B03: Elucidation of the dynamics of the autophagosomal membrane-associated protein GABARAP by NMR spectroscopy

Although (macro-)autophagy is one of the fundamental cellular mechanisms for non-selective as well as selective protein degradation in health and disease, the regulation of the assembly of cellular membranes into autophagosomal compartments is still not fully understood. The GABARAP/MAP1LC3/Atg8 family of proteins is implicated in membrane trafficking and fusion events crucial to autophagosome biogenesis, synaptic plasticity and apoptosis. To this end, the soluble form of the proteins belonging to the GABARAP/MAP1LC3/Atg8 family is enzymatically lipidated to allow membrane anchoring, which in turn has been reported to mediate membrane tethering and hemifusion upon oligomerization. GABARAP undergoes pronounced conformational dynamics on the micro- to millisecond time-scale, a highly conspicuous structural feature of the GABARAP/MAP1LC3/Atg8 family that is evolutionarily conserved from yeast to mammals but remains poorly understood. It is now widely accepted that the nature and efficiency of the function of a protein is determined not only by its static three-dimensional structure, but also by the sum of all its dynamic properties. NMR spectroscopy offers the unique capability of obtaining information about both, structure and dynamics on virtually all time-scales, at atomic resolution and under near-physiological solution conditions. Recent methodological advances now allow not only accurate quantification of the kinetics and thermodynamics of dynamic processes on the biochemically particularly relevant micro- to millisecond time-scale by NMR relaxation dispersion spectroscopy, but also high-resolution structure determination of the low and transiently populated states involved. In principle these experimental strategies, although demonstrated on moderately sized soluble model proteins, are also applicable to membrane-associated proteins. This, however, poses certain additional challenges, which will be addressed in the present project. We will systematically delineate and quantify the conformational dynamics of GABARAP using state-of-the-art NMR spectroscopy supplemented by other biophysical and biochemical methods, as well as determine the structure – to atomic resolution whenever possible – of the various monomeric and oligomeric conformational states involved. To reveal the functional interplay between conformational dynamics, oligomerization, membrane anchoring and tethering/hemifusion on the sub-molecular level, we will compare GABARAP in its soluble identity on the one hand and its membrane-anchored identity on the other hand, with the goal of obtaining a detailed understanding of the mechanism how switching between different conformations allows this protein to execute its functional roles in mediating autophagosomal membrane biogenesis and trafficking. Elucidating the functional dynamics of GABARAP will also serve as a case study to establish general experimental and bioinformatic strategies for studying membrane protein dynamics by NMR spectroscopy in concert with complementary biophysical techniques like FRET, for the first funding period of the SFB 1208 and beyond.

Figure 1: Preliminary experiments demonstrate that GABARAP is highly dynamic on a wide range of time-scales. In particular, CPMG NMR relaxation dispersion experiments (left) reveal two separate conformational exchange processes on the millisecond time-scale in the two highlighted regions of the protein (right). To elucidate the functional interplay between conformational dynamics and membrane-anchoring of GABARAP in atomic detail we are investigating GABARAP both in its soluble cytosolic form as well as anchored to nanodiscs (right, schematic model not to scale), a particularly promising membrane mimetic for solution NMR spectroscopy.

Project leader:
Dr. Philipp Neudecker, undefined email, undefinedInstitute of Physical Biology

Researcher: Irina Apanasenkoundefined email

Responsible for the content: E-MailRedaktionsteam SFB 1208