Comparative analysis of anti-polyglutamine Fab crystals grown on Earth and in microgravity.

Huntington's disease is one of nine neurodegenerative diseases caused by a polyglutamine (polyQ)-repeat expansion. An anti-polyQ antigen-binding fragment, MW1 Fab, was crystallized both on Earth and on the International Space Station, a microgravity environment where convection is limited. Once the crystals returned to Earth, the number, size and morphology of all crystals were recorded, and X-ray data were collected from representative crystals. The results generally agreed with previous microgravity crystallization studies. On average, microgravity-grown crystals were 20% larger than control crystals grown on Earth, and microgravity-grown crystals had a slightly improved mosaicity (decreased by 0.03°) and diffraction resolution (decreased by 0.2 Å) compared with control crystals grown on Earth. However, the highest resolution and lowest mosaicity crystals were formed on Earth, and the highest-quality crystal overall was formed on Earth after return from microgravity.

Anti-PolyQ Antibodies Recognize a Short PolyQ Stretch in Both Normal and Mutant Huntingtin Exon 1.

Huntington's disease is caused by expansion of a polyglutamine (polyQ) repeat in the huntingtin protein. A structural basis for the apparent transition between normal and disease-causing expanded polyQ repeats of huntingtin is unknown. The "linear lattice" model proposed random-coil structures for both normal and expanded polyQ in the preaggregation state. Consistent with this model, the affinity and stoichiometry of the anti-polyQ antibody MW1 increased with the number of glutamines. An opposing "structural toxic threshold" model proposed a conformational change above the pathogenic polyQ threshold resulting in a specific toxic conformation for expanded polyQ. Support for this model was provided by the anti-polyQ antibody 3B5H10, which was reported to specifically recognize a distinct pathologic conformation of soluble expanded polyQ. To distinguish between these models, we directly compared binding of MW1 and 3B5H10 to normal and expanded polyQ repeats within huntingtin exon 1 fusion proteins. We found similar binding characteristics for both antibodies. First, both antibodies bound to normal, as well as expanded, polyQ in huntingtin exon 1 fusion proteins. Second, an expanded polyQ tract contained multiple epitopes for fragments antigen-binding (Fabs) of both antibodies, demonstrating that 3B5H10 does not recognize a single epitope specific to expanded polyQ. Finally, small-angle X-ray scattering and dynamic light scattering revealed similar binding modes for MW1 and 3B5H10 Fab-huntingtin exon 1 complexes. Together, these results support the linear lattice model for polyQ binding proteins, suggesting that the hypothesized pathologic conformation of soluble expanded polyQ is not a valid target for drug design.