A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

Abstract

Homorepeat (HR) proteins are involved in key biological processes and multiple pathologies, however their high‐resolution characterization has been impaired due to their homotypic nature. To overcome this problem, we have developed a strategy to isotopically label individual glutamines within HRs by combining nonsense suppression and cell‐free expression. Our method has enabled the NMR investigation of huntingtin exon1 with a 16‐residue polyglutamine (poly‐Q) tract, and the results indicate the presence of an N‐terminal α‐helix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing a labeled glutamine into a pathological version of huntingtin with 46 glutamines. This methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly‐Q‐related diseases. Our approach can be extended to other amino acids to investigate biological processes involving proteins containing low‐complexity regions (LCRs).

Interaction of Huntingtin Exon-1 Peptides with Lipid-Based Micellar Nanoparticles Probed by Solution NMR and Q-Band Pulsed EPR

“…1 The presence of a long polyQ stretch results in the rapid formation of polymorphic fibrils, 2 the rate of which is modulated by the presence of flanking regions (htt NT and the proline rich domain C-terminal to the polyQ sequence), as well as additional factors including binding to lipid membranes and the presence of reactive oxygen species. 3 Htt NT is known to target a variety of membrane-containing structures within cells, 4 undergoes a random coil to α -helix transition upon binding lipid membranes, 5 and mediates aggregation of polyQ on membrane surfaces.…”

Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

“…with amino acids, nucleotides, salts and an energy regeneration system ( Figure 2). [20,21,28,30,31] It is important to mention that, once used, the tRNA CUA cannotb er eloaded inside the CF reaction, making this process ao ne-off reaction.…”

“…Finally, atRNA with the appropriate anticodon (CUA) and the isotopically labeled canonicalamino acid (represented by agreen dot) is also added to the CF reaction.T his figure was adapted from ref. [20].C opyright 2018,Wiley-VCH. Figure 3.…”

“…[34] As aconsequence of this lability,t he yield of suppressed sample with respect to the total input of loaded tRNA CUA is relativelyl ow. [20,31] This inherent limitation has to be addressed in the future fort he efficient application of SSIL.…”

“…[18] This is the case for the 1 H, 15 Nh eteronuclear single quantum correlation ( 15 NHSQC) experiment in ah untingtin construct with 16 consecutive glutamines ( Figure 1C,E )a nd the androgen receptor,w here an elongated density of peaks appears in the 15 NHSQC due to the formation of at ransient a-helix. [18][19][20] Even under these circumstances, when the number of consecutive residues increases further,t he dispersion induced by structure formationi sn ot enough to yield isolated peaks ( Figure 1D,Ffor ah untingtin construct with 46 consecutive glutamines). As ac onsequence of this degeneracy of frequencies, it is impossible to obtain high-resolution information of LCRs and the structural bases of their biological function cannot be unveiled.…”