Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Ventricular Rupture due to Myocardial Infarction without Obstructive Coronary Artery Disease.

An 87-year-old woman presenting with myocardial infarction and ST-segment elevation in the electrocardiogram suffered from pericardial effusion due to left ventricular rupture. After ruling out obstructive coronary artery disease and aortic dissection, she underwent cardiac surgery showing typical infarct-macerated myocardial tissue in situ. This case shows that even etiologically unclear and small-sized myocardial infarctions can cause life-threatening mechanical complications.

Incorporation of an Unnatural Amino Acid as a Domain-Specific Fluorescence Probe in a Two-Domain Protein.

The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid p-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein.

Structural and Functional Characterization of a Novel Family of Cyclophilins, the AquaCyps.

Cyclophilins are ubiquitous cis-trans-prolyl isomerases (PPIases) found in all kingdoms of life. Here, we identify a novel family of cyclophilins, termed AquaCyps, which specifically occurs in marine Alphaproteobacteria, but not in related terrestric species. In addition to a canonical PPIase domain, AquaCyps contain large extensions and insertions. The crystal structures of two representatives from Hirschia baltica, AquaCyp293 and AquaCyp300, reveal the formation of a compact domain, the NIC domain, by the N- and C-terminal extensions together with a central insertion. The NIC domain adopts a novel mixed alpha-helical, beta-sheet fold that is linked to the cyclophilin domain via a conserved disulfide bond. In its overall fold, AquaCyp293 resembles AquaCyp300, but the two proteins utilize distinct sets of active site residues, consistent with differences in their PPIase catalytic properties. While AquaCyp293 is a highly active general PPIase, AquaCyp300 is specific for hydrophobic substrate peptides and exhibits lower overall activity.

Long-Range Energetic Changes Triggered by a Proline Switch in the Signal Adapter Protein c-CrkII.

The signal adapter protein c-CrkII from chicken but not from human uses isomerization at Pro238 in the SH3C domain to regulate the activity of the SH3N domain. The different behavior of human and chicken c-CrkII originates from only two differences in sequence, at positions 239 after Pro238 and 272 in the N-Src loop of SH3C. We analyzed the kinetics of substrate binding to SH3N and an assay for its coupling with Pro238 isomerization in SH3C to identify the molecular path from Pro238 to the substrate binding site of SH3N. The trans→cis isomerization at Pro238 and a relocation of Phe239 re-organize the energetics of a hydrophobic cluster in the N-Src loop of SH3C and re-shape this region to optimize its interactions with SH3N. Concomitantly, the backbone becomes strained at Met272. We suggest that, in human c-CrkII, movement at position 239 and strain at position 272 are not tolerated because the ß-branched residues Ile239 and Val272 restrain the backbone mobility and thus destabilize the cis Pro238 form.

Prolyl isomerization and its catalysis in protein folding and protein function.

Prolyl isomerizations are intrinsically slow processes. They determine the rates of many protein folding reactions and control regulatory events in folded proteins. Prolyl isomerases are able to catalyze these isomerizations, and thus, they have the potential to assist protein folding and to modulate protein function. Here, we provide examples for how prolyl isomerizations limit protein folding and are accelerated by prolyl isomerases and how native-state prolyl isomerizations regulate protein functions. The roles of prolines in protein folding and protein function are closely interrelated because both of them depend on the coupling between cis/trans isomerization and conformational changes that can involve extended regions of a protein.

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures.

Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Prolyl isomerization as a molecular memory in the allosteric regulation of the signal adapter protein c-CrkII.

c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans → cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction.

Dimeric Structure of the Bacterial Extracellular Foldase PrsA.

Secretion of proteins into the membrane-cell wall space is essential for cell wall biosynthesis and pathogenicity in Gram-positive bacteria. Folding and maturation of many secreted proteins depend on a single extracellular foldase, the PrsA protein. PrsA is a 30-kDa protein, lipid anchored to the outer leaflet of the cell membrane. The crystal structure of Bacillus subtilis PrsA reveals a central catalytic parvulin-type prolyl isomerase domain, which is inserted into a larger composite NC domain formed by the N- and C-terminal regions. This domain architecture resembles, despite a lack of sequence conservation, both trigger factor, a ribosome-binding bacterial chaperone, and SurA, a periplasmic chaperone in Gram-negative bacteria. Two main structural differences are observed in that the N-terminal arm of PrsA is substantially shortened relative to the trigger factor and SurA and in that PrsA is found to dimerize in a unique fashion via its NC domain. Dimerization leads to a large, bowl-shaped crevice, which might be involved in vivo in protecting substrate proteins from aggregation. NMR experiments reveal a direct, dynamic interaction of both the parvulin and the NC domain with secretion propeptides, which have been implicated in substrate targeting to PrsA.

