Chemical shift assignment; Conformational properties; Focal adhesion kinase; Homodimerics; Tyrosine kinase; Focal Adhesion Protein-Tyrosine Kinases; Humans; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Phase Separation; Protein Conformation; Protein Domains; Biochemistry
Abstract :
[en] Protein tyrosine kinase 2 (Pyk2), also known as focal adhesion kinase 2, and focal adhesion kinase 1 (Fak1) are two related nonreceptor tyrosine kinases (hereafter referred to as FAKs). Here, we focused on characterizing a linker region of the FAK proteins (hereafter referred to as FAK KFL for Kinase FAT Linker), which in the case of Pyk2 has previously been shown to play a functional role in calcium sensing through its interaction with calmodulin. Using structural nuclear magnetic resonance spectroscopy, we provide chemical shift assignments for the FAK KFLs, defining their conformational properties. Analysis of the FAK KFL conformations revealed their predominantly disordered nature, except for well-defined segments with a significant tendency to form α-helices, which were modeled to form homodimeric interfaces. In addition, we showed that the FAK KFL segments form condensates in vitro under high crowding conditions. By directly comparing the conformational properties of the Pyk2 and Fak1 KFL domains and providing structural data, this study provides valuable insights into the structural basis of FAK KFL interactions. Furthermore, the results show that disordered segments in proteins within the focal adhesion complex undergo phase separation, a process of potential biological significance due to protein clustering.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Martin, Lucy ; CNRS EMR9002-BSI─Integrative Structural Biology, Lille, France ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Freitas, Daniela P. ; BioISI─Instituto de Biosistemas e Ciências Integrativas, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal ; Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal
Boll, Emmanuelle; CNRS EMR9002-BSI─Integrative Structural Biology, Lille, France ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Brans, Alain ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'Ingénierie des Protéines (CIP)
Mortelecque, Justine; CNRS EMR9002-BSI─Integrative Structural Biology, Lille, France ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Cantrelle, François-Xavier ; CNRS EMR9002-BSI─Integrative Structural Biology, Lille, France ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Dourlen, Pierre ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Gomes, Cláudio M. ; BioISI─Instituto de Biosistemas e Ciências Integrativas, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal ; Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal
Danis, Clément ; CNRS EMR9002-BSI─Integrative Structural Biology, Lille, France ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Landrieu, Isabelle ; CNRS EMR9002-BSI─Integrative Structural Biology, Lille, France ; Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, Lille, France
Language :
English
Title :
Conformation-Driven Phase Separation in the Linker Domain of Focal Adhesion Kinases
Schaller, M. D. Cellular Functions of FAK Kinases: Insight into Molecular Mechanisms and Novel Functions. J. Cell Sci. 2010, 123 (Pt 7), 1007–1013, 10.1242/jcs.045112
Girault, J. A.; Costa, A.; Derkinderen, P.; Studler, J. M.; Toutant, M. FAK and PYK2/CAKbeta in the Nervous System: A Link between Neuronal Activity, Plasticity and Survival?. Trends Neurosci 1999, 22 (6), 257–263, 10.1016/S0166-2236(98)01358-7
Lev, S.; Moreno, H.; Martinez, R.; Canoll, P.; Peles, E.; Musacchio, J. M.; Plowman, G. D.; Rudy, B.; Schlessinger, J. Protein Tyrosine Kinase PYK2 Involved in Ca(2+)-Induced Regulation of Ion Channel and MAP Kinase Functions. Nature 1995, 376 (6543), 737–745, 10.1038/376737a0
de Pins, B.; Mendes, T.; Giralt, A.; Girault, J.-A. The Non-Receptor Tyrosine Kinase Pyk2 in Brain Function and Neurological and Psychiatric Diseases. Front Synaptic Neurosci 2021, 13, 749001 10.3389/fnsyn.2021.749001
Giralt, A.; de Pins, B.; Cifuentes-Díaz, C.; López-Molina, L.; Farah, A. T.; Tible, M.; Deramecourt, V.; Arold, S. T.; Ginés, S.; Hugon, J.; Girault, J.-A. PTK2B/Pyk2 Overexpression Improves a Mouse Model of Alzheimer’s Disease. Exp. Neurol. 2018, 307, 62–73, 10.1016/j.expneurol.2018.05.020
