Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (Le-Nguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.     

The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.     

Acyclic permutants of naturally occurring cyclic proteins. Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple-stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the beta-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.     

Intracellular folding pathway of the cystine knot-containing glycoprotein hormone alpha-subunit

Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.     

Novel Inhibitor Cystine Knot Peptides from Momordica charantia

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.     

Oxidative folding and structural analyses of a Kunitz-related inhibitor and its disulfide intermediates: functional implications

Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase β with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The rate-limiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of two-disulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding reaction and the inhibition of tryptase β. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications.     

Disulfide bond rearrangement during formation of the chorionic gonadotropin beta-subunit cystine knot in vivo

The intracellular kinetic folding pathway of the human chorionic gonadotropin beta-subunit (hCG-beta) reveals the presence of a disulfide between Cys residues 38-57 that is not detected by X-ray analysis of secreted hCG-beta. This led us to propose that disulfide rearrangement is an essential feature of cystine knot formation during CG-beta folding. To test this, we used disulfide bond formation to monitor progression of intracellular folding intermediates of a previously uncharacterized protein, the CG-beta subunit of cynomolgous macaque (Macaca fascicularis). Like its human counterpart hCG-beta with which it shares 81% identity, macaque (m)CG-beta is a cystine knot-containing subunit that assembles with an alpha-subunit common to all glycoprotein hormone members of its species to form a biologically active heterodimer, mCG, which, like hCG, is required for pregnancy maintenance. An early mCG-beta folding intermediate, mpbeta1, contained two disulfide bonds, one between Cys34 and Cys88 and the other between Cys38 and Cys57. The subsequent folding intermediate, mpbeta2-early, was represented by an ensemble of folding forms that, in addition to the two disulfides mentioned above, included disulfide linkages between Cys9 and Cys57 and between Cys38 and Cys90. These latter two disulfides are those contained within the beta-subunit cystine knot and reveal that a disulfide exchange occurred during the mpbeta2-early folding step leading to formation of the mCG-beta knot. Thus, while defining the intracellular kinetic protein folding pathway of a monkey homologue of CG-beta, we detected the previously predicted disulfide exchange event crucial for CG-beta cystine knot formation and attainment of CG-beta assembly competence.     

Pathway of oxidative folding of bovine alpha-interferon: predominance of native disulfide-bonded folding intermediates

Bovine alpha-interferon (BoINF-alpha) is a single polypeptide protein containing 166 amino acids, two disulfide bonds (Cys1-Cys99 and Cys29-Cys138), and five stretches of alpha-helical structure. The pathway of oxidative folding of BoINF-alpha has been investigated here. Of the eight possible one- and two-disulfide isomers, only two nativelike one-disulfide isomers, BoINF-alpha (Cys1-Cys99) and BoINF-alpha (Cys29-Cys138), predominate as intermediates along the folding pathway. More strikingly, alpha-helical structures formed almost quantitatively before any detectable formation of a disulfide bond. This is demonstrated by the observation that fully reduced BoINF-alpha (starting material of oxidative folding) and reduced carboxymethylated BoINF-alpha both exhibit alpha-helical structure content indistinguishable form that of native BoINF-alpha. The folding mechanism of BoINF-alpha appears to be compatible with the framework model, in which secondary structures fold first, followed by docking (compaction) of preformed secondary structural elements yielding the native structure.     

The underlying mechanism for the diversity of disulfide folding pathways

The disulfide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding intermediates with native-like structures. Our laboratory has recently analyzed the folding pathway(s) of four 3-disulfide-containing proteins, including hirudin, potato carboxypeptidase inhibitor, epidermal growth factor, and tick anticoagulant peptide. Their folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1- and 2-disulfide intermediates and 2) the presence of 3-disulfide scrambled isomers as folding intermediates. To search for the underlying causes of these diversities, we conducted kinetic analyses of the reductive unfolding of these five proteins. The experiment of reductive unfolding was designed to evaluate the relative stability and interdependence of disulfide bonds in the native protein. It is demonstrated here that among these five proteins, there exists a striking correlation between the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway. A sequential reduction of the native disulfide bonds is associated with the presence of predominant intermediates with native- like structures.     

