Monitoring intracellular protein interactions using NanoLuc ® Binary T echnology (NanoBiT™) Brock Binkowski, Christopher Eggers, Braeden Butler, Marie Schwinn, Michael Slater, Thomas Machleidt, Mei Cong, Keith Wood & Frank Fan Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399 1. Introduction 4. GPCR interaction with β-arrestin-2 7. Agonist-induced RTK dimerization 2. NanoBiT overview 5. RAF dimerization using stable expression 7. Quantifying agonist binding via dimerization 3. Real time kinetics of PPI interaction dynamics 6. Nuclear hormone receptor dimerization 9. Summary NanoBiT is extremely bright • Fusion partners can be expressed at very low levels, minimizing potential artifacts • 100-1,000 fold brighter than split firefly luciferase NanoBiT components are small & stable • LgBiT, 18 kDa; SmBiT, 11 amino acids • LgBiT evolved for increased structural stability, providing a stable fusion partner NanoBiT is reversible • Monitor both protein association and dissociation events in real time NanoBiT offers experimental flexibility • Monitor protein interaction dynamics at a single time point or continuously for 1-2 hours • Room temperature or 37 °C measurements • Validated in 96-, 384- & 1536-well formats Check http://www.promega.com/nanobit for new NanoBiT PPI expression vectors For more information on NanoBiT, please see ACS Chemical Biology, 11(2), p. 400-408 www.promega.com 5/2016 Corresponding author: [email protected] • LgBiT and SmBiT are fused to proteins A & B • A:B interaction facilitates LgBiT:SmBiT interaction, generating a bright luminescent enzyme • LgBiT:SmBiT with low affinity (K D = 190 μM), limiting non-specific association and reducing assay background • LgBiT:SmBiT interaction is reversible (kon = 500 M -1 sec -1 ; koff = 0.2 sec -1 ) • LgBiT evolved for increased structural stability making it a better fusion partner • Non-lytic assay format allows real-time measurements of protein interaction dynamics for 1-2 hrs 0 20 40 0 25 50 75 100 0.1 1 10 100 1,000 Time (min) NanoBiT, norm GloSensor, norm NanoBiT GloSensor ISO PRO FSK • Transient expression of SmBiT-PRKACA & LgBiT-PRKAR2A in HEK293 • Modulators of intracellular cAMP added sequentially at indicated time points • Inverse correlation for NanoBiT vs. GloSensor cAMP 22F (cAMP biosensor) • NanoBiT can monitor reversible PPIs in real time 0 20 40 0 5 10 15 20 Time (min) Normalized response ADRB2:ARRB2 AVPR2:ARRB2 2 2 Inactive PKA ATP Agonist 2 1 CA R2A CA R2A 1 2 R2A R2A cAMP 1 = SmBiT 2 = LgBiT 1 CA CA 1 Active PKA LgBiT SmBiT PM ARRB2 P P PM ARRB2 Agonist LgBiT SmBiT Internalization Transient or stable association for GPCR:ARRB2 ARRB2 P P SmBiT LgBiT • Transient expression of ADRB2-LgBiT/SmBiT- ARRB2 or AVPR2-SmBiT/LgBiT-ARRB2 in HEK293 cells • Saturating ISO or AVP added at time zero • Expected transient interaction seen for ADRB2:ARRB2 (class A receptor) • Expected stable interaction seen for AVPR2:ARRB2 (class B receptor) • NanoBiT does not interfere with endogenous biology + LgBiT 18 kDa SmBiT 11 amino acids Protein A Protein B A:B interaction Structural complementation gives a bright, luminescent enzyme • Stable expression of BRAF-LgBiT & CRAF-SmBiT via bi-directional CMV promoter • Recombinase-mediated, single copy integration into HEK293 genome • Expected rank order potency observed for BRAF inhibitors that promote dimerization • Z’ values >0.5 in 384-well format using manual dispensing • NanoBiT can be miniaturized to 384- & 1536- well formats Protein:protein interactions (PPIs) are essential to the cellular signal transduction pathways that contribute to cancer. Although numerous approaches exist to monitor PPIs in vitro, methods for intracellular detection have been more limited. We developed NanoLuc® Binary Technology (NanoBiT), a two-subunit system based on NanoLuc® luciferase that can be applied to the intracellular detection of PPIs. