Abstract
Cyclophilin A (CypA) is a peptidyl-prolyl cis/trans-isomerase that is involved in multiple signaling events of eukaryotic cells. It might either act as a catalyst for prolyl bond isomerization, or it can form stoichiometric complexes with target proteins. We have investigated the linear sequence recognition code for CypA by phage display and found the consensus motif FGPXLp to be selected after five rounds of panning. The peptide FGPDLPAGD showed inhibition of the isomerase reaction and NMR chemical shift mapping experiments highlight the CypA interaction epitope. Ligand docking suggests that the peptide was able to bind to CypA in the cis- and trans-conformation. Protein Data Bank searches reveal that many human proteins contain the consensus motif, and several of these protein motifs are shown to interact with CypA in vitro. These sequences represent putative target sites for binding of CypA to intracellular proteins.
Highlights
Cyclophilin A (CypA)1 is a ubiquitously expressed protein that has been found in a variety of functional contexts
We have investigated the linear sequence recognition code for CypA by phage display and found the consensus motif FGPXLp to be selected after five rounds of panning
The peptide FGPDLPAGD was further investigated for its potential to inhibit catalysis of cis/trans-prolyl isomerization of the model substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide by CypA
Summary
CypA, cyclophilin A; GST, glutathione S-transferase; PPII helix, the left-handed polyproline II helix; Itk, interleukin-2-inducible T cell kinase; HIV-1, human immunodeficiency virus, type 1; PPIase, peptidyl-prolyl cis/trans-isomerase; SH3, Src homology 3. Binding of the X-P-dipeptide bond may result in stable complex formation or transient interaction and catalysis, depending on the sequence or conformational context of the critical proline. The sequence context for catalysis by CypA has been investigated in detail [8] and revealed no stringent requirements for the nature of the amino acid flanking the central proline. We identify the consensus sequence as FGPXLp and confirm the importance of the individual amino acids for the peptide FGPDLPAGD. The latter peptide is an active site inhibitor, and NMR spectroscopy shows that the binding epitope overlaps with the interaction site of cyclosporin A and other CypA ligands. We show that a phage display-derived peptide motif is bound by CypA in the context of the entire cytoplasmic domain of the T cell adhesion molecule CD2
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