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Deep mutagenesis of PDGF-B reveals sequence constraints for homodimerization and high affinity receptor interactions

ABSTRACT: Platelet-derived growth factors (PDGF) are disulfide-bonded dimers that act on PDGF receptors (PDGFR) to stimulate cell proliferation, differentiation, migration, and/or function. There are multiple isoforms of both the ligand and receptor, which combined with the potential for heterodimerization, gives the system great complexity. Different ligands may be promiscuous or specific for receptor partners. To better understand how ligand sequence relates to receptor recognition, the mutational landscape of PDGF-B for binding to the high affinity receptor PDGFRbeta was experimentally determined by deep mutational scanning. A PDGF-B library containing all possible single amino acid substitutions was displayed on the surface of yeast and sorted for binding to the PDGFRbeta ectodomain. Following deep sequencing, the enrichment or depletion of all mutations was calculated as a proxy for relative activity. All PDGF-B cysteines forming three intra- and two interchain disulfides are highly conserved, indicating that PDGF-B is correctly folding into disulfide-linked dimers on the yeast surface. Indeed, several mutations at the ligand dimer interface are enriched and may enhance dimerization through increased hydrophobic packing or reduced electrostatic repulsion. Residues from the protruding PDGF-B subunit in contact with the receptor are tightly conserved, whereas interfacial residues from the receding subunit are mutationally tolerant. There are multiple enriched mutations at the receptor interface that the scan predicts increase PDGF-BB–PDGFRbeta affinity. The data may prove insightful for engineering PDGF ligands biased towards forming homodimers with increased receptor affinity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE129019 | GEO | 2024/11/11

REPOSITORIES: GEO

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Project description:Proteins function in crowded cellular environments in which they must bind to specific target proteins but also avoid binding to many other off-target proteins. In large protein families this task is particularly challenging because many off-target proteins have very similar structures. How this specificity of physical protein-protein interactions in cellular networks is encoded and evolves is not very well understood. Here we address the question of specificity-encoding by comprehensively quantifying the effects of all mutations in one protein, JUN, on its binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. Fitting a global thermodynamic model to the data reveals that most affinity changing mutations equally affect JUN’s propensity to bind to all its interaction partners. Mutations that alter the specificity of binding are much rarer. These specificity-altering mutations are, however, distributed throughout the JUN interaction interface. JUN’s interaction specificity is encoded by both positive determinants that promote on-target interactions and negative determinants that prevent off-target interactions. Indeed, about half of the specificity-defining residues in JUN have dual functions and both promote on-target binding and prevent off-target binding. Whereas nearly all mutations that alter specificity are pleiotropic and also alter the affinity of binding to all interaction partners, the converse is not true with mutations outside of the interface able to tune affinity without affecting specificity. Our results provide the first global view of how mutations in a protein affect binding to all its potential interaction partners and reveal the distributed encoding of specificity and affinity in an interaction interface. They also show how the modular architecture of coiled-coils provides an elegant solution to the challenge of optimising specificity and affinity in a large protein family.

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Deep mutagenesis of PDGF-B reveals sequence constraints for homodimerization and high affinity receptor interactions

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