The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2Mguanidine, compared to 5.6Mfor the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation.
P22 tailspike; protein folding; stability; mutation; ionic interaction
The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39°C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41°C, and at 42°C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.
Myelination of the central nervous system (CNS) is critical to vertebrate nervous systems for efficient neural signaling. CNS myelination occurs as oligodendrocytes terminally differentiate, a process regulated in part by the myelin regulatory factor, MYRF. Using bioinformatics and extensive biochemical and functional assays, we find that MYRF is generated as an integral membrane protein that must be processed to release its transcription factor domain from the membrane. In contrast to most membrane-bound transcription factors, MYRF proteolysis seems constitutive and independent of cell- and tissue-type, as we demonstrate by reconstitution in E. coli and yeast. The apparent absence of physiological cues raises the question as to how and why MYRF is processed. By using computational methods capable of recognizing extremely divergent sequence homology, we identified a MYRF protein domain distantly related to bacteriophage tailspike proteins. Although occurring in otherwise unrelated proteins, the phage domains are known to chaperone the tailspike proteins' trimerization and auto-cleavage, raising the hypothesis that the MYRF domain might contribute to a novel activation method for a membrane-bound transcription factor. We find that the MYRF domain indeed serves as an intramolecular chaperone that facilitates MYRF trimerization and proteolysis. Functional assays confirm that the chaperone domain-mediated auto-proteolysis is essential both for MYRF's transcriptional activity and its ability to promote oligodendrocyte maturation. This work thus reveals a previously unknown key step in CNS myelination. These data also reconcile conflicting observations of this protein family, different members of which have been identified as transmembrane or nuclear proteins. Finally, our data illustrate a remarkable evolutionary repurposing between bacteriophages and eukaryotes, with a chaperone domain capable of catalyzing trimerization-dependent auto-proteolysis in two entirely distinct protein and cellular contexts, in one case participating in bacteriophage tailspike maturation and in the other activating a key transcription factor for CNS myelination.
Membrane-bound transcription factors are synthesized as integral membrane proteins, but are proteolytically cleaved in response to relevant cues, untethering their transcription factor domains from the membrane to control gene expression in the nucleus. Here, we find that the myelin regulatory factor MYRF, a major transcriptional regulator of oligodendrocyte differentiation and central nervous system myelination, is also a membrane-bound transcription factor. In marked contrast to most well-known membrane-bound transcription factors, cleavage of MYRF appears to be unconditional. Surprisingly, this processing is performed by a protein domain shared with bacteriophages in otherwise unrelated proteins, where the domain is critical to the folding and proteolytic maturation of virus tailspikes. In addition to revealing a previously unknown key step in central nervous system myelination, this work also illustrates a remarkable example of evolutionary repurposing between bacteriophages and eukaryotes, with the same protein domain capable of catalyzing trimerization-dependent auto-proteolysis in two completely distinct protein and cellular contexts.
Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS–gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.
P22 tailspike protein (TSP); transient disulfide bond; assembly; disulfide shuffling; SDS
Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome, or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central β-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess co-translational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire β-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone β-helix domain occurs co-translationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.