Control of protein function by prolyl isomerization.

BACKGROUND: Prolyl cis/trans isomerizations have long been known as critical and rate-limiting steps in protein folding. RESULTS: Now it is clear that they are also used as slow conformational switches and molecular timers in the regulation of protein activity. Here we describe several such proline switches and how they are regulated. CONCLUSIONS AND GENERAL SIGNIFICANCE: Prolyl isomerizations can function as attenuators and provide allosteric systems with a molecular memory. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Mia40 combines thiol oxidase and disulfide isomerase activity to efficiently catalyze oxidative folding in mitochondria.

Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein.

Phosphorylation and prolyl isomerization independently regulate the signal adapter function of CrkII.

The signaling protein CrkII switches between forms with high or low binding affinity. Both phosphorylation and native-state prolyl isomerization were suggested to regulate the transition between these forms. Here we analyzed how phosphorylation at Tyr222 and Tyr252 and the Pro238Ala substitution affect signal transfer of human and chicken CrkII to a downstream target. Human CrkII is regulated by phosphorylation only, but chicken CrkII is regulated by Pro238 trans→cis isomerization and by Tyr222 phosphorylation. Surprisingly, they act in an independent fashion. Apparently, the allosteric transition to a low-activity form can be induced by phosphorylation or prolyl isomerization located at distant sites in CrkII.

Mia40 is optimized for function in mitochondrial oxidative protein folding and import.

Mia40 catalyzes oxidative protein folding in mitochondria. It contains a unique catalytic CPC dithiol flanked by a hydrophobic groove, and unlike other oxidoreductases, it forms long-lived mixed disulfides with substrates. We show that this distinctive property originates neither from particular properties of mitochondrial substrates nor from the CPC motif of Mia40. The catalytic cysteines of Mia40 display unusually low chemical reactivity, as expressed in conventional pK values and reduction potentials. The stability of the mixed disulfide intermediate is coupled energetically with hydrophobic interactions between Mia40 and the substrate. Based on these properties, we suggest a mechanism for Mia40, where the hydrophobic binding site is employed to select a substrate thiol for forming the initial mixed disulfide. Its long lifetime is used to retain partially folded proteins in the mitochondria and to direct folding toward forming the native disulfide bonds.

Molecular determinants of a regulatory prolyl isomerization in the signal adapter protein c-CrkII.

The cellular CT10 regulator of kinase protein (c-CrkII) transmits signals from oncogenic tyrosine kinases to cellular targets. Nuclear magnetic resonance studies had suggested that in chicken c-CrkII a native state prolyl cis-trans isomerization is involved in signal propagation. Corresponding evidence for the closely related human c-CrkII was not obtained. Here we analyzed the kinetics of folding and substrate binding of the two homologues and found that cis-trans isomerization of Pro238 determines target binding in chicken but not in human c-CrkII. A reciprocal mutational analysis uncovered residues that determine the isomeric state at Pro238 and transmit it to the binding site for downstream target proteins. The transfer of these key residues to human c-CrkII established a regulatory proline switch in this protein, as well. We suggest that Pro238 isomerization extends the lifetime of the signaling-active state of c-CrkII and thereby functions as a long-term molecular storage device.

Mia40 targets cysteines in a hydrophobic environment to direct oxidative protein folding in the mitochondria.

Mia40 catalyses the oxidative folding of disulphide-containing proteins in the mitochondria. The folding pathway is directed by the formation of the first mixed disulphide between Mia40 and its substrate. Here, we employ Cox17 to elucidate the molecular determinants of this reaction. Mia40 engages initially in a dynamic non-covalent enzyme-substrate complex that forms and dissociates within milliseconds. Cys36 of Cox17 forms the mixed disulphide in an extremely rapid reaction that is limited by the preceding complex formation with Mia40. Cys36 reacts much faster than the three other cysteines of Cox17, because it neighbours three hydrophobic residues. Mia40 binds preferentially to hydrophobic regions and the dynamic nature of the non-covalent complex allows rapid reorientation for an optimal positioning of the reactive cysteine. Mia40 thus uses the unique proximity between its substrate-binding site and the catalytic disulphide to select a particular cysteine for forming the critical initial mixed disulphide.

