 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 11, 2024 |
| Title |
Deep mutagenesis of PDGF-B reveals sequence constraints for homodimerization and high affinity receptor interactions |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
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.
|
| |
|
| Overall design |
The mature protein sequence of PDGF-B (S1 to A104; synthetic codon-optimized sequence) was expressed on the yeast surface fused downstream of Aga2p, with a C-terminal c-myc epitope tag. A single site-saturation mutagenesis (SSM) library was generated by overlap extension PCR, encoding all possible PDGF-B single amino acid substitutions. Yeast cultures expressing the library were incubated with a soluble extracellular fragment of PDGFRbeta fused to IgG Fc. Surface expressed ligand and bound receptor were detected with fluorescent antibodies targeting the c-myc tag and IgG Fc moiety. Yeast positive for expressed PDGF-B and showing either high or low bound PDGFRbeta were collected by fluorescence-activated cell sorting (FACS). Plasmid DNA was extracted from the cultures before and after sorting, from which the enrichment or depletion of all PDGF-B sequence variants was determined following Illumina sequencing.
|
| |
|
| Contributor(s) |
Procko E, Young HJ |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| |
| Submission date |
Mar 29, 2019 |
| Last update date |
Nov 11, 2024 |
| Contact name |
Erik Procko |
| E-mail(s) |
procko@illinois.edu
|
| Phone |
217-300-1454
|
| Organization name |
University of Illinois
|
| Department |
Biochemistry
|
| Lab |
RAL 318G
|
| Street address |
601 S Goodwin Ave
|
| City |
Urbana |
| State/province |
IL |
| ZIP/Postal code |
61801 |
| Country |
USA |
| |
|
| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
|
| Samples (6)
|
| GSM3690819 |
Naive PDGF-B library |
| GSM3690820 |
Wildtype PDGF-B: Control for sequencing errors |
| GSM3690821 |
PDGF-B library sorted for weak or absent binding to 30 nM PDGFRbeta-Fc |
| GSM3690822 |
PDGF-B library sorted for high binding to 30 nM PDGFRbeta-Fc |
| GSM3690823 |
PDGF-B library sorted for weak or absent binding to 50 nM PDGFRbeta-Fc |
| GSM3690824 |
PDGF-B library sorted for high binding to 50 nM PDGFRbeta-Fc |
|
| Relations |
| BioProject |
PRJNA529784 |
| SRA |
SRP189837 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE129019_enrichment_ratios_PDGF-B.xlsx |
226.5 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
Less...
More...