Bacteriophage tailspike proteins act as primary receptors, often possessing endoglycosidase activity toward bacterial lipopolysaccharides or other exopolysaccharides, which enable phage absorption and subsequent DNA injection into the host. Phage CBA120, a contractile long-tailed Viunalikevirus phage infects the virulent Escherichia coli O157:H7. This phage encodes four putative tailspike proteins exhibiting little amino acid sequence identity, whose biological roles and substrate specificities are unknown. Here we focus on the first tailspike, TSP1, encoded by the orf210 gene. We have discovered that TSP1 is resistant to protease degradation, exhibits high thermal stability, but does not cleave the O157 antigen. An immune-dot blot has shown that TSP1 binds strongly to non-O157:H7 E. coli cells and more weakly to K. pneumoniae cells, but exhibits little binding to E. coli O157:H7 strains. To facilitate structure-function studies, we have determined the crystal structure of TSP1 to a resolution limit of 1.8 Å. Similar to other tailspikes proteins, TSP1 assembles into elongated homotrimers. The receptor binding region of each subunit adopts a right-handed parallel β helix, reminiscent yet not identical to several known tailspike structures. The structure of the N-terminal domain that binds to the virion particle has not been seen previously. Potential endoglycosidase catalytic sites at the three subunit interfaces contain two adjacent glutamic acids, unlike any catalytic machinery observed in other tailspikes. To identify potential sugar binding sites, the crystal structures of TSP1 in complexes with glucose, α-maltose, or α-lactose were determined. These structures revealed that each sugar binds in a different location and none of the environments appears consistent with an endoglycosidase catalytic site. Such sites may serve to bind sugar units of a yet to be identified bacterial exopolysaccharide.
The P22-like bacteriophages have short tails. Their virions bind to their polysaccharide receptors through six trimeric tailspike proteins that surround the tail tip. These short tails also have a trimeric needle protein that extends beyond the tailspikes from the center of the tail tip, in a position that suggests that it should make first contact with the host’s outer membrane during the infection process. The base of the needle serves as a plug that keeps the DNA in the virion, but role of the needle during adsorption and DNA injection is not well understood. Among the P22-like phages are needle types with two completely different C-terminal distal tip domains. In the phage Sf6-type needle, unlike the other P22-type needle, the distal tip folds into a “knob” with a TNF-like fold, similar to the fiber knobs of bacteriophage PRD1 and Adenovirus. The phage HS1 knob is very similar to that of Sf6, and we report here its crystal structure which, like the Sf6 knob, contains three bound L-glutamate molecules. A chimeric P22 phage with a tail needle that contains the HS1 terminal knob efficiently infects the P22 host, Salmonella enterica, suggesting the knob does not confer host specificity. Likewise, mutations that should abrogate the binding of L-glutamate to the needle do not appear to affect virion function, but several different other genetic changes to the tip of the needle slow down potassium release from the host during infection. These findings suggest that the needle plays a role in phage P22 DNA delivery by controlling the kinetics of DNA ejection into the host.
An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies. Amyloid deposits represent a related state associated with a variety of human diseases. The critical folding intermediates at the juncture of productive folding and the off-pathway aggregation reaction have been identified for the phage P22 tailspike and coat proteins. Though the parallel β coil tailspike is thermostable, an early intracellular folding intermediate is thermolabile. As the temperature of intracellular folding is increased, this species partitions to inclusion bodies, a kinetic trap within the cell. The earliest intermediates along the in vitro aggregation pathway, sequential multimers of the thermolabile folding intermediates, have been directly identified by native gel electrophoresis. Temperature-sensitive folding (tsf) mutations identify sites in the β coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pathway to inclusion bodies, rescuing the mutant chains. These mutants identify sites important for avoiding aggregation. Coat folding intermediates also partition to inclusion bodies as temperature is increased. Coat tsf mutants are suppressed by overexpression of the GroE chaperonin, indicating that the thermolabile intermediate is a physiological substrate for GroE. We suggest that many proteins are likely to have thermolabile intermediates in their intracellular folding pathways, which will be precursors to inclusion body formation at elevated temperatures and therefore substrates for heat shock chaperonins.
inclusion body; protein folding; chaperones; aggregation
The tailed bacteriophage φ29 has 12 “appendages” (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an “auto-chaperone” that aids trimerization. We also show that auto-cleavage of the C-terminal domain is a post-trimerization event that is followed by a unique ATP-dependent release. The post-translationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have similar maturation mechanism as is performed by φ29 gp12 for Bacillus subtilis.
