Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting

• Published in: Molecular cell 2021-01, Vol.81 (2), p.323-339.e11
• Main Authors: Komolov, Konstantin E., Sulon, Sarah M., Bhardwaj, Anshul, van Keulen, Siri C., Duc, Nguyen Minh, Laurinavichyute, Daniela K., Lou, Hua Jane, Turk, Benjamin E., Chung, Ka Young, Dror, Ron O., Benovic, Jeffrey L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Baculoviridae - genetics; Baculoviridae - metabolism; Binding Sites; calcium; Calcium - metabolism; Calmodulin - chemistry; Calmodulin - genetics; Calmodulin - metabolism; Cloning, Molecular; Crystallography, X-Ray; G protein-coupled receptor; G-Protein-Coupled Receptor Kinase 5 - chemistry; G-Protein-Coupled Receptor Kinase 5 - genetics; G-Protein-Coupled Receptor Kinase 5 - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; HEK293 Cells; Humans; hydrogen/deuterium exchange mass spectrometry; Kinetics; molecular dynamics; Molecular Dynamics Simulation; Phosphorylation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; protein kinase; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Sf9 Cells; Spodoptera; Substrate Specificity; Thermodynamics; X-ray crystallography
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2020.11.026

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5. [Display omitted] •CaM and GRK5 form a bipartite interface within a 1:1 complex•CaM triggers large conformational changes in GRK5•CaM activates GRK5-mediated phosphorylation of non-receptor substrates•The mechanism of GRK5 activation by CaM and GPCRs is largely conserved Komolov et al. use structural, computational, and functional approaches to characterize the mechanism of calmodulin-mediated activation of the G protein-coupled receptor kinase GRK5. Additional findings reveal that GRK5 activation and localization cooperate under control of calmodulin for selective targeting of substrates at the plasma membrane and cytoplasm.