Structural and biochemical characterisation of small GTPase Rab23 and clinical mutants

Rab23 is a small GTPase that plays critical roles in vesicle transport in cells, primary
cilia formation, and development. Mutations, such as M12K, C85R, and Y79del, on Rab23
are linked to Carpenter Syndrome, a developmental disorder characterized by deformed
skull, abnormal development of fingers and toes, and mental retardation. High levels of
Rab23 expression are also known to be associated with several forms of cancers.
Therefore, Rab23 is a potential drug target. Despite its importance, the molecular
mechanisms of Rab23 function and how its malfunctioning lead to disease development
remain unclear.
Like other small GTPases, Rab23 acts as a molecular switch. It is switched off when
GDP is bound and switched on when the bound GDP is exchanged with GTP. The
nucleotide exchange requires a guanine exchange factor (GEF). When switched on,
Rab23 can bind effectors for downstream signalling activation. To switch Rab23 off, a
GTPase activating protein (GAP) will stimulate the hydrolysis of GTP back to GDP.
Although these interactors have been biochemically identified, structural
characterisation of Rab23 structural complexes are still limited. To date, only 2
structures of the mouse Rab23-GDP complexes have been reported. The lack of
structural information on Rab23 limits thorough understanding of the molecular
mechanisms of Rab23 and disease development.
Preliminary data from our high-resolution crystal structures of the Carpenter
Syndrome-linked Y79del mutant showed structural distortion on the Switch II region,
and its inability to adopt an ‘on’ state conformation despite GTP analogue binding. Our
in vitro GTPase assay results showed that Rab23 Y79del had impaired GEF- and GAPstimulated activities while functional assays in cells demonstrated a loss of function.
Based on these data, we hypothesize that the alpha 2 helix of Switch II is essential for
the GTPase function and the Carpenter Syndrome mutations distorted the structural
integrity of this region, resulting in loss of function.
We propose to combine structural biology, biochemistry, and cell functional assays to
thoroughly characterize the structures and molecular mechanisms of Rab23 and its
clinical mutants. In this project, we will (1) perform structural studies on Rab23 mutants
and Rab23-interactor complexes; (2) analyse the in vitro biochemical and biophysical
activities of Rab23 mutants; and (3) verify the functional activities of Rab23 mutants in
cells. Results from this study will allow us to unveil the molecular mechanisms of Rab23,
which plays roles in oncogenesis and development, and aid future therapeutic design.