Crystallography of lamin A facilitated by chimeric fusions

Abstract

AbstractAll proteins of the intermediate filament (IF) family contain the signature central α-helical domain which forms a coiled-coil dimer. Because of its length, past structural studies relied on a ‘divide-and-conquer’ strategy whereby fragments of this domain were recombinantly produced, crystallized and analysed using X-rays. Here we describe a further development of this approach towards structural studies of nuclear IF protein lamin. To this end, we have fused lamin A fragments to short N- and C-terminal capping motifs which provide for the correct formation of parallel, in-register coiled-coil dimers. As the result, a chimeric construct containing lamin A residues 17-70 C-terminally capped by the Eb1 domain was solved to 1.83 Å resolution. Another chimera containing lamin A residues 327-403 N-terminally capped by the Gp7 domain was solved to 2.9 Å. In the latter case the capping motif was additionally modified to include a disulphide bridge at the dimer interface. We discuss multiple benefits of fusing coiled-coil dimers with such capping motifs, including a convenient crystallographic phasing by either molecular replacement or sulphur single-wavelength anomalous dispersion (S-SAD) measurements.