Write here any specific comment you might have about
experimental approaches and methods used in the study.
HTT is a very difficult molecule to work with and its large size
precludes use of traditional methods for conformational and structural
analysis i.e. NMR and X-ray crystallography. The methods used to assess
huntingtin structure and conformational flexibility in this study are
similar to those previously described inVijayvargia
et al 2016 eLIFE with marginal gains in resolution i.e. EM maps
at 30 A compared 9 A give only marginal gains with respect to revealing
structural details of this molecule.
It is not clear how GFP linked to the mobile exon1 region of the
huntingtin protein would be located in a single position in relation to
the globular structure of HTT, nor how this could be determined to be
GFP in a low resolution (15 A) volume. Therefore, reliance on this
method for volume orientation prior to modelling is risky in our opinion
and the authors should justify this methodology in more detail. Without
access to the data in the EMdb and PDB it is hard to comment further.
The SAXS analysis is not a novel approach and was recentlypublished by our
group. Although the in-line size-exclusion chromatography would help
to separate different oligomer species, the monomer and dimer forms of
HTT form overlapping peaks on a Superose6 column which would still
render the sample a mixture of oligomeric states. This could account for
the high Dmax values compared to Rg. NB: Dmax values are similar to our
own experimentally determined values for HTT and HTT-HAP40 where samples
were analysed without cross-linking. The inclusion of HTT-HAP40 complex
samples in the work by Jung et al would be a good control.
The phosphosite specific antibodies are a fantastic resource and we hope
they will be shared widely with the HD community, in addition to details
of their validation. A missed opportunity is mapping of the PTMs onto
the models generated to see if this would give any insight into
potential mechanisms of conformational changes in the protein molecule.
For example, S2116 resides close to the interface of C-HEAT and BRIDGE
domains, so phosphorylation could lead to a change in electrostatic
interaction of the domains and conformational change. Again, without
access to the data in the EMdb and PDB it is hard to comment further.
Phosphorylation mimic mutations for S2116 could also be investigated to
determine the effects of phosphorylation.
For the other phosphorylation sites (S434, S1181, S1201, S2114) that
were mutated to A, the authors should ensure that there are no
structural or stability changes in HTT as a result of the amino acid
changes. These studies would ensure that they are probing only changes
related to phosphorylation.