Giralt, A.; Brito, V.; Chevy, Q.; Simonnet, C.; Otsu, Y.; Cifuentes-Díaz, C.; de Pins, B.; Coura, R.; Alberch, J.; Ginés, S.; Poncer, J.-C.; Girault, J.-A. Pyk2 Modulates Hippocampal Excitatory Synapses and Contributes to Cognitive Deficits in a Huntington’s Disease Model. Nat. Commun. 2017, 8, 15592, 10.1038/ncomms15592
Dourlen, P.; Fernandez-Gomez, F. J.; Dupont, C.; Grenier-Boley, B.; Bellenguez, C.; Obriot, H.; Caillierez, R.; Sottejeau, Y.; Chapuis, J.; Bretteville, A.; Abdelfettah, F.; Delay, C.; Malmanche, N.; Soininen, H.; Hiltunen, M.; Galas, M.-C.; Amouyel, P.; Sergeant, N.; Buée, L.; Lambert, J.-C.; Dermaut, B. Functional Screening of Alzheimer Risk Loci Identifies PTK2B as an in Vivo Modulator and Early Marker of Tau Pathology. Mol. Psychiatry 2017, 22 (6), 874–883, 10.1038/mp.2016.59
Hall, J. E.; Fu, W.; Schaller, M. D. Focal Adhesion Kinase: Exploring Fak Structure to Gain Insight into Function. Int. Rev. Cell Mol. Biol. 2011, 288, 185–225, 10.1016/B978-0-12-386041-5.00005-4
Schlaepfer, D. D.; Hauck, C. R.; Sieg, D. J. Signaling through Focal Adhesion Kinase. Prog. Biophys. Mol. Biol. 1999, 71 (3–4), 435–478, 10.1016/S0079-6107(98)00052-2
Momin, A. A.; Mendes, T.; Barthe, P.; Faure, C.; Hong, S.; Yu, P.; Kadaré, G.; Jaremko, M.; Girault, J.-A.; Jaremko, Ł.; Arold, S. T. PYK2 Senses Calcium through a Disordered Dimerization and Calmodulin-Binding Element. Commun. Biol. 2022, 5 (1), 800, 10.1038/s42003-022-03760-8
Dignon, G. L.; Best, R. B.; Mittal, J. Biomolecular Phase Separation: From Molecular Driving Forces to Macroscopic Properties. Annu. Rev. Phys. Chem. 2020, 71, 53–75, 10.1146/annurev-physchem-071819-113553
Wei, M.-T.; Elbaum-Garfinkle, S.; Holehouse, A. S.; Chen, C. C.-H.; Feric, M.; Arnold, C. B.; Priestley, R. D.; Pappu, R. V.; Brangwynne, C. P. Phase Behaviour of Disordered Proteins Underlying Low Density and High Permeability of Liquid Organelles. Nat. Chem. 2017, 9 (11), 1118–1125, 10.1038/nchem.2803
Case, L. B.; De Pasquale, M.; Henry, L.; Rosen, M. K. Synergistic Phase Separation of Two Pathways Promotes Integrin Clustering and Nascent Adhesion Formation. Elife 2022, 11, e72588 10.7554/eLife.72588
Wang, Y.; Zhang, C.; Yang, W.; Shao, S.; Xu, X.; Sun, Y.; Li, P.; Liang, L.; Wu, C. LIMD1 Phase Separation Contributes to Cellular Mechanics and Durotaxis by Regulating Focal Adhesion Dynamics in Response to Force. Dev. Cell 2021, 56 (9), 1313–1325.e7, 10.1016/j.devcel.2021.04.002
Luna-Vargas, M. P. A.; Christodoulou, E.; Alfieri, A.; van Dijk, W. J.; Stadnik, M.; Hibbert, R. G.; Sahtoe, D. D.; Clerici, M.; Marco, V. D.; Littler, D.; Celie, P. H. N.; Sixma, T. K.; Perrakis, A. Enabling High-Throughput Ligation-Independent Cloning and Protein Expression for the Family of Ubiquitin Specific Proteases. J. Struct Biol. 2011, 175 (2), 113–119, 10.1016/j.jsb.2011.03.017
Camilloni, C.; De Simone, A.; Vranken, W. F.; Vendruscolo, M. Determination of Secondary Structure Populations in Disordered States of Proteins Using Nuclear Magnetic Resonance Chemical Shifts. Biochemistry 2012, 51 (11), 2224–2231, 10.1021/bi3001825
Shen, Y.; Delaglio, F.; Cornilescu, G.; Bax, A. TALOS+: A Hybrid Method for Predicting Protein Backbone Torsion Angles from NMR Chemical Shifts. J. Biomol NMR 2009, 44 (4), 213–223, 10.1007/s10858-009-9333-z
Berjanskii, M. V.; Wishart, D. S. A Simple Method to Measure Protein Side-Chain Mobility Using NMR Chemical Shifts. J. Am. Chem. Soc. 2013, 135 (39), 14536–14539, 10.1021/ja407509z
Rekhi, S.; Garcia, C. G.; Barai, M.; Rizuan, A.; Schuster, B. S.; Kiick, K. L.; Mittal, J. Expanding the Molecular Language of Protein Liquid-Liquid Phase Separation. Nat. Chem. 2024, 16 (7), 1113–1124, 10.1038/s41557-024-01489-x
Garabedian, M. V.; Su, Z.; Dabdoub, J.; Tong, M.; Deiters, A.; Hammer, D. A.; Good, M. C. Protein Condensate Formation via Controlled Multimerization of Intrinsically Disordered Sequences. Biochemistry 2022, 61 (22), 2470–2481, 10.1021/acs.biochem.2c00250
Ramšak, M.; Ramirez, D. A.; Hough, L. E.; Shirts, M. R.; Vidmar, S.; Eleršič Filipič, K.; Anderluh, G.; Jerala, R. Programmable de Novo Designed Coiled Coil-Mediated Phase Separation in Mammalian Cells. Nat. Commun. 2023, 14 (1), 7973, 10.1038/s41467-023-43742-w
Ramirez, D. A.; Hough, L. E.; Shirts, M. R. Coiled-Coil Domains Are Sufficient to Drive Liquid-Liquid Phase Separation in Protein Models. Biophys. J. 2024, 123 (6), 703–717, 10.1016/j.bpj.2024.02.007
