Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads

Abstract

Weak, transient binding of globular protein domains to Short Linear Motifs (SLiMs) in disordered regions of other proteins drive cellular signaling. Mapping the energy landscape of such interactions is essential for deciphering signaling networks and developing therapeutic inhibitors, but is complicated by technical challenges associated with quantitatively measuring weak interactions in high-throughput. Here, we synthesize peptide libraries on spectrally encoded beads with each peptide sequence uniquely linked to a spectral code (MRBLE-pep), allowing high-throughput quantification of protein-peptide binding via imaging. Using computational modeling and MRBLEpep assays, we map the affinity landscape for human calcineurin (CN), a conserved protein phosphatase essential for the immune response and target of immunosuppressants, interacting with a known SLiM (PxIxIT). We find that PxIxIT recognition depends critically on flanking residues and is regulated by post-translational modifications. Using this information, we designed PxIxIT peptides with unprecedented affinity and therapeutic potential that strongly inhibit CN function in vivo.

Identifying New Substrates and Functions for an Old Enzyme: Calcineurin

“…Compromising the PxIxIT sequence or adding a peptide or small molecule that competes with native PxIxIT sequences for binding to calcineurin inhibits dephosphorylation of many substrates in vitro and in vivo (Aramburu et al 1999;Roy and Cyert 2009;Matsoukas et al 2015;Nguyen et al 2018).…”

“…Changing the PxIxIT affinity alters the Ca 2+ -concentration dependence of substrate dephosphorylation, showing that fine-tuning of this single interaction can modulate the output of Ca 2+ signaling in vivo (Czirják and Enyedi 2006;Roy et al 2007;Muller et al 2009). Recent analyses of PxIxIT peptide affinity using a novel microfluidic technology identified residues outside the PxIxIT core that influence affinity for calcineurin: basic residues at position 2 increase affinity; hydrophobic or acidic residues at position -1 increase or decrease affinity, respectively; and acidic or phosphorylated residues at position 9 increase affinity (Nguyen et al 2018). These insights promise to improve PxIxIT discovery and, ultimately, identification of new calcineurin substrates, anchoring proteins and regulators.…”

“…First, information about how specific sequence elements either positively or negatively affect affinity of these motifs would improve evaluation of potential motif instances. A recent quantitative study of PxIxIT-calcineurin affinities revealed the impact of nonconsensus residues, and provides valuable insights that can be applied to proteome-wide PxIxIT identification (see above; Nguyen et al 2018). Second, discovery of more calcineurin-binding peptides of both PxIxIT and LxVP types would expand the repertoires of these SLiMs and allow construction of a statistical model for each motif (i.e., a positionspecific scoring matrix (PSSM)), which includes weighted information for each residue in the motif.…”

Logomaker: Beautiful sequence logos in python

“…Logomaker thus fills a major need in the Python community for flexible logo-generating software. Indeed, preliminary versions of Logomaker have already been used to generate logos for multiple publications (Belliveau et al, 2018;Wong et al, 2018;Forcier et al, 2018;Nguyen et al, 2018;Barnes et al, 2019;Kinney and McCandlish, 2019). Logomaker is thoroughly tested, has minimal dependencies, and can be installed from PyPI by executing pip install logomaker at the command line.…”

Single point mutations can potentially enhance infectivity of SARS-CoV-2 revealed by in silico affinity maturation and SPR assay

“…The Pearson correlation coefficient matched well with the original benchmark. Moreover, 22 Huy et al used powerful technology (MRBLE-pep) with Rosetta Flex ddG protocol to correctly predict that I3 and I5 mutations decrease affinity of CN-binding peptides.…”