Molecular design and optimization of hepatic cancer SLP76‐derived PLCγ1 SH3‐binding peptide with the systematic N‐substitution of peptide PXXP motif. Issue 12 (8th August 2019)

• Record Type: Journal Article
• Authors: Tang, Wenqing; Zhao, Zhiying; Wang, Chen; Ye, Tao; Yang, Biwei
• Is Part Of: Journal of molecular recognition. Volume 32:Issue 12(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2019-08-08
• Subjects: hepatic cancer -- N‐substituted amino acid -- peptide -- phospholipase Cγ1 -- protein‐peptide recognition -- rational design -- SH3 domain; Molecular recognition -- Periodicals; Models, Molecular -- Periodicals; Molecular Conformation -- Periodicals; Molecular Sequence Data -- Periodicals; Molecular Structure -- Periodicals; Carrier Proteins -- Periodicals; 572.8
• DOI: 10.1002/jmr.2806
• Languages: English
• ISSNs: 0952-3499

Abstract

Abstract: The phospholipase Cγ1 (PLCγ1) is essential for T‐cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP‐containing decapeptide segment ( 185 QPP VPP QRPM 194, termed as SLP76 185–194 peptide) of adaptor protein SLP76 following T‐cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76 185–194 PXXP motif with nonnatural N‐substituted amino acids, as the proline is the only endogenous N‐substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate‐sized N‐substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N‐substituted peptides as well as native SLP76 185–194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence‐based assays. Two N‐substituted peptides, SLP76 185–194 (N‐Leu187/N‐Gln190) and SLP76 185–194 (N‐Thr187/N‐Gln190), are designed to have high potency ( K d = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5‐fold and 3.4‐fold relative to native peptide ( K d = 5.7 ± 1.2 μM). Abstract : A PXXP‐containing decapeptide segment SLP76185–194 is derived from the hepatic cancer protein SLP76, which can only bind to PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. The SLP76185–194 peptide affinity can be improved by replacing its two key proline residues with non‐natural N‐substituted amino acids. The replacement would increase peptide flexibility, but can restore peptide activity by establishing additional interactions with the domain.