X-ray crystallography; infection; tailspike; receptor
The Dr hemagglutinin of uropathogenic Escherichia coli is a fimbrial homopolymer of DraE subunits encoded by the dra operon. The dra operon includes the draB and draC genes, whose products exhibit homology to chaperone-usher proteins involved in the biogenesis of surface-located polymeric structures. DraB is one of the periplasmic proteins belonging to the superfamily of PapD-like chaperones. It possesses two conserved cysteine residues characteristic of the FGL subfamily of Caf1M-like chaperones. In this study we obtained evidence that DraB cysteines form a disulfide bond in a mature chaperone and have the crucial function of forming the DraB-DraE binary complex. Expression experiments showed that the DraB protein is indispensable in the folding of the DraE subunit to a form capable of polymerization. Accumulation of DraB-DraEn oligomers, composed of head-to-tail subunits and the chaperone DraB, was observed in the periplasm of a recombinant E. coli strain which expressed DraB and DraE (but not DraC). To investigate the donor strand exchange mechanism during the formation of DraE oligomers, we constructed a series of DraE N-terminal deletion mutants. Deletion of the first three N-terminal residues of a potential donor strand resulted in a DraE protein lacking an oligomerization function. In vitro data showed that the DraE disulfide bond was not needed to form a binary complex with the DraB chaperone but was essential in the polymerization process. Our data suggest that assembly of Dr fimbriae requires a chaperone-usher pathway and the donor strand exchange mechanism.
The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.
The host cell recognition protein of the Escherichia coli bacteriophage HK620 is a large homotrimeric tailspike that cleaves the O18A1 type O antigen. The crystal structure of HK620 tailspike determined in the apo and substrate-bound form is reported by Barbirz et al. in this issue of Molecular Microbiology. Lacking detectable sequence similarity, the fold and overall organization of the HK620 tailspike are similar to those of the tailspikes of the related phages P22 and Sf6. The substrate-binding site is intra-subunit in P22 and HK620 tailspikes, but inter-subunit in Sf6, demonstrating how phages can adapt the same protein fold to recognize different substrates.
tailspike; evolution; polysaccharide; hydrolase; bacteriophage
Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single-domain large pore, while a two-domain narrow pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large pore conformation requires that the protein be isolated from outer membranes with an intact disulfide bond and then adequately incubated in lipids at temperatures ≥ 26 °C to overcome the high energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines in the eighth β-barrel of the N-terminal domain by oligo-(R)-3-hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post-translational secretion across the SEC translocase in the inner membrane. After cleavage of the signal sequence, protein chaperones, such a Skp, DegP and SurA, guide OmpA across the periplasm to the BAM complex in the outer membrane. During this passage, a disulfide bond is formed between C290 and C302 by DsbA, and the hydrophobicity of segments of the C-terminal domain which are destined for incorporation as β-barrels in the outer membrane bilayer is increased by covalent attachment of oligo-(R)-3-hydroxybutyrates. With the aid of the BAM complex, OmpA is then assembled into the outer membrane as a single-domain large pore.
outer membrane protein A; protein folding; oligo-(R)-3-hydroxybutyrates; disulfide bond; protein targeting; Escherichia coli
Human γ-crystallins are long-lived unusually stable proteins of the eye lens exhibiting duplicated, double Greek key domains. The lens also contains high concentrations of the small heat shock chaperone α-crystallin, which suppresses aggregation of model substrates in vitro. Mature-onset cataract is believed to represent an aggregated state of partially-unfolded and covalently damaged crystallins. Nonetheless, the lack of cell or tissue culture for anucleate lens fibers and the insoluble state of cataract proteins has made it difficult to identify the conformation of the human γ-crystallin substrate species recognized by human α-crystallin. The three major human lens monomeric γ-crystallins, γD, γC, and γS, all refold in vitro in the absence of chaperones, on dilution from denaturant into buffer. However, off-pathway aggregation of the partially folded intermediates competes with productive refolding. Incubation with human αB-Crystallin chaperone during refolding suppressed the aggregation pathways of the three human γ-crystallin proteins. The chaperone did not dissociate or refold the aggregated chains under these conditions. The αB-crystallin oligomers formed long-lived, stable complexes with its γD-crystallin substrates. Using α-crystallin chaperone variants lacking tryptophans, we obtained fluorescence spectra of the chaperone/substrate complex. Binding of substrate γ-crystallins with two or three of the four buried tryptophans replaced by phenylalanines, showed that the bound substrate remained in a partially folded state with neither domain native-like. These in vitro results provide support for protein unfolding/protein aggregation models for cataract, with α-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood, but becoming saturated with advancing age.
