Using thermal scanning assays to test protein-protein interactions of inner-ear cadherins

Abstract

Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (KD) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for inner-ear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (Tm) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These Tm shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the Tm shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)- pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly two-fold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (KD from nM up to 100 μM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.

The Dynamic Strength of the Hair-Cell Tip Link Reveals Mechanisms of Hearing and Deafness

“…We found the mean lifetime of the interaction (1/koff) to be 71.1 ± 5.3 s (SEM, n = 18) and the on-rate to be 1.32 ± 0.5 x 10 5 M -1 s -1 (Fig. 2b) in reasonable agreement with our estimates and with published single-bond on-rates 32 . These values provide an empirical means of determining the effective concentration Ceff in the B1 state, yielding Ceff ~800 ± 400 µM, three orders of magnitude more concentrated than the equilibrium KD of a single-bond interaction 8,23 and sufficient for promoting rapid rebinding to the B2 state (Supplementary Discussion).…”

“…Our measurements of tip link single-bond kinetics at zero force match well with previously published results 8,23,32 , and appear to be independent of whether the protein was expressed in bacteria and re-folded (allowing no posttranslational modifications) or expressed in mammalian cells. Barring post-translational modifications that occur in hair cells and not in our HEK cells, and that might act to change the affinity of the bond interface, the tip link in hair cells is likely regulated by the same intrinsic single-bond kinetics as that measured in vitro.…”

“…The effective local concentration Ceff can be approximated by calculating the volume of a sphere with a radius equal to the length an individual EC1-2 binding domain (~10 nm) 8 , yielding Ceff ~400 µM (Supplementary Discussion). Using a single-bond kon of 6.2 ± 2.8 x 10 4 M -1 s -1 32 and a singlebond koff of 0.6 s -1 (Fig. 1g), we estimate the zero-force rebinding rate (B1B2) to be ~25 ± 14 s -1 , and the overall lifetime of the connection to be 17-52 seconds, much longer than the single-bond lifetime calculated above of 1.6 ± 0.4 seconds.…”

“…The human PCDH15 R113G mutation causes hearing loss without a vestibular or visual phenotype 49 . The R113 residue participates in the binding interface, and the R113G mutation reduces bond affinity in vitro 8,32 . To determine how this mutation disrupts function, we used polarized Fc-domain dimerization to introduce this mutation either heterozygously (R113G +/-) or homozygously (R113G +/+ ), and measured its strength with force spectroscopy (Fig.…”

An Adhesive Interface for the Non-Clustered δ1 Protocadherin-1 Involved in Respiratory Diseases

“…The bacterially expressed PCDH1 EC1-5 C375S C548S, EC1-5 C375S C548S K398E, and EC1-5 C375S C548S V115R fragments were purified as described above and used for differential scanning fluorimetry (DSF) 77–79 . Experiments were repeated three times using protein at 0.2 mg/ml for all constructs in buffer (20 mM TrisHCl [pH 8.0], 150 mM KCl, 50 mM NaCl, and 2 mM CaCl 2 ) mixed with SYPRO Orange dye (Invitrogen).…”