Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism.
ABSTRACT: Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.
Project description:Peptidyl prolyl cis-trans isomerases (PPIases) are ubiquitous enzymes in biology that catalyze the cis-trans isomerization of the proline imide peptide bond in many cell signaling pathways. The local change of the isomeric state of the prolyl peptide bond acts as a switching mechanism in altering the conformation of proteins. A complete understanding of the mechanism of PPIases is still lacking, and current experimental techniques have not been able to provide a detailed atomistic picture. Here we have carried out several accelerated molecular dynamics simulations with explicit solvent, and we have provided a detailed description of cis-trans isomerization of the free and cyclophilin A-catalyzed process. We show that the catalytic mechanism of cyclophilin is due mainly to the stabilization and preferential binding of the transition state that is achieved by a favorable hydrogen bond interaction with a backbone NH group. We also show that the substrate in the transition state interacts more favorably with the enzyme than the cis isomer, which in turn interacts more favorably than the trans isomer. The stability of the enzyme-substrate complex is directly correlated with the interaction the substrate makes with a highly conserved arginine residue. Finally, we show that catalysis is achieved through the rotation of the carbonyl oxygen on the N-terminal of the prolyl peptide bond in a predominately unidirectional fashion.
Project description:Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases in vitro using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the PPIB gene), FKBP13 (FKBP2), FKBP19 (FKBP11), FKBP22 (FKBP14), FKBP23 (FKBP7), and FKBP65 (FKBP10), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen in vitro and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER.
Project description: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.
Project description:Initially discovered in the context of immunomodulation, peptidyl-prolyl cis/trans isomerases (PPIases) were soon identified as enzymes catalyzing the rate-limiting protein folding step at peptidyl bonds preceding proline residues. Intense searches revealed that PPIases are a superfamily of proteins consisting of three structurally distinguishable families with representatives in every described species of prokaryote and eukaryote and, recently, even in some giant viruses. Despite the clear-cut enzymatic activity and ubiquitous distribution of PPIases, reports on solely PPIase-dependent biological roles remain scarce. Nevertheless, they have been found to be involved in a plethora of biological processes, such as gene expression, signal transduction, protein secretion, development, and tissue regeneration, underscoring their general importance. Hence, it is not surprising that PPIases have also been identified as virulence-associated proteins. The extent of contribution to virulence is highly variable and dependent on the pleiotropic roles of a single PPIase in the respective pathogen. The main objective of this review is to discuss this variety in virulence-related bacterial and protozoan PPIases as well as the involvement of host PPIases in infectious processes. Moreover, a special focus is given to Legionella pneumophila macrophage infectivity potentiator (Mip) and Mip-like PPIases of other pathogens, as the best-characterized virulence-related representatives of this family. Finally, the potential of PPIases as alternative drug targets and first tangible results are highlighted.
Project description:Peptidyl-proline isomerases (PPIases) are a chaperone superfamily comprising the FK506-binding proteins (FKBPs), cyclophilins, and parvulins. PPIases catalyze the cis/trans isomerization of proline, acting as a regulatory switch during folding, activation, and/or degradation of many proteins. These "clients" include proteins with key roles in cancer, neurodegeneration, and psychiatric disorders, suggesting that PPIase inhibitors could be important therapeutics. However, the active site of PPIases is shallow, solvent-exposed, and well conserved between family members, making selective inhibitor design challenging. Despite these hurdles, macrocyclic natural products, including FK506, rapamycin, and cyclosporin, bind PPIases with nanomolar or better affinity. De novo attempts to derive new classes of inhibitors have been somewhat less successful, often showcasing the "undruggable" features of PPIases. Interestingly, the most potent of these next-generation molecules tend to integrate features of the natural products, including macrocyclization or proline mimicry strategies. Here, we review recent developments and ongoing challenges in the inhibition of PPIases, with a focus on how natural products might inform the creation of potent and selective inhibitors.
Project description:The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.
Project description:AIP56 (apoptosis inducing protein of 56?kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-?B p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30?min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.
Project description:FK 506 and cyclosporin A are potent immunosuppressive compounds that inhibit T-cell activation by interfering with signal transduction. In vitro, FK 506 binds and inhibits the activity of FK 506-binding protein (FKBP), a peptidylprolyl rotamase (cis-trans isomerase). Cyclosporin A acts similarly on a different proline rotamase, cyclophilin. Experiments described here demonstrate genetically that FKBP is a target for FK 506 in vivo. We have isolated the gene encoding the FKBP proline rotamase (FPR1) from Saccharomyces cerevisiae. The encoded yeast protein is highly homologous with bovine and human FKBP and shares no homology with cyclophilin. Disruption of FPR1 and CPR1 (encoding cyclophilin) individually or in combination is not lethal; thus, either enzymatic proline rotamerization is not essential for life or an unknown proline rotamase can substitute for the missing enzymes. Overexpression or disruption of FPR1 confers resistance to growth inhibition by FK 506, suggesting that FKBP is a target for FK 506 in yeast. However, FKBP is only one of at least two targets because strains lacking FKBP are only partially resistant to FK 506.
Project description:Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by peptidylprolyl isomerases, a superfamily of molecular chaperones. Here, we provide atomic insight into a tug-of-war between cis/trans isomerization and molecular chaperone activity. Catalysis of proline isomerization by cyclophilin A lowers the energy barrier for ?-synuclein misfolding, while isomerase-binding to a separate, disease-associated protein region opposes aggregation. We further show that cis/trans isomerization outpowers the holding activity of cyclophilin A. Removal of the proline isomerization barrier through posttranslational truncation of ?-synuclein reverses the action of the proline isomerase and turns it into a potent molecular chaperone that inhibits protein misfolding. The data reveal a conserved mechanism of dual functionality in cis/trans isomerases and define its molecular determinants acting on intrinsically disordered proteins.
Project description:Peptidyl-prolyl isomerases (PPIases) catalyze intrinsically slow and often rate-limiting isomerization of prolyl-peptide bonds in unfolded or partially folded proteins, thereby speeding up the folding process and preventing misfolding. They often possess binding and chaperone domains in addition to the domain carrying the isomerization activity. Although generally, their substrates display no identity in their amino acid sequence upstream and downstream of the proline with 20 possibilities for each residue, PPIases are efficient enzymes. SlyD is a highly efficient PPIase consisting of an isomerase domain and an additional chaperone domain. The binding of peptide substrates to SlyD and its enzymatic activity depend to some extend on the proline-proximal residues, however, the impact of proline-distant residues has not been investigated so far. Here, we introduce a label-free NMR-based method to measure SlyD activity on different peptide substrates and analysed the data in the context of obtained binding affinities and several co-crystal structures. We show that especially charged and aromatic residues up to eight positions downstream and three positions upstream of the proline and outside the canonical region of similar conformations affect the activity and binding, although they rarely display distinct conformations in our crystal structures. We hypothesize that these positions primarily influence the association reaction. In the absence of the chaperone domain the isomerase activity strongly correlates with substrate affinity, whereas additional factors play a role in its presence. The mutual orientation of isomerase and chaperone domains depends on the presence of substrates in both binding sites, implying allosteric regulation of enzymatic activity.