Biochemical and structural studies on LRRK2 regulation.

• Applicant: Professor Dr. Stefan Knapp
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 407661177

• Final Report Year: 2023

Final Report Abstract

This project resulted in a comprehensive structure and function description of the C-terminal domains (RCKW) of the human protein kinase LRRK2, a central large signalling molecule deregulated in Parkinsons disease (PD). Our efforts crystallizing this protein failed using more homogeneous protein preparations, small molecules as well as protein based high affinity binders (DARPINs) that were identified in the framework of this project. However, the highly optimized recombinant protein resulted in structure determination by EM providing first insights into LRRK2 regulation on the structural level as well as the role of PD mutations. This triggered additional collaboration with the community working on LRRK2 and the combined effort led to 9 high profile publications with our group as authors. Phosphorylation studies showed no influence of PKA phosphorylation on recombinant truncated LRRK2. Using LRRK1 as a model for full length LRRK2, we identified phosphorylation sites leading to LRRK1 activation by releasing an inhibitors block. However, this mechanism seems LRRK1 specific as similar phosphorylation sites are absent in LRRK2. Further studies on full length LRRK2 and interaction partners such as 14-3-3 proteins are need to clarify the mechanism of LRRK2 activation in the future. We established a network of collaborating laboratories that will now take research on LRRK2 on the translational level. EM was now also established in our laboratory and we solved already a number of LRRK2 structures with small molecules for future rational optimizations of LRRK2 inhibitors, in particular small molecules with type-II binding modes that beaks interaction with LRRK2 binding partners in cells.

Publications

• Crystallizing the Parkinson’s Disease Protein LRRK2 Under Microgravity Conditions. Cold Spring Harbor Laboratory.
  Mathea, Sebastian; Baptista, Marco; Reichert, Paul; Spinale, April; Wu, Jian; Allaire, Marc; Fiske, Brian & Knapp, Stefan
  
• Kinase Domain Is a Dynamic Hub for Driving LRRK2 Allostery. Frontiers in Molecular Neuroscience, 13.
  Taylor, Susan S.; Kaila-Sharma, Pallavi; Weng, Jui-Hung; Aoto, Phillip; Schmidt, Sven H.; Knapp, Stefan; Mathea, Sebastian & Herberg, Friedrich W.
  
• Structure of LRRK2 in Parkinson’s disease and model for microtubule interaction. Nature, 588(7837), 344-349.
  Deniston, C. K.; Salogiannis, J.; Mathea, S.; Snead, D. M.; Lahiri, I.; Matyszewski, M.; Donosa, O.; Watanabe, R.; Böhning, J.; Shiau, A. K.; Knapp, S.; Villa, E.; Reck-Peterson, S. L. & Leschziner, A. E.
  
• Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2. Proceedings of the National Academy of Sciences, 118(23).
  Schmidt, Sven H.; Weng, Jui-Hung; Aoto, Phillip C.; Boassa, Daniela; Mathea, Sebastian; Silletti, Steve; Torres-Paris, Constanza; Hu, Junru; Wallbott, Maximilian; Komives, Elizabeth A.; Knapp, Stefan; Herberg, Friedrich W. & Taylor, Susan S.
  
• Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms. Biochemical Journal, 478(3), 553-578.
  Malik, Asad U.; Karapetsas, Athanasios; Nirujogi, Raja S.; Mathea, Sebastian; Chatterjee, Deep; Pal, Prosenjit; Lis, Pawel; Taylor, Matthew; Purlyte, Elena; Gourlay, Robert; Dorward, Mark; Weidlich, Simone; Toth, Rachel; Polinski, Nicole K.; Knapp, Stefan; Tonelli, Francesca & Alessi, Dario R.
  
• LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains. PLOS Biology, 20(2), e3001427.
  Weng, Jui-Hung; Aoto, Phillip C.; Lorenz, Robin; Wu, Jian; Schmidt, Sven H.; Manschwetus, Jascha T.; Kaila-Sharma, Pallavi; Silletti, Steve; Mathea, Sebastian; Chatterjee, Deep; Knapp, Stefan; Herberg, Friedrich W. & Taylor, Susan S.
  
• Nanobodies as allosteric modulators of Parkinson’s disease–associated LRRK2. Proceedings of the National Academy of Sciences, 119(9).
  Singh, Ranjan K.; Soliman, Ahmed; Guaitoli, Giambattista; Störmer, Eliza; von Zweydorf, Felix; Dal Maso, Thomas; Oun, Asmaa; Van Rillaer, Laura; Schmidt, Sven H.; Chatterjee, Deep; David, Joshua A.; Pardon, Els; Schwartz, Thomas U.; Knapp, Stefan; Kennedy, Eileen J.; Steyaert, Jan; Herberg, Friedrich W.; Kortholt, Arjan; Gloeckner, Christian Johannes & Versées, Wim
  
• PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain. Biochemical Journal, 479(18), 1941-1965.
  Malik, Asad U.; Karapetsas, Athanasios; Nirujogi, Raja S.; Chatterjee, Deep; Phung, Toan K.; Wightman, Melanie; Gourlay, Robert; Morrice, Nick; Mathea, Sebastian; Knapp, Stefan & Alessi, Dario R.
  
• Structure of LRRK1 and mechanisms of autoinhibition and activation. Cold Spring Harbor Laboratory.
  Reimer, Janice M.; Dickey, Andrea M.; Lin, Yu Xuan; Abrisch, Robert G.; Mathea, Sebastian; Chatterjee, Deep; Fay, Elizabeth J.; Knapp, Stefan; Daugherty, Matthew D.; Reck-Peterson, Samara L. & Leschziner, Andres E.