Structure of a Single-Chain Fv Bound to the 17 N-Terminal Residues of Huntingtin Provides Insights into Pathogenic Amyloid Formation and Suppression

Erwin De Genst,Dimitri Y Chirgadze,Fabrice A.C Klein,David C Butler,Dijana Matak-Vinković,Yvon Trottier,James S Huston,Anne Messer,Christopher M Dobson

doi:10.1016/j.jmb.2015.03.021

Abstract

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3–11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12–17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a β-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv–peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.

Highlights

Huntington's disease (HD) is the most prevalent of a set of human neurodegenerative disorders linked to expansion of polyglutamine tracts in specific proteins [1,2]
The antibody fragment C4 single-chain Fv antibody (scFv) has been shown to inhibit strongly the formation of intracellular inclusions of mutant forms of the huntingtin protein (mHTT)-exon1 fragments of huntingtin in cellular and animal models of HD [23,24,25]. These experiments were, conducted in complex cellular environments, and so we investigated the in vitro ability of the isolated C4 scFv protein to inhibit the aggregation of mHTT-exon1 protein fragments
When C4 scFv was added after the proteolytic cleavage of maltose binding protein (MBP), effectively all of the HTT-Ex1-Q46 peptide remained soluble for incubation times of at least 76 h, an effect similar to that of the well-known peptide inhibitor of polyQ aggregation, polyglutamine binding peptide 1, QBP1 [27], which we included in our experimental strategy as a positive control (Fig. 1)

Summary

Introduction

Huntington's disease (HD) is the most prevalent of a set of human neurodegenerative disorders linked to expansion of polyglutamine (polyQ) tracts in specific proteins [1,2]. The full-length protein associated with HD, huntingtin (HTT), is found predominantly in the cytosol. N-terminal proteolytic fragments of mutant forms of HTT (mHTT), which are characterized by aberrant expansions of the wild-type polyQ tract of N 36 residues, are found to accumulate as insoluble inclusions in cellular nuclei [3,4,5,6]. The sequence of the HTT-exon fragment can be divided into three regions: a 17-residue N-terminal region [HTT(1-17)], immediately followed by the polyQ tract of variable length and a proline-rich region at the C-terminal end of the peptide [11].

Methods

Results

Conclusion