Diversity in the disulfide folding pathways of cystine knot peptides

Summary The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence in directing the folding pathway.     

Optimal oxidative folding of the novel antimicrobial cyclotide from Hedyotis biflora requires high alcohol concentrations

Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol-disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria.     

NMR structural characterization and computational predictions of the major intermediate in oxidative folding of leech carboxypeptidase inhibitor

The III-A intermediate constitutes the major rate-determining step in the oxidative folding of leech carboxypeptidase inhibitor (LCI). In this work, III-A has been directly purified from the folding reaction and structurally characterized by NMR spectroscopy. This species, containing three native disulfides, displays a highly native-like structure; however, it lacks some secondary structure elements, making it more flexible than native LCI. III-A represents a structurally determined example of a disulfide-insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We also show that theoretical approaches based on topological constraints predict with good accuracy the presence of this folding intermediate. Overall, the derived results suggest that, as it occurs with non-disulfide bonded proteins, native-like interactions between segments of secondary structure rather than the crosslinking of disulfide bonds direct the folding of LCI.     

Characterizing the unstructured intermediates in oxidative folding

A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-[Pt(en)(2)Cl(2)](2+) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.     

Oxidative folding of the cystine knot motif in cyclotide proteins

The cyclotides are a large family of plant proteins that have a cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Despite the apparent complexity of their cystine knot motif it is possible to efficiently fold these proteins, as exemplified by oxidative folding studies on the prototypic cyclotide, kalata B1. This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates.     

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a K_d of ca. 10⁻⁵ M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI''s interaction with a partly-folded protein, and the first to analyze this folding catalyst''s changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.     

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase.

The oxidative refolding of hen lysozyme has been studied by a variety of time-resolved biophysical methods in conjunction with analysis of folding intermediates using reverse-phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four-disulfide-containing fully native protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (approximately 30%) appears to fold more quickly via other three-disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des-[76-94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.     

Acceleration of oxidative protein folding by curcumin through novel non-redox chemistry

Curcumin, the major constituent of turmeric is a known antioxidant. We have examined the oxidative folding of the model four-disulfide-bond-containing protein bovine pancreatic ribonuclease A (RNase A) in its presence; results indicate that RNase A regeneration rate increases in a curcumin-dependent manner. Examination of the native tendency of the fully-reduced polypeptide and the stability of key folding intermediates suggests that the increased oxidative folding rate can be attributed to native-like elements induced within the fully-reduced polypeptide and the stabilization of native-like species by this non-redox-active natural product. Our results provide a template for the design of curcuminoid-based synthetic small-molecule fold catalysts that accelerate the folding of ER-processed proteins; this assumes significance given that nitrosative stress and dysfunction of the ER-resident oxidoreductase protein disulfide isomerise due to S-nitrosylation are factors associated with the pathogenesis of Alzheimer’s and Parkinson’s diseases.     

Major kinetic traps for the oxidative folding of leech carboxypeptidase inhibitor

The leech carboxypeptidase inhibitor (LCI) is a 66-amino acid protein, containing four disulfides that stabilize its structure. This polypeptide represents an excellent model for the study and understanding of the diversity of folding pathways in small, cysteine-rich proteins. The pathway of oxidative folding of LCI has been elucidated in this work, using structural and kinetic analysis of the folding intermediates trapped by acid quenching. Reduced and denatured LCI refolds through a rapid, sequential flow of one- and two-disulfide intermediates and reaches a rate-limiting step in which a mixture of three major three-disulfide species and a heterogeneous population of non-native four-disulfide (scrambled) isomers coexist. The three three-disulfide intermediates have been identified as major kinetic traps along the folding pathway of LCI, and their disulfide structures have been elucidated in this work. Two of them contain only native disulfide pairings, and one contains one native and two non-native disulfide bonds. The coexistence of three-disulfide kinetic traps adopting native disulfide bonds together with a significant proportion of fully oxidized scrambled isomers shows that the folding pathway of LCI features properties exhibited by both the bovine pancreatic trypsin inhibitor and hirudin, two diverse models with extreme folding characteristics. The results further demonstrate the large diversity of disulfide folding pathways.     

The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys(A20) and Cys(B19). We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.     

Protein folding and misfolding: mechanism and principles

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding.