SCIENTIFIC ORGANIZATION:
Saint-Petersburg State Polytechnical University, Laboratory of Molecular Neurodegeneration

REPORT FORM:
«Poster report»

AUTHOR(S)
OF THE REPORT:
Zhemkov V.A., Vali S., Kulminskaya A.A., Vlasova O.L., Bezprozvanny I.B. and Kim M.W.

TALKING POINTS:

Huntington’s disease (OMIM #143100) is a severe autosomal-dominant neurodegenerative disorder, which is caused by CAG triplet expansion in the first exon of huntingtin gene1. On the protein level this leads to polyglutamine elongation in the N-terminus of the protein. Currently there is a little understanding about normal function of huntingtin in the cell and the exact mechanisms of its cytotoxicity.

One of the hypotheses states that mutant huntingtin undergo a conformational switch and adopts a novel conformation that differs from a native one. This form is aggregation-prone and lead to deposition of intranuclear huntingtin inclusions2. The other possible alteration caused by expanded huntingtin is deranges in the interactions with huntingtin partners in cell3.

Studies of huntingtin aggregation in vitro and in vivo have shown that amino acid sequences flanking the polyglutamine tract can modulate its aggregation properties and cytotoxicity4. The very N-terminal 17 amino acid domain N17 is likely to enhance the aggregation of mutant huntingtin5–7. Design based on studies of protein-ligand interactions is an effective way for identification and optimization of molecules with drug-like properties. Recently we have identified a peptoid compound HNP1 that selectively binds to N17. The structural studies of N17/HNP1 complex will give valuable information and clues for optimization of the chemical structure of HNP1. However, crystallization and X-ray analysis of small peptides faces a lot of difficulties.

The purpose of this work was development of the system for co-crystallization of biotinylated huntingtin N17 (BioHttN17) domain with streptavidin (SA). For this purpose we tested different forms of streptavidin such as wild-type tetrameric streptavidin8 (tSA) and its chimeric monomeric form9 (mSA). We prepared tSA/BioHttN17 and mSA/BioHttN17 complexes and were able to obtain crystals of tSA/BioHttN17 of high diffraction quality. Based on experimental electron density map the partial model of BioHttN17 was built but the map was not well resolved. It can be said that HttN17 adopts a different from native conformation which can be explained by distortions due to crystal packing or the linker sequence between biotin group and peptide itself should be optimized to reduce conformational flexibility of the latter. As an alternative approach we co-crystallized BioHttN17 with monomeric streptavidin and were able to obtain first diffracting crystals of this complex.Recent structure of mSA have shown that molecules of monomeric streptavidin in crystals are less densely packed10.

To conclude, we have crystallized biotinylated N17 domain of huntingtin in complex with different forms of streptavidin. Further studies will show whether this method allows structural studies of N17 domain in complex with therapeutic ligands. In future the same technique can be applied to crystallization of other peptides of interest.

References:

1. Huntington, T. et al. A Novel Gene Containing a Trinucleotide That Is Expanded and Unstable on Huntington ’ s Disease Chromosomes. 72, 971–983 (1993).
   
   
2. Ross, C. A. Intranuclear Neuronal Inclusions : A Common Pathogenic Mechanism for Glutamine-Repeat Neurodegenerative Diseases ? Neuron 19, 1147–1150 (1997).
   
   
3. Williams, A. J. & Paulson, H. L. Polyglutamine neurodegeneration: protein misfolding revisited. Trends Neurosci. 31, 521–8 (2008).
   
   
4. Duennwald, M. L., Jagadish, S., Muchowski, P. J. & Lindquist, S. Flanking sequences profoundly alter polyglutamine toxicity in yeast. Proc. Natl. Acad. Sci. U. S. A. 103, 11045–50 (2006).
   
   
5. Rockabrand, E. et al. The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis. Hum. Mol. Genet. 16, 61–77 (2007).
   
   
6. Liebman, S. W. & Meredith, S. C. Protein folding: sticky N17 speeds huntingtin pile-up. Nat. Chem. Biol. 6, 7–8 (2010).
   
   
7. Morimoto, RI, Christen, Y. Protein Quality Control in Neurodegenerative Diseases. Springer 144 (Springer Berlin Heidelberg, 2013). doi:10.1007/978-3-642-27928-7
   
   
8. Wolf, J. The properties of Streptavidin, a Biotin-Binding Protein produced by Streptomycetes. Arch. Biochem. 106, 1–5 (1964).
   
   
9. Lim, K. H., Huang, H., Pralle, A. & Park, S. Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol. Bioeng. 110, 57–67 (2013).
   
   
10. Demonte, D., Drake, E. J., Lim, K. H., Gulick, A. M. & Park, S. Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins 81, 1621–33 (2013).