of the Max - MDC

Structure and Membrane
Interaction of G-proteins
Oliver Daumke
(Helmholtz Fellow)
Guanine nucleotide binding proteins (G-proteins) are involved in a diverse range of cellular processes
including protein synthesis, sensual perception, vesicular transport and signal transduction
cascades. Whereas small G-proteins are molecular switches that cycle between an active GTPbound
form and an inactive GDP-bound form, large G-proteins of the dynamin superfamily are mechano-chemical
enzymes that use the energy of GTP hydrolysis to actively remodel membranes. Members
of both groups bind to membranes, and this interaction is crucial for their function. Our projects aim
to elucidate the interaction and reciprocal modulation of membranes and G-proteins using structural,
biochemical and cell-biological methods.
EHD as a molecular model for membrane
remodelling GTPases
Members of the dynamin superfamily are multi-domain proteins
with an N-terminal G-domain. Its founding member
dynamin oligomerises around the neck of clathrin-coated
vesicles and induces vesicle scission in a GTP hydrolysisdependent
manner. How this is achieved at the molecular
level is, however, completely unclear.
In this project, we want to establish the less characterised
EHD family as a model system to understand principles of
membrane remodelling in the dynamin superfamily.
EHDs comprise a highly conserved eukaryotic protein family
with four members (EHD1-4) in mammals and a single member
in C. elegans, D. melanogaster and many eukaryotic parasites.
The proteins have an N-terminal G-domain, followed
by a helical domain and a C-terminal EH-domain. The EHdomain
is known to interact with asparagine-proline-phenylalanine
(NPF) motifs of proteins involved in endocytosis.
EHDs can be found at vesicular and tubular structures in
vivo, and EHD family members have been shown to regulate
several trafficking pathways including the exit of cargo proteins
from the endocytic recycling compartment.
In the laboratory of Harvey McMahon at the LMB in
Cambridge,UK, we could show that mouse EHD2 binds with
low affinity to nucleotides, like other members of the
dynamin superfamily. Surprisingly, ATP rather than GTP was
bound. We demonstrated that EHD2 could bind to negatively
charged liposomes, and this binding resulted in the deformation
of the liposomes into long tubular structures (Figure
1a). EHD2 oligomerised in ring-like structures around the
tubulated liposomes. Furthermore, in the presence of liposomes,
the slow ATPase reaction of EHD2 was enhanced, which
is another typical feature of dynamin-related G-proteins.
We solved the crystal structure of EHD2 in the presence of a
non-hydrolysable ATP analogue (Figure 1b) and found structural
similarities to the G-domain of dynamin. EHD2 crystallised
as a dimer, in agreement with previous ultracentrifugation
analysis results, where dimerisation is mediated
via a highly conserved surface area in the G-domain. The
helical domains of the two EHD monomers are facing each
other, and we could show that the lipid-binding site is at
the tip of the helical domains. Thus, by dimerisation of the
G-domains, both helical domains create a highly curved
lipid interaction site. We further predicted the architecture
of the EHD2 oligomeric ring (Figure 1c). In this model,
approximately 20 EHD2 dimers assemble across the G-
domain with the lipid-binding site oriented towards the
tubulated liposome surface.
We continue work on this project to understand the exact
function of EHD2 at the membrane and the role of ATP
hydrolysis. Furthermore, we want to develop an inhibitor
molecule for EHD proteins to identify and inhibit the cellular
pathways in which EHD proteins are involved.
Structure and function of the GIMAP family
GIMAP GTPases comprise seven members in humans which
are predominantly expressed in cells of the immune system.
Some of the members localise to the mitochondrial membrane
and are proposed to regulate apoptosis by regulating
the entry of cytochrome c from the mitochondria into the
cytosol. We will clarify the exact function of this protein
family at the mitochondria and the interaction with membranes
using structural, biochemical and cell-biological
methods. These results will have implications for several
types of leukaemia in which GIMAP members are overexpressed.
Cancer Research 115