Fig. 4: HTT and HAP40 form a very stable non-covalent complex that withstands dissociation. | Communications Biology

Fig. 4: HTT and HAP40 form a very stable non-covalent complex that withstands dissociation.

From: Huntingtin structure is orchestrated by HAP40 and shows a polyglutamine expansion-specific interaction with exon 1

Fig. 4

a Raw native (left) and a deconvoluted zero-charged (right) spectrum of the HTT-HAP40 Q23 complex. b Mass profile of HTT-HAP40 complex obtained using mass photometry, showing that the complex is monodisperse. c Composite native top–down mass spectrum of the HTT-HAP40 complex demonstrating large (right of the precursor) and small (left of the precursor) dissociation products produced at the highest activation energy. The data reveal that N- and C-terminal fragments of HTT are eliminated from the HTT-HAP40 complex upon collisional activation, whereas the intact HAP40 remains bound. Small fragment peaks are coloured following domain colour convention for the HTT-HAP40 complex. d Upper mass profile represents deconvoluted masses (red) of the large dissociation products (c in red) experimentally obtained by activating HTT-HAP40 complexes in the gas phase using HCD. Mirrored fragment masses (purple) are obtained by subtracting masses of the small experimental fragments (c in purple) from the precursor mass. e Annotation of small fragments obtained at high-resolution settings and mapping to the sequence of HTT Q23. Red stars, diamonds, triangles, and squares in panels (c–e) denote distinct dissociation products of HTT-HAP40 and link them from c (raw peaks) to d (deconvoluted masses) to e (complementary low-mass fragments). f Energy-resolved plot of fragment abundances: HTT with HAP40 ejected (yellow), HTT upon release of C-terminal fragment y311 or y148 (purple). g Structure of HTT-HAP40 complex with eliminated regions highlighted and represented as mesh. Colour-coding is in accordance with the domain colour convention for HTT-HAP40. h Assessing HTT-HAP40 Q23 complex stability by measuring transition temperature using DSF in different buffer conditions with 300 mM NaCl. i Caspase6 digestion of HTT-HAP40 Q23 proteins assessed by SDS-PAGE and j analytical gel filtration. Peak fractions from gel filtration run on SDS-PAGE are indicated and SDS-PAGE identified cleavage products are indicated (*).

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