title: Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.
abstract: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.
authors.affiliation: Department of Chemistry, Katholieke Universiteit Leuven, Belgium.
authors.forename: Renée
authors.initials: R
authors.lastname: Vancraenenbroeck
authors.forename: Evy
authors.initials: E
authors.lastname: Lobbestael
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Weeks
authors.forename: Sergei V
authors.initials: SV
authors.lastname: Strelkov
authors.forename: Veerle
authors.initials: V
authors.lastname: Baekelandt
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Taymans
authors.forename: Marc
authors.initials: M
authors.lastname: De Maeyer
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D009930:Organic Chemicals; D010300:Parkinson Disease / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D016415:Sequence Alignment
chemical_list: D009930:Organic Chemicals; D011994:Recombinant Proteins; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; C041772:empigen BB