α-crystallin; cataracts; small heat shock protein; chaperone; aggregation
Mammalian heat shock protein gp96 is an obligate chaperone for multiple integrins and TLRs, the mechanism of which is largely unknown. We have identified gp93 in Drosophila having high sequence homology to gp96. However, no functions were previously attributed to gp93. To determine whether gp93 and gp96 are functionally conserved, we have expressed gp93 in gp96-deficient mouse cells. Remarkably, the Drosophila gp93 is able to chaperone multiple murine gp96 clients including integrins α4, αL, and β2 and TLR2 and TLR9. This observation has led us to examine the structural basis of the chaperone function of gp96 by a close comparison between gp96 and gp93. We report that whereas gp96 undergoes intermolecular disulfide bond formation via Cys138, gp93 is unable to do so due to the absence of a cysteine near the same region. However, abrogation of disulfide bond formation by substituting C with A (C138A) in gp96 via site-directed mutagenesis did not compromise its chaperone function. Likewise, gp93 chaperone ability could not be improved by forcing intermolecular bond formation between gp93 N termini. We conclude that gp93 is the Drosophila ortholog of gp96 and that the chaperone function of the two molecules is conserved. Moreover, gp96 N-terminal disulfide bond formation is not critical for its function, underscoring the importance of N-terminal dimerization via non-disulfide bond-mediated interactions in client protein folding by gp96. Further study of gp96 from an evolutionary angle shall be informative to uncover the detailed mechanism of its chaperone function of client proteins in the secretory pathway.
The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D) have been implicated in the regulation of the innate immune system within the lung. In particular, SP-D appears to have both pro- and anti-inflammatory signaling functions. At present, the molecular mechanisms involved in switching between these functions remain unclear. SP-D differs in its quaternary structure from SP-A and the other members of the collectin family, such as C1q, in that it forms large multimers held together by the N-terminal domain, rather than aligning the triple helix domains in the traditional “bunch of flowers” arrangement. There are two cysteine residues within the hydrophobic N terminus of SP-D that are critical for multimer assembly and have been proposed to be involved in stabilizing disulfide bonds. Here we show that these cysteines exist within the reduced state in dodecameric SP-D and form a specific target for S-nitrosylation both in vitro and by endogenous, pulmonary derived nitric oxide (NO) within a rodent acute lung injury model. S-nitrosylation is becoming increasingly recognized as an important post-translational modification with signaling consequences. The formation of S-nitrosothiol (SNO)-SP-D both in vivo and in vitro results in a disruption of SP-D multimers such that trimers become evident. SNO-SP-D but not SP-D, either dodecameric or trimeric, is chemoattractive for macrophages and induces p38 MAPK phosphorylation. The signaling capacity of SNO-SP-D appears to be mediated by binding to calreticulin/CD91. We propose that NO controls the dichotomous nature of this pulmonary collectin and that posttranslational modification by S-nitrosylation causes quaternary structural alterations in SP-D, causing it to switch its inflammatory signaling role. This represents new insight into both the regulation of protein function by S-nitrosylation and NO's role in innate immunity.
