The filamin A actin binding domain structure and function: implications for a gain-of-function mechanism for the otopalatodigital syndrome: a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
The filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is
homologous to that found in other F-actin binding proteins such as dystrophin.
Mutations in the filamin A gene cause a wide range of disease symptoms in humans
reflecting the diversity of the roles that filamin A has in cell structure and signalling
pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular
heterotopia (PVNH) generally results from the complete loss of filamin A protein, and
affects the central nervous system. The clinically separate otopalatodigital disorders
(OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of
function phenotype diseases. At the beginning of this work, there was very little
structural data available for the human filamins, and none for the crucial highly
conserved actin binding domain. This lack of structural data limited the interpretation of
the biochemical and genetic data and constrained our understanding of the disease
associated mutations that cluster in this domain. These studies aimed to provide insights
into the structure and mechanism of actin binding domains, and thus provide a better
understanding of the diseases caused when this domain is mutated.
A secondary structural analysis and crystal structures of the wildtype and OPD2
associated mutant ABDs were obtained. The overall fold of the three proteins was
equivalent as determined by circular dichroism spectroscopy and x-ray crystallography.
The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity,
accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2,
frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed
similar levels of filamin A compared to the control cells. In addition the OPD and
PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an
equivalent rate to control cells.
Together these results have allowed correlations to be developed between structure,
protein stability, actin affinity, cellular phenotype and the overall clinical phenotype.
Showing that, at least in one example, OPD2 may be due to an increased actin affinity
providing further evidence for a gain of function mechanism of OPD2.