between bacterial ancestors and eukaryotic reggies/flotillins and several other SPFH proteins. These relationships had been invisible to previous sequence analyses that had been restricted to eukaryotic proteins. We have furthermore characterized the substrate spectrum of the acetyl transferase YuaI which is a reggie-associated protein from the soil bacterium B. subtilis. YuaI acetylates the primary amino groups of polyamines, preferentially spermin and spermidin. YuaI is upregulated under cellular stress and seems to contribute to polyamine homeostasis under these conditions. The most important results of this project have been obtained regarding the interaction of reggie-like with reggie-associated proteins, namely of the proteins YuaG and YuaF from B. subtilis. TIRF (total internal reflection fluorescence) microscopy studies show that both proteins colocalize into focal structures within the plasma membrane. YuaG is responsible for forming these foci, and YuaG recruits YuaF into these foci mediated by its C-terminal domain. Interaction studies by NMR turned out to be extremely difficult and failed when one or both putative binding partners were presented as soluble constructs. However, we have strong evidence for a direct, physical interaction between YuaG and YuaF, when both proteins are simultaneously embedded in the lipid bilayer of a lipid nanodisc. This suggests that the interaction between both proteins is rather weak, and that the lipid bilayer environment is required for formation of a stable complex.
Projektbezogene Publikationen (Auswahl)
- (2008) Resonance Assignment of the Soluble Domain of the NfeD Protein YqiJ from Eschericia coli. BMRB database entry 15664
Hinderhofer, M., Walker, C.A., Wolf, J., Friemel, A., Boos, W., & Möller, H.M.
- (2009) Evolution of prokaryotic SPFH proteins. BMC Evolutionary Biology 9
Hinderhofer, M., Walker, C.A., Friemel, A., Stuermer, C.A.O., Möller, H.M. & Reuter, A.
- (2012) Synthetic motility and cell shape defects for deletions of flotillin/reggie paralogs in Bacillus subtilis and interplay with NfeD proteins. J Bacteriol 194: 4652-4661
Dempwolff, F., Möller, H.M. & Graumann, P.L.
