RACK1 (receptor for activated C-kinase 1) interactions with spectrin repeat elements.
Receptor for activated C-kinase 1 (RACK1) is an intracellular scaffolding protein involved in a multitude of signalling pathways. The cytoskeleton is fundamental for intracellular cell signalling as it forms an interconnected network of regulatory proteins. Here, spectrin is a central component as it forms the actin-spectrin network that serves as docking surfaces for cellular components. The interaction between RACK1 and components of spectrin, the single spectrin repeats R16, R17 and the double spectrin repeat R1617 from the α-spectrin chain were investigated by biosensor technology and docking analysis. RACK1 associated only weakly to R16 (KD  = 1.0 ± 0.5 × 10(-6)  M), about 20 times stronger to R1617 (KD  = 5.3 ± 0.7 × 10(-8)  M) and 100 times stronger to R17 (KD  = 0.9 ± 0.3 × 10(-8)  M). Docking analysis showed that while R16 alone preferentially docked with its B-helix, R17 docked through its A-helix and BC loop. The double repeat and RACK1 mainly formed two different complex conformations. R1617 docked tangentially to the N/C-terminal of RACK1 or radially along a groove on the outer surface of RACK1. These configurations could account for the slight increase in entropic and the decrease in enthalpic interactions for the R1617-RACK1 interaction, compared with the interactions of RACK1 to the two single repeats. Our results suggest a mode of interaction that allows spectrin to attach to the N/C part of RACK through the inter-helical AB and BC loops and adopt a multitude of configurations in between the two limiting configurations.
Department of Molecular Biology, University of Bergen, HIB, Thormoehlens gt. 55, N-5020, Bergen, Norway.
Line M
LM
Myklebust
Department of Molecular Biology, University of Bergen, HIB, Thormoehlens gt. 55, N-5020, Bergen, Norway.
Ole
O
Horvli
Department of Molecular Biology, University of Bergen, HIB, Thormoehlens gt. 55, N-5020, Bergen, Norway.
Arnt J
AJ
Raae
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D062105:Molecular Docking Simulation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism*; D013816:Thermodynamics
D000596:Amino Acids; D009363:Neoplasm Proteins; D010455:Peptides; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D013049:Spectrin; D019204:GTP-Binding Proteins
RACK1; SPR (surface plasmon resonance); SR (spectrin repeats); binding kinetics; biosensor; thermodynamic analysis and molecular docking