Structural and Biochemical Characterization of the Human Cyclophilin Family of Peptidyl-Prolyl Isomerases

Abstract

Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure∶function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3-D representations and animated transitions. Please note that a Web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins

“…Isothermal titration calorimetry experiments were performed on a VP-ITC Microcal calorimeter at 25°C, according to Davis et al (2010). Purified CsCyp was extensively dialyzed against 50 mM HEPES, pH 8.0, and 200 mM NaCl.…”

Antiviral drug discovery: broad-spectrum drugs from nature

“…Cyclophilin A (CypA) belongs to the family of peptidyl-prolyl-cis-trans isomerases (PPIase) and is involved in protein folding, trafficking, formation of multiprotein complexes (MPC) and other cellular functions 103,104 . CypA interacts with viral proteins supporting viral replication 69 .…”

Interaction of cyclophilin A with a novel binding protein, SR-25, and characterization of their expression pattern in Chinese hepatocellular carcinoma patients

“…Cyclophilins (Cyps) are a group of cellular proteins known as immunophilins, which display the enzymatic activity of peptidyl-prolyl isomerases (PPIases) (1). These enzymes catalyze the cis to trans conversion of proline-containing peptides (1,2).…”

“…These enzymes catalyze the cis to trans conversion of proline-containing peptides (1,2). CypA is a key member of the Cyp family, and was first shown to mediate the immunosuppressive function of cyclosporine (Cs) A through the formation of a CsA-CypA complex (3).…”