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acid peptide) subunits are expressed as fusions to proteins of interest, where PPI facilitates subunit complementation to give a bright, luminescent enzyme. Unlike related approaches where an enzyme or protein is simply split, LgBiT was independently optimized for structural stability and SmBiT was selected from a peptide library specifically for the PPI application. The result is a subunit pair that weakly associates (K D = 190 μM) yet still maintains 30% of the activity of full-length NanoLuc at saturation. In contrast to many split systems, the LgBiT:SmBiT interaction is reversible, allowing the detection of rapidly dissociating proteins. PPI dynamics can be followed in real-time in living cells using the Nano-Glo® Live Cell Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate. Advantages over split systems include better sensitivity, reversibility, fusion to a peptide or a small, structurally stable protein domain, real-time measurements using a non-lytic assay format, and subunits with reduced affinity for self-association. 0 10 20 30 0 2 4 6 8 Time (min) Normalized Response HER1:HER1 NRG1 EGF Vehicle 0 10 20 30 0 2 4 6 Time (min) Normalized Response HER1:HER2 NRG1 EGF Vehicle 0 10 20 30 0 2 4 6 8 Time (min) Normalized Response HER1:HER3 NRG1 EGF Vehicle 0 10 20 30 0 2 4 6 8 Time (min) Normalized Response HER2:HER3 NRG1 EGF Vehicle • The cytoplasmic domain of HER1, 2 & 3 was replaced with linker-BiT • Transient expression in U2OS cells • Saturating EGF or NRG1 added at time zero • The expected selectivity profile was seen for EGF and NRG1 • NanoBiT can monitor RTK homo- or heterodimerization in real time HER1 EGF HER1 EGF 0 10 20 30 0 1 2 3 4 Time (min) Normalized Response 1E-6 3.33E-7 1E-7 3.33E-8 1E-8 3.33E-9 1E-9 3.33E-10 1E-10 Vehicle [EGF] (g/ml) HER1:HER1 -11 -10 -9 -8 -7 -6 -5 0 1 2 3 4 log[EGF] (g/ml) Normalized Response EC50 = 4.0 ng/ml HER1:HER1 0 10 20 30 0 2 4 6 Time (min) Normalized Response 1E-6 3.33E-7 1E-7 3.33E-8 1E-8 3.33E-9 1E-9 3.33E-10 1E-10 Vehicle [NRG1] (g/ml) HER2:HER3 -11 -10 -9 -8 -7 -6 -5 0 2 4 6 log[NRG1] (g/ml) Normalized Response EC50 = 9.3 ng/ml HER2:HER3 • HER truncations used as described above • Transient expression in U2OS cells • Varying EGF or NRG1 added at time zero • CRC data at 7 minutes • Quantify agonist interaction with RTK via dimerization 0 20 40 60 80 10 3 10 4 10 5 10 6 Time (min) Luminescence (RLU) R1881 (100 nM) Vehicle R1881 • Transient expression of LgBiT-AR & AR-SmBiT in HEK293 cells • R1881 agonist added at time zero, which induces AR dimerization • CRC data plotted at 30 minutes • Validated for GR homodimerization as well • NanoBiT can monitor nuclear hormone receptor dimerization in real time 10 0 10 1 10 2 10 3 10 4 0 2 10 5 4 10 5 6 10 5 8 10 5 [Inhibitor] (nM) Luminescence (RLU) GDC0879 AZ628 PLX4720 Sorafenib ML786 1 2 3 10 3 10 4 10 5 10 6 Plate Luminescence (RLU) GDC0879 Vehicle Ras Ras P B-Raf C-Raf B-Raf C-Raf Active Inhibitor HER1 EGF HER1 EGF N D L N D L N D L Ag N D L -12 -11 -10 -9 -8 -7 -6 0 20 40 60 80 log[R1881] (M) Normalized Response