Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant. Instead of increasing the concentration of diffusing molecules, which is inevitably limited by the optical diffraction limit, we employed an increased density of surface bait protein. The low occupancy of the surface baits permitted determination of the kinetics with single-molecule resolution. We used this approach to study a PPI network consisting of Ras and its downstream proteins including full-length Rafs and catalytic subunits of phosphoinositide 3-kinase.

Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.

G309 or S310 mutations on the HER2 extracellular domain II induce receptor activation. Clinically, S310F is most frequent among HER2 extracellular domain mutations and patients with the S310F mutation without HER2 amplification responded to trastuzumab with or without the pertuzumab combination. However, the ability of S310F mutant to form homodimers or heterodimers with wild-type HER2 and other HER receptors, or their reactivity to trastuzumab and pertuzumab treatments, has not been reported. We overexpressed S310F as well as G309A, G309E and S310Y HER2 mutants and tested their reactivity to trastuzumab and pertuzumab. All mutants reacted to trastuzumab, but S310F mutant did not react to pertuzumab along with S310Y or G309E mutants. Thereafter, we tested the effects of trastuzumab and pertuzumab on 5637 cell line expressing both wild-type HER2 and S310F mutant. The ligand-independent HER2 homodimerization blocking antibody, trastuzumab, did not inhibit the activation of the HER2 receptor, suggesting that the S310F HER2 mutant did not form homodimers or heterodimers with wild-type HER2. Because 5637 cells overexpressed the EGFR, the effects of cetuximab and gefitinib were determined, and both inhibited the activation of HER2 and significantly reduced cell growth. Because pertuzumab did not inhibit the phosphorylation of HER2 while it bound to wild-type HER2, EGFR-mediated phosphorylation is expected to occur on the S310F mutant. To confirm whether the S310F mutant HER2 retained its affinity to the EGFR, single molecule interaction analyses using TIRF microscopy were performed, which showed that S310F mutant successfully formed complexes with EGFR. In conclusion, HER2 S310F mutant can form an active heterodimer with the EGFR and it can be inhibited by cetuximab, but not by trastuzumab in combination with pertuzumab.

BH3 mimetics are protein-protein interaction (PPI) inhibitors that saturate anti-apoptotic proteins in the BCL2 family to lift the resistance of cancer cells to apoptosis, a prominent example of protein complex-targeting therapies1–4. Despite the remarkable success of the BH3 mimetic ABT-199 in treating hematological malignancies5–7, only a fraction of patients respond to ABT-1998–10and eventually develop resistance11, 12, necessitating the usage of predictive biomarkers for both initial responses and resistance development. We here used the single-molecule pull-down13, 14and co-immunoprecipitation (co-IP)15, 16platform to quantify more than 20 different types of PPI complexes using ∼1.2x106cells in total, revealing the latest rewired status of the BCL2-family PPI network. By comparing the obtained multi-dimensional data with BH3 mimetic efficacies determinedex vivo, we constructed an analysis model for ABT-199 efficacy. Our model designates the BCL2-BAX and BCLxL-BAK complexes to contrasting roles, with the former complex driving ABT- 199 efficacy and the latter contributing to resistance development. We then applied this model to assist in therapeutic decision-making for acute myeloid leukemia patients in a prospective manner. Our work demonstrates a capability for the extensive characterization of PPI complexes in clinical specimens, potentially opening up a new avenue of precision medicine for protein complex-targeting therapies.