Design, synthesis, and evaluation of linear and cyclic peptide ligands for PDZ10 of the multi-PDZ domain protein MUPP1.
PDZ10 is the 10th of 13 PDZ domains found within MUPP1, a cytoplasmic scaffolding protein first identified as an endogenous binding partner of serotonin receptor type 2c (5HT2c). This association, as with those of several other interacting proteins that have subsequently been identified, is mediated through the C-terminal tail of the PDZ domain partner. Using isothermal titration calorimetry (ITC), we measured the thermodynamic binding parameters [changes in Gibbs free energy (DeltaG), enthalpy (DeltaH) and entropy (TDeltaS)] of the isolated PDZ10 domain for variable-length N-acetylated peptides from the 5HT2c serotonin receptor C-terminal sequence, as well as for octapeptides of eight other putative partner proteins of PDZ10 (5HT2a, hc-kit, hTapp1, mTapp2, TARP, NG2, claudin-1, and HPV-18 E6). In length dependence studies of the 5HT2c sequence, the maximal affinity of the peptides leveled off rapidly and further elongation did not significantly improve the dissociation constant (Kd) of 11 microM observed with the pentapeptide. Among the native partners of PDZ10, octapeptides derived from the hc-kit and 5HT2c proteins were the strongest binders, with Kd values of 5.2 and 8.5 microM, respectively. The heat capacity change (DeltaCp) for the 5HT2c octapeptide was determined to be -94 cal/mol, and a calculated estimate indicates burial of polar and apolar surface areas in equal measure upon ligand binding. Peptides with phosphoserine at either the P-1 or P-2 position experienced decreased affinity, which is in accord with the hypothesis that reversible phosphorylation is a possible mechanism for regulating PDZ domain-mediated interactions. Additionally, two conformationally constrained side chain-bridged cyclic peptide ligands were also designed, prepared, evaluated by ITC, and shown to bind PDZ10 primarily through a favorable change in entropy.
Sharma
Sudhir C
SC
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Rupasinghe
Chamila N
CN
Parisien
Rachel B
RB
Spaller
Mark R
MR
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D016384:Consensus Sequence; D015195:Drug Design; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D054731:PDZ Domains / Q000187:drug effects; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D044405:Receptor, Serotonin, 5-HT2C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; C491749:Mpdz protein, rat; D010455:Peptides; D010456:Peptides, Cyclic; D044405:Receptor, Serotonin, 5-HT2C; D011993:Recombinant Fusion Proteins