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,h owever their high-resolution characterization has been impaired due to their homotypic nature.T oo vercome this problem, we have developed astrategy 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 a1 6-residue polyglutamine (poly-Q) tract, and the results indicate the presence of an N-terminal ahelix at near neutral pH that vanishes towards the end of the HR. The generality of the strategy was demonstrated by introducing alabeled glutamine into apathological version of huntingtin with 46 glutamines.T his methodology paves the way to decipher the structural and dynamic perturbations induced by HR extensions in poly-Q-related diseases.O ur approach can be extended to other amino acids to investigate biological processes involving proteins containing low-complexity regions (LCRs).

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

“…When LCRs adopt a fully or partially folded structure, the chemical environment experienced by the nuclei is less efficiently averaged and some chemical dispersion is observed . This is the case for the 1 H, 15 N heteronuclear single quantum correlation ( 15 N HSQC) experiment in a huntingtin construct with 16 consecutive glutamines (Figure C, E) and the androgen receptor, where an elongated density of peaks appears in the 15 N HSQC due to the formation of a transient α‐helix . Even under these circumstances, when the number of consecutive residues increases further, the dispersion induced by structure formation is not enough to yield isolated peaks (Figure D, F for a huntingtin construct with 46 consecutive glutamines).…”

“…The loaded tRNA CUA is then added to a cell‐free (CF) system where the target protein is produced. CF is an in vitro protein synthesis method composed of a lysate containing the transcription‐translation machinery, normally obtained from E. coli cultures, which is supplemented with amino acids, nucleotides, salts and an energy regeneration system (Figure ) . It is important to mention that, once used, the tRNA CUA cannot be reloaded inside the CF reaction, making this process a one‐off reaction.…”

“…An important chemical feature of loaded tRNA CUA is the high susceptibility to hydrolysis of the phosphoester bond between the tRNA CUA and the amino acid . As a consequence of this lability, the yield of suppressed sample with respect to the total input of loaded tRNA CUA is relatively low . This inherent limitation has to be addressed in the future for the efficient application of SSIL.…”

“…We have recently demonstrated the use of SSIL to structurally characterize low‐complexity regions using the huntingtin protein as model protein . The N‐terminal region of huntingtin, also known as exon1 (httex1), is the causative agent of Huntington's disease (HD), a deadly neurodegenerative pathology .…”

“…The structural bases of this pathological threshold remain poorly understood due to the inherent problems that homo‐repeats pose to traditional NMR approaches. Using a GlnRS/tRNA CUA pair derived from yeast, five glutamines of an httex1 construct with 16 consecutive glutamines (H16) were isotopically labeled with yields that allowed the NMR investigation (≈10 μ m from 5 mL of CF reaction) . The resulting samples provided isolated peaks in the highly congested Poly‐Q region of the 15 N HSQC, as well as in the side chain region (Figure A, B).…”