Cells of the lung lining secrete a microbe-binding molecule called surfactant protein D (SP-D) that helps activate the inflammatory system against invading pathogens. In the absence of infection, SP-D is important in limiting inflammation, demonstrated by the fact that mice lacking the SP-D gene have chronic inflammation and emphysema. SP-D has two structural features—a lectin-like head domain and a collagenous tail domain—that, respectively, inhibit and stimulate inflammation. Here we define a mechanism for generating the active “inflammatory” version of SP-D. SP-D is held together in its multimeric state by interacting cysteine residues, which are susceptible to modification by the gaseous second messenger, nitric oxide, to form S-nitrosothiols. In this multimeric state, the tail domains are buried, limiting the ability of SP-D to activate inflammation. S-nitrosylation causes the multimers to fall apart into trimers, exposing the tail domain. S-nitrosylated SP-D induces inflammatory cell activation as determined by chemotaxis, calcium influx, and phosphorylation state. This activity is dependent upon both the S-nitrosothiol and the disruption of SP-D's multimeric structure. These modifications are observed in an in vivo model of inflammation and form a critical part of the process. A model is proposed in which nitric oxide operates as a molecular switch for SP-D.
Nitric oxide is shown to control the pro- and anti-inflammatory functions of surfactant protein D by altering its quaternary structure viaS-nitrosylation.
Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.
The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]1/2 at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys36-Cys49 and two disulfide bonds formed by two pair of consecutive cysteines, Cys22-Cys23 and Cys56-Cys57, a unique disulfide structure of polypeptide that has not been documented previously.
Conformational stability of C3a; Denaturation of C3a; Unfolding of C3a; Method of disulfide scrambling; Scrambled isomers of C3a; Reductive unfolding of native C3a
High molecular weight thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). It is Trx that is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize NADPH as the source of reducing equivalents to reduce a bound flavin prosthetic group, which in turn reduces an N-terminal redox center that has the conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol and CCT is the thiol involved in charge-transfer complexation. The reduced N-terminal redox center reduces a C-terminal redox center on the opposite subunit of the head-to-tail homodimer. It is the C-terminal redox center that catalyzes the reduction of the Trx-disulfide. Variations in the amino acid sequence of the C-terminal redox center differentiate high molecular weight TRs into different types. Type Ia TRs have tetrapeptide C-terminal redox centers of sequence GCUG, where U is the rare amino acid selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs replace the Sec residue with a conventional cysteine (Cys) residue and can use small polar amino acids such as serine and threonine in place of the flanking glycine residues. The TR from P. falciparum (PfTR) is similar in structure and mechanism to type Ia and type Ib TRs except that the C-terminal redox center is different in its amino acid sequence. The C-terminal redox center of PfTR has the sequence G534CGGGKCG541 and we classify it as a type II high molecular weight TR. The oxidized type II redox motif will form a 20-membered disulfide ring, while the absence of spacer amino acids in the type I motif results in the formation of a rare 8-membered ring. We used site-directed mutagenesis and protein semisynthesis to investigate features of the distinctive type II C-terminal redox motif that help it perform catalysis. Deletion of Gly541 reduces thioredoxin-reductase activity by ~50-fold, most likely due to disruption of an important hydrogen bond between the amide N-H of Gly541 and the carbonyl of Gly534 that helps to stabilize the β-turn-β motif. Alterations of the 20-membered disulfide ring by amino acid deletion or substitution resulted in impaired catalytic activity. Subtle changes in the ring structure and size via homocysteine for cysteine substitution using semisynthesis also caused significant reductions in catalytic activity, demonstrating the importance of the disulfide ring’s geometry in making the C-terminal redox center reactive for thiol/disulfide exchange. The data suggested to us that the transfer of electrons from the N-terminal redox center to the C-terminal redox center may be rate limiting. We propose that the transfer of electrons from the N-terminal redox center in PfTR to the type II C-terminal disulfide is accelerated by the use of an “electrophilic activation” mechanism. In this electrophilic activation mechanism, the type II C-terminal disulfide is polarized, making the sulfur atom of Cys540 electron deficient, highly electrophilic, and activated for thiol/disulfide exchange with the N-terminal redox center. This hypothesis was investigated by constructing chimeric PfTR mutant enzymes containing C-terminal type I sequences GCCG and GCUG, respectively. The PfTR-GCCG chimera had 500-fold less thioredoxin-reductase activity than the native enzyme, but still reduced selenocystine and lipoic acid efficiently. The PfTR-GCUG chimera had higher catalytic activity than the native enzyme with Trx, selenocystine, and lipoic acid as substrates. The results suggested to us that: (i) Sec in the mutant enzyme accelerated the rate of thiol/disulfide exchange between the N- and C-terminal redox centers, (ii) the type II redox center evolved for efficient catalysis utilizing Cys instead of Sec, and the type II redox center of PfTR is partly responsible for substrate recognition of the cognate PfTrx substrate relative to non-cognate thioredoxins.