Energetic communication between functional sites of the gene-3-protein during infection by phage fd.

To initiate infection of Escherichia coli, phage fd uses its gene-3-protein (G3P) to bind first to an F pilus and then to the TolA protein at the cell surface. G3P is normally auto-inhibited because a tight interaction between the two N-terminal domains N1 and N2 buries the TolA binding site. Binding of N2 to the pilus activates G3P by initiating long-range conformational changes that are relayed to the domain interface and to a proline timer. We discovered that the 23-28 loop of the N1 domain is critical for propagating these conformational signals. The analysis of the stability and the folding dynamics of G3P variants with a shortened loop combined with TolA interaction studies and phage infection experiments reveal how the contact between the N2 domain and the 23-28 loop of N1 is energetically linked with the interdomain region and the proline timer and how it affects phage infectivity. Our results illustrate how conformational transitions and prolyl cis/trans isomerization can be coupled energetically and how conformational signals to and from prolines can be propagated over long distances in proteins.

Generation of a highly active folding enzyme by combining a parvulin-type prolyl isomerase from SurA with an unrelated chaperone domain.

Parvulins are small prolyl isomerases and serve as catalytic domains of folding enzymes. SurA (survival protein A) from the periplasm of Escherichia coli consists of an inactive (Par1) and an active (Par2) parvulin domain as well as a chaperone domain. In the absence of the chaperone domain, the folding activity of Par2 is virtually abolished. We created a chimeric protein by inserting the chaperone domain of SlyD, an unrelated folding enzyme from the FKBP family, into a loop of the isolated Par2 domain of SurA. This increased its folding activity 450-fold to a value higher than the activity of SurA, in which Par2 is linked with its natural chaperone domain. In the presence of both the natural and the foreign chaperone domain, the folding activity of Par2 was 1500-fold increased. Related and unrelated chaperone domains thus are similarly efficient in enhancing the folding activity of the prolyl isomerase Par2. A sequence analysis of various chaperone domains suggests that clusters of exposed methionine residues in mobile chain regions might be important for a generic interaction with unfolded protein chains. This binding is highly dynamic to allow frequent transfer of folding protein chains between chaperone and catalytic domains.

Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD.

SlyD is a bacterial two-domain protein that functions as a molecular chaperone, a prolyl cis/trans isomerase, and a nickel-binding protein. This review summarizes recent findings about the molecular enzyme mechanism of SlyD. The chaperone function located in one domain of SlyD is involved in twin-arginine translocation and increases the catalytic efficiency of the prolyl cis/trans isomerase domain in protein folding by two orders of magnitude. The C-terminal tail of SlyD binds Ni2+ ions and supplies them for the maturation of [NiFe] hydrogenases. A combined biochemical and biophysical analysis revealed the molecular basis of the delicate interplay of the different domains of SlyD for optimal function.

The prolyl isomerase SlyD is a highly efficient enzyme but decelerates the conformational folding of a client protein.

Folding enzymes often use distinct domains for the interaction with a folding protein chain and for the catalysis of intrinsically slow reactions such as prolyl cis/trans isomerization. Here, we investigated the refolding reaction of ribonuclease T1 in the presence of the prolyl isomerase SlyD from Escherichia coli to examine how this enzyme catalyzes the folding of molecules with an incorrect trans proline isomer and how it modulates the conformational folding of the molecules with the correct cis proline. The kinetic analysis suggests that prolyl cis → trans isomerization in the SlyD-bound state shows a rate near 100 s(-1) and is thus more than 10(4)-fold accelerated, relative to the uncatalyzed reaction. As a consequence of its fast binding and efficient catalysis, SlyD retards the conformational folding of the protein molecules with the correct cis isomer, because it promotes the formation of the species with the incorrect trans isomer. In the presence of ≥1 µM SlyD, protein molecules with cis and trans prolyl isomers refold with identical rates, because SlyD-catalyzed cis/trans equilibration is faster than conformational folding. The cis or trans state of a particular proline is thus no longer a determinant for the rate of folding.