Structural studies of Huntington's Disease

Huntington’s disease (HD) is a dominantly-inherited neuropsychiatric disorder. Typically the symptoms begin in adulthood, slowly progressing from movement disorder to behavioural and cognitive disturbances, often manifested in depression and dementia. It has been known since 1993 that the disease is due to mutation of a single gene coding for huntingtin (HTT) that extends the poly-glutamine (poly-Q) repeats in the protein. Aggregation of poly-Q repeat fragments is considered to be the crucial step in a series of events that lead to neurodegeneration. Inhibiting the enzyme responsible for the cleavage of mutant HTT - thereby reducing the generation of the poly-Q fragments - could be one way of preventing neurodegeneration. Caspase-6 was identified as this processing enzyme. Structures of other members of the caspase family have been studied in great detail, yet caspase-6 had proven resistant to structural studies with only the crystal structure of the apo-enzyme in a pH-inactivated state solved.

CHDI Foundation, Inc., a biomedical research organization exclusively focused on developing disease-modifying therapies for HD, initiated a collaboration with BioFocus, a drug discovery provider, to solve the structure of the active caspase-6 and in complex with inhibitors. The collaboration set out to gain insights into features unique to caspase-6, with a view towards developing caspase-6 specific drugs. Using mosquito^® Crystal for automated low-volume crystal screening, BioFocus identified crystallization conditions for apo-caspase-6 in the active state (PDB ID 3P45), in complex with the reversible inhibitor Ac-VEID-CHO (PDB ID 3P4U), as well as in complex with the irreversible inhibitor Z-VAD-FMK (PDB ID 3QNW).

Optimization of the crystallization conditions of the irreversible inhibitor yielded plate-like crystals typically only 10-20 μm thick, which made the use of synchrotron light at Diamond Light Source crucial to gain sufficient optical resolution into the enzyme-inhibitor interaction.

The image shows an overlay of the structure of caspase-6 in complex with Z-VAD-FMK (yellow) and Ac-VEID-CHO (blue) shows the large rearrangement of the highlighted surface loops between the different complexes.

“The rapid access to the MX stations at the Diamond synchrotron helps us to deliver final structures to our clients within a few weeks of obtaining the first crystals.” said Dr Ilka Müller, Senior Scientist at BioFocus.

Surprisingly, and unlike the reversible inhibitor Ac-VEID-CHO, the irreversible inhibitor Z-VAD-FMK binds to caspase-6 in the inactivated conformational state, and in a binding mode not previously observed for any other caspase-inhibitor complex.

“The application of Diamond Light Source facilities streamlined our caspase-6 structural biology efforts. We were able to get a better insight into its unique overall topology at physiological pH. This information may prove to be useful in structure-based inhibitor design for caspase-6.” said Dr Alex Kiselyov, Director at CHDI Foundation.

Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation

Müller, I., Lamers, M.B., Ritchie, A.J., Dominguez, C., Munoz-Sanjuan, I. and Kiselyov, A.

Bioorg Med Chem Lett. (2011) 21, 5244-5247.