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. 2024 Jul 12;13:RP95397. doi: 10.7554/eLife.95397

Figure 3. Affinity measurements between the SH3 domain of BIN1 and full-length proteins from cell extracts using nHU-MS.

(A) Outline of the holdup assay and benchmarking of nHU. Holdup is a simple tool to measure the fraction bound of prey molecules. This prey solution can be either a single purified protein, or a complex mixture of molecules and the prey depletion can be monitored with a multitude of analytical approches, such as western blot, or mass spectrometry. Titration holdup experiments were used to further characterize the interactions of BIN1_SH3 and full-length DNM2 using either recombinant, purified DNM2, or total myoblast extract containing endogenous DNM2 as a prey. The two experiments show nearly identical binding affinity and partial activity. (The average and standard deviation of three nHU western blot experiments are shown.) (B) Results of single point nHU-MS experiments carried out with the SH3 domain of BIN1 and total Jurkat extracts. Interaction partners above the significance threshold (tan line) are colored in orange if putative PRMs were identified in their sequence and blue if not. (C) Measured depletion values were converted to affinities using the functions indicated below panel B, assuming a simple binding mechanism and 10 μM estimated bait concentration. The number of unique affinity measurements performed and the identified BIN1 interaction partners found in a single experiment/in all measurements are indicated below panel C. (D, E) We also performed nHU-MS experiments with a set of closely or distantly related SH3 domains and compared their affinity profiles with BIN1. This way, we could quantify that related SH3-domains, for example the one found in AMPH, show similarities in their affinity interactomes, displaying statistically significant correlation between the measured affinity constants. In contrast, unrelated SH3 domains, such as the one found in ABL1, bind targets with dissimilar affinities. A linear fit (grey line) and a 95% confidence band (black line) is shown on all affinity comparisons. The statistical significance of correlation was determined by two-tailed, unpaired T-test. See Figure 3—figure supplements 1–3 and Supplementary file 1 for further details. Mass spectrometry experiments were performed with three injection replicates.

Figure 3.

Figure 3—figure supplement 1. Quality control of purified DNM2.

Figure 3—figure supplement 1.

(A) FL human DNM2 was produced in insect cells and was purified on GST column, preloaded with GST-fused BIN1_SH3. DNM2 was eluted at acidic pH and the pooled elution fractions were dialized to a basic pH. (B) Malachite green GTPase activity assay was used to verify the catalytic activity of recombinant DNM2 (n = 3).
Figure 3—figure supplement 1—source data 1. Original SDS-PAGE for panel A.
Figure 3—figure supplement 2. Raw results of the titration nHU and titration HU experiments.

Figure 3—figure supplement 2.

(A) Titration HU-WB experiments were carried out using purified DNM2. (B) Titration nHU-WB experiments were carried out using myoblast extracts. The recovered supernatants were assayed using western blot with DNM2, PRC1 and GAPDH antibodies. The recovered supernatants were assayed using western blot with DNM2 antibody. All western blots were repeated three times and the measured luminescent signals overlaid with colorimetric images are shown.
Figure 3—figure supplement 2—source data 1. Original western blot image (overlayed with colorimetric image) of Membrane 1.
Figure 3—figure supplement 2—source data 2. Original western blot image (overlayed with colorimetric image) of Membrane 2.
Figure 3—figure supplement 2—source data 3. Original western blot image (overlayed with colorimetric image) of Membrane 3.
Figure 3—figure supplement 2—source data 4. Original western blot image (overlayed with colorimetric image) of Membrane 4, DNM2.
Figure 3—figure supplement 2—source data 5. Original western blot image (overlayed with colorimetric image) of Membrane 4, GAPDH.
Figure 3—figure supplement 2—source data 6. Original western blot image (overlayed with colorimetric image) of Membrane 5, DNM2.
Figure 3—figure supplement 2—source data 7. Original western blot image (overlayed with colorimetric image) of Membrane 5, GAPDH.
Figure 3—figure supplement 2—source data 8. Original western blot image (overlayed with colorimetric image) of Membrane 6, DNM2.
Figure 3—figure supplement 2—source data 9. Original western blot image (overlayed with colorimetric image) of Membrane 6, GAPDH.
Figure 3—figure supplement 2—source data 10. Original western blot image (overlayed with colorimetric image) of Membrane 7, PRC1.
Figure 3—figure supplement 2—source data 11. Original western blot image (overlayed with colorimetric image) of Membrane 7, GAPDH.
Figure 3—figure supplement 2—source data 12. Original Western blot image (overlayed with colorimetric image) of Membrane 8, PRC1.
Figure 3—figure supplement 2—source data 13. Original western blot image (overlayed with colorimetric image) of Membrane 8, GAPDH.
Figure 3—figure supplement 2—source data 14. Original western blot image (overlayed with colorimetric image) of Membrane 9, PRC1.
Figure 3—figure supplement 2—source data 15. Original western blot image (overlayed with colorimetric image) of Membrane 9, GAPDH.
Figure 3—figure supplement 3. Additional results of nHU-MS experiments.

Figure 3—figure supplement 3.

(A) Result of an independent single point nHU-MS experiment carried out with the SH3 domain of BIN1 measured on a different mass spectrometer (Orbitrap Elite). Below the volcano plot, the mapped proteomic space is shown of the two nHU measurements that were done using the two instruments (Orbitrap Elite and Orbitrap Exploris 480). On the top right, the statistical overlap of significant binders is shown, as well as their recall calculations. Below, the comparison of the independently measured BIN1 affinities is shown in the two measurements, differently coloring partners that were found to be significant in both measurement from partners that were found to be significant in only one measurement. (B–F) Results of single point nHU-MS experiments carried out with the SH3 domains of AMPH, ABL1, ARHGEF7, PRMT2, and OBSCN using total Jurkat extracts (left). Interaction partners that show deficiency in abundance above the significance threshold (tan line) are colored in orange in case we could identify putative class 1/2 PxxP motifs in their sequence and blue in case we cannot. Measured depletion values were converted to affinities using a 10 μM estimated bait concentration assuming simple binding mechanism (middle/right). (C, D, E) On panel C, D and E, an additional comparison is shown with the affinities of BIN1 (extreme right panel). A linear fit (grey line) and a 95% confidence band (black line) is shown on all affinity comparisons.