Native holdup (nHU) to measure binding affinities from cell extracts

Abstract

Characterizing macromolecular interactions is essential for understanding cellular processes, yet nearly all methods used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to quantify equilibrium binding constants and explore binding mechanisms of protein interactions from cell extracts. Compared to other pulldown-based assays, nHU requires less sample preparation and can be coupled to any analytical methods, such as western blotting (nHU-WB) or mass spectrometry (nHU-MS) as readouts. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. This challenges the unwritten paradigm stating that biophysical parameters like binding constants cannot be accurately determined from cells or cellular extracts. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

The Holdup Multiplex, an assay for high-throughput measurement of protein-ligand affinity constants using a mass-spectrometry readout

“…We also performed more than 600 Kd measurements either by split-Nanoluciferase Protein Complementation Assay, by SPR or by competitive fluorescent polarization, and the remarkable quantitative correlations between the dissociation constants measured by Holdup and by these orthogonal techniques demonstrated the accuracy of the Holdup assay 9,29,31,38 . In addition, we observed quantitative correlation between the Holdup measurements obtained on domain-motif interactions, and the enrichment of full-length proteins from cellular extracts in AP-MS experiments 9,33 .…”

“…Actually, the Holdup Multiplex could be applied to any kind of PPI involving motifs, domains or full-length proteins, provided that the polypeptides of the library can be individually quantified by mass-spectrometry. Alternatively, full-length protein binders can be quantified by massspectrometry from cellular extracts 33 . Here, we used a library of 1000 peptides, but in practice the achievable throughput is only limited by the LC-MS/MS setup, which is routinely able to quantify tens of thousands of peptides in classical bottom-up proteomics experiments.…”

“…The Holdup Multiplex is a quantitative comparative chromatographic retention assay based on our previously developed Holdup assay 9,[29][30][31][32][33]35 , and designed to drastically increase the achievable throughput by quantifying in one single experiment the affinities of thousands of protein-ligand interactions, spanning several orders of magnitude (Figure 1). Briefly, a library of n different ligands of interest is incubated either with a resin-bound polypeptide (whole protein or fragment), or with an empty, control resin.…”

“…We previously developed the Holdup assay that quantifies equilibrium binding affinities of PPIs with extremely high precision and dynamic range 9,[29][30][31][32] , and can reach unmatched experimental throughput when combined with mass spectrometry 33,34 . Here, we demonstrate how the method can be scaled to measure hundreds of protein-peptide affinities at unprecedented accuracy with only one single experiment, by developing and benchmarking a multiplexed version of this assay that uses a label-free quantitative mass-spectrometry strategy.…”

Uncovering the BIN1-SH3 interactome underpinning centronuclear myopathy

“…Here we investigate the biophysical consequences of several previously uncharacterized natural BIN1 SH3 domain variants and show that tentatively pathological variants are not only affecting the previously well-characterized interaction with DNM2, but also hundreds of other previously unknown BIN1 interactions. We showed this by charting an unbiased affinity interactomic map of the SH3 domain of BIN1 using a top-down affinity interactomic strategy, exploiting full advantages of our innovative experimental approaches (Gogl et al, 2022 ;Zambo et al, 2022 ). Using our recently developed native holdup approach we investigated the binding of BIN1 SH3 to nearly 7,000 FL proteins from total cell extracts, out of which we could quantify apparent dissociations constants for ca.…”