This title appears in the Scientific Report : 2014 

Conformational dynamics of the autophagy-related protein GABARAP on multiple time-scales
Möller, Christina
Ma, Peixiang / Schwarten, Melanie / Willbold, Dieter / Neudecker, Philipp (Corresponding Author)
Strukturbiochemie ; ICS-6
2014
2014
EUROMAR 2014, Zürich (Schweiz), 2014-06-29 - 2014-07-03
Poster
Signalling Pathways and Mechanisms in the Nervous System
Understanding the function of a protein usually requires knowledge of its tertiary structure and conformationaldynamics. NMR spectroscopy is a powerful tool for studying structure and dynamics on virtually all time-scales frompicoseconds to real time at atomic resolution. In particular, sub-nanosecond dynamics determine spin relaxationrates, whereas the biochemically often more relevant dynamics on the micro- to millisecond time-scale causes linebroadening, which can be quantified by Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments[1].The 117-residue GABAA receptor-associated protein (GABARAP) from H. sapiens is known to mediate vesicletransport and fusion events in autophagy and possibly also apoptosis [2]. To this end, GABARAP is enzymaticallylipid-conjugated to allow membrane anchoring, which has been reported to facilitate hemifusion of membranes uponoligomerization. Structure determination of GABARAP by NMR [3] and X-ray crystallography [4] suggested significantconformational heterogeneity, which is conserved in the yeast homologue Atg8 [2]. Intriguingly, crystallizationunder high salt conditions resulted in an alternate conformation in which the N-terminal region is associated withthe hydrophobic binding pockets of an adjacent subunit in the crystal [4]. Unfortunately, it remains unclear whetherthis alternate conformation indeed facilitates oligomerization during membrane fusion and/or tubulin polymerizationor is merely a crystallization artifact, and the conformational dynamics of GABARAP is still poorly understood.To gain insight, we have measured 15N longitudinal and transverse relaxation rates, {1H}15N heteronuclear NOEs,and 15N relaxation dispersion profiles at several different temperatures, which reveal conformational dynamics invarious regions of the tertiary structure. The C-terminal region is highly mobile on the nanosecond time-scale asindicated by low order parameters S2. CPMG RD experiments show two distinct conformational exchange processeson the millisecond time-scale. Specifically, resonances in the N-terminal helical subdomain exhibit two separate resonances(plus exchange cross-peaks in multidimensional experiments) as a result of slow to intermediate exchangeon a time-scale of several milliseconds between two conformations with similar equilibrium populations. By contrast,residues lining the hydrophobic binding pockets reveal a slightly faster exchange process with an excited statepopulation of about 1%. Investigation of the structural details of these conformational exchange processes via CPMGRD experiments on a variety of different nuclei is currently underway.References: [1] A. G. Palmer 2004, Chem. Rev. 104, 3623-3640. [2] M. Schwarten et al. 2010, Biochem. Biophys. Res. Commun.395, 426-431. [3] T. Stangler et al. 2002, J. Biol. Chem. 277, 13363-13366. [4] J. E. Coyle et al. 2002, Neuron 33, 63-74.(RD 717, Poster S. 334 von 438)