title: Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes.
abstract: 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.
authors.affiliation: Structural Biology Brussels, Free University of Brussels, Brussels, Belgium.
authors.forename: Romany N N
authors.initials: RN
authors.lastname: Abskharon
authors.forename: Sameh H
authors.initials: SH
authors.lastname: Soror
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Hassan
authors.initials: H
authors.lastname: El Hassan
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Legname
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: Alexandre
authors.initials: A
authors.lastname: Wohlkonig
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000378:metabolism; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011328:Prions / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis
chemical_list: D000936:Antigen-Antibody Complex; D010446:Peptide Fragments; D011328:Prions