Verlag des Forschungszentrums Jülich
JUEL-4022
In the hereby presented work, the first available three-dimensional solution structure
of GABARAP was determined by multidimensional, heteronuclear NMR spectroscopy an
isotopically 15N and 15N/13C labeled protein. The structure is based an almost complete
assignment of all 1H, 15N and 13C resonances and 4577 experimental proton-proton
distance restraints. In the context of this work, a purification protocol for recombinant
GABARAP was established .
The three-dimensional fold of GABARAP is described by a four-stranded β-sheet,
wich two helices an either side . The β-sheet together with the two helices an the concave
side of the sheet correspond to an ubiquitin-like fold. The structure of GABARAP in
solution was determined in an ensemble of structures with a precision of 0.28 ± 0.04 Ä
r.m.s.-deviation of the protein backbone coordinates .
One part of the molecule exists in at least two different conformations that interchange
with each other an a time scale between 10 and 25 Hz.
Based an the special dynamical properties and topological position of Ile41, a new
putative ligand-bindung site could be proposed .
The structural information about GABARAP could be related to biological functions
of the protein . Amino- and carboxyl-terminal ends of the protein directly interact with
each other . The sidechain of Tyr115 ist integrated in a hydrophobic pocket and the
hydroxyl oxygen of this tyrosine phenolic ring is hydrogen-bonded to the backbone amide
nitrogen of Lys2 . In contrast to the crystal structures of GABARAP and the homologous
GATE-16 protein, the carboxyl terminus is an integral part of the globular and compact
fold of GABARAP. Modulation of the conformation of the carboxyl-terminus might be
of importance for ubiquitin-like processing and modification of the carboxyl-terminal
residues . Moreover, the conformation of the carboxyl-terminus might be modulated by
phosphorylation of Tyr115 or interaction with a PDZ domain containing protein, and
thereby possibly regulate the function of GABARAP.
Employing (15N-1H)-HSQC titration studies, it was shown, that a part of the
TM3/TM4 loop of GABAA receptor γ2L subunit, a phage display selected peptide,
and a part of the ShcB protein, interact with GABARAP wich low affinities at exactly
the Same region of the molecular surface of GABARAP. The results of this work suggest
that these interactions are based an formation of a pseudo-continuous β-sheet .
Stangler, Thomas
Die Lösungsstruktur des humanen GABAa-Rezeptor assoziierten Proteins GABARAP
XII, 102 S., 2003
The GABAA-receptor-associated protein (GABARAP) is a member of a family of intracellular
membrane trafficking and fusion proteins and has been implicated in postsynaptic
membrane targeting of GABAA receptors and modulation of GABAergic synapses.
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