Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes.

TitleCombining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes.
Publication TypeJournal Article
Year of Publication2011
AuthorsAbskharon, R. N. N., S. H. Soror, E. Pardon, H. El Hassan, G. Legname, J. Steyaert, and A. Wohlkonig
JournalProtein Eng Des Sel
Date Published2011 Sep
KeywordsAnimals, Antigen-Antibody Complex, Crystallization, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Humans, Mice, Peptide Fragments, Prions, Proteolysis

Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP(C)-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb_PrP_01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb_PrP_01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 Å, β = 101.96° and diffracted to 2.1 Å resolution using synchrotron radiation. Human PrP (90-231)/Nb_PrP_01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 Å, β = 96.23° and diffracted to 1.5 Å resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes.

Alternate JournalProtein Eng. Des. Sel.
PubMed ID21536542