New insights into the structure and assembly of nuclear lamins from chemical cross-linking and mass spectrometry
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Date
30/11/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Makarov, Alexandr
Metadata
Abstract
Now that the functioning of microtubules and the actin cytoskeleton has been worked out in
enormous detail, the next important task is defining the structure of intermediate filaments
that are far behind the other two major skeletal networks due to their inherent resistance to
most structural techniques. The evolution of novel structural approaches for flexible proteins
is making this possible now. In my thesis I will aim to elucidate the structure and assembly
principles of lamin A nuclear intermediate filament protein.
To study lamin A, I principally employed chemical cross-linking that allows the capturing of
full-length protein structures in solution. I combined this with mass spectrometry approaches
to identify cross-linked residues at the various stages of lamin A assembly that were
additionally tracked with SILAC labelling and rotary metal shadowing TEM.
Unlike
previous cross-linking studies on intermediate filaments I use a zero-length self-excluding
cross-linking agent EDC that is better tailored for investigation of the polar interactions
between multiple unstructured or otherwise flexible charged sequences of lamins.
Using this composite approach I interrogated lamin A dimeric and tetrameric assemblies. I
elucidated hinge-like properties of the L12 and found indications that L1 and the region
containing coil 2A and L2 and the beginning of coil 2B possess properties of linker-like
flexibility and of predicted linear α-helical bundle and could act as molecular springs or
compression buffers for the nuclear intermediate filaments.
Further I confirm the role of the N-terminal unstructured region in lamin A assembly and for
the first time show similar role for the C-terminal unstructured region flanking the rod
domain of lamin A. Collected data strongly supports the model where both positively
charged unstructured regions participate in extensive interaction with acidic rod termini and
act as molecular bridges between these in the head-to-tail interface, confirming the
uniformity of this principle between cytoplasmic and nuclear intermediate filaments.
Formation of these bridges requires conformational change likely happening due to proline
residues in the mitotic phosphorylation sites. Finally I suggest a mechanism of regulation of
the order of assembly unique to the nuclear intermediate filament where C-terminal
unstructured region blocks lateral interactions until it is tethered to the head-to-tail interface.
Collected data on the dynamic behaviour of the C-terminal unstructured region and its ability
to tether lamin A Ig domain may have far reaching implications for filament assembly and
regulation of binding of hundreds of lamin A partner proteins presenting an important step in
our understanding of relationship between lamin A structure and function and how altering
the former could lead to disease.