2023 Structural and biochemical analysis of SynDLP, a bacterial dynamin-like protein with eukaryotic features

Authors:
Lucas Gewehr

Journal:
Dissertation

Institute:

Johannes Gutenberg-Universität Mainz

Abstract:

Light energy is converted into chemical energy through the process of photosynthesis, which is the basis for complex life on earth. In most oxygenic photosynthetic organisms, a specialized internal membrane system, the thylakoid membrane (TM), harbors protein complexes that enable the conversion of light into chemical energy. There are still many unanswered questions about TM biogenesis and dynamics. Dynamin-like proteins (DLPs) are large mechanochemical GTPases with various membrane-active functions in eukaryotes. Recently, a bacterial DLP (BDLP) was identified in the cyanobacterial model organism Synechocystis sp. PCC 6803. The protein was termed SynDLP and is possibly involved in TM remodeling in the cyanobacterium. The physiological function of most BDLPs identified to date is unclear. Isolated SynDLP was found to be an active GTPase that forms oligomers in solution and interacts with negatively charged TM lipids. Moreover, SynDLP does not appear to be essential for Synechocystis sp.PCC 6803, at least under standard growth conditions.
My studies clearly confirm that SynDLP is a BDLP, as shown by the determined structure of SynDLP oligomers. The monomers consist of typical DLP domains. The arrangement of monomers within the oligomer resembles the architecture of some eukaryotic DLPs, here observed for the first time in a BDLP. Thus, SynDLP is the closest known ancestor of a group of eukaryotic DLPs. Moreover, SynDLP exhibits features unique for DLPs, such as an intramolecular disulfide bridge in the bundle signaling element (BSE) domain and a large intermolecular interface between the BSE and GTPase domains. Both influence the GTPase activity of SynDLP. To further study the membrane interaction properties of SynDLP, nucleotide-dependent membrane binding of SynDLP was visualized via fluorescence microscopy using a fluorescently labeled SynDLP variant. Surface plasmon resonance spectroscopy was established to quantify the membrane interaction propensity of SynDLP.
Finally, a membrane activity of SynDLP was detected as it fuses TM-mimicking model membranes in vitro. Importantly, expression of native SynDLP in Synechocystis sp. PCC 6803 was detected using SynDLP-specific antibodies.
In summary, the results of this work provide new insights into the structure and function of SynDLP, the BDLP of Synechocystis sp. PCC 6803. The new information classifies SynDLP as a BDLP that is closely related to eukaryotic DLPs.