mass thioredoxin reductases (TRs) are pyridine nucleotide
disulfide oxidoreductases that catalyze the reduction of the disulfide
bond of thioredoxin (Trx). Trx is responsible for reducing multiple
protein disulfide targets in the cell. TRs utilize reduced β-nicotinamide
adenine dinucleotide phosphate to reduce a bound flavin prosthetic
group, which in turn reduces an N-terminal redox center that has the
conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol while the thiol involved in
charge-transfer complexation is denoted as CCT. The reduced
N-terminal redox center reduces a C-terminal redox center on the opposite
subunit of the head-to-tail homodimer, the C-terminal redox center
that catalyzes the reduction of the Trx-disulfide. Variations in the
amino acid sequence of the C-terminal redox center differentiate high-molecular
mass TRs into different types. Type Ia TRs have tetrapeptide C-terminal
redox centers of with a GCUG sequence, where U is the rare amino acid
selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs
has its Sec residue replaced with a conventional cysteine (Cys) residue
and can use small polar amino acids such as serine and threonine in
place of the flanking glycine residues. The TR from Plasmodium
falciparum (PfTR) is similar in structure and mechanism to
type Ia and type Ib TRs except that the C-terminal redox center is
different in its amino acid sequence. The C-terminal redox center
of PfTR has the sequence G534CGGGKCG541, and
we classify it as a type II high-molecular mass TR. The oxidized type
II redox motif will form a 20-membered disulfide ring, whereas the
absence of spacer amino acids in the type I motif results in the formation
of a rare eight-membered ring. We used site-directed mutagenesis and
protein semisynthesis to investigate features of the distinctive type
II C-terminal redox motif that help it perform catalysis. Deletion
of Gly541 reduces thioredoxin reductase activity by ∼50-fold,
most likely because of disruption of an important hydrogen bond between
the amide NH group of Gly541 and the carbonyl of Gly534 that helps
the β–turn−β motif. Alterations of the 20-membered
disulfide ring either by amino acid deletion or by substitution resulted
in impaired catalytic activity. Subtle changes in the ring structure
and size caused by using semisynthesis to substitute homocysteine
for cysteine also caused significant reductions in catalytic activity,
demonstrating the importance of the disulfide ring’s geometry
in making the C-terminal redox center reactive for thiol–disulfide
exchange. The data suggested to us that the transfer of electrons
from the N-terminal redox center to the C-terminal redox center may
be rate-limiting. We propose that the transfer of electrons from the
N-terminal redox center in PfTR to the type II C-terminal disulfide
is accelerated by the use of an “electrophilic activation”
mechanism. In this mechanism, the type II C-terminal disulfide is
polarized, making the sulfur atom of Cys540 electron deficient, highly
electrophilic, and activated for thiol–disulfide exchange with
the N-terminal redox center. This hypothesis was investigated by constructing
chimeric PfTR mutant enzymes containing C-terminal type I sequences
GCCG and GCUG, respectively. The PfTR-GCCG chimera had 500-fold less
thioredoxin reductase activity than the native enzyme but still reduced
selenocystine and lipoic acid efficiently. The PfTR-GCUG chimera had
higher catalytic activity than the native enzyme with Trx, selenocystine,
and lipoic acid as substrates. The results suggested to us that (i)
Sec in the mutant enzyme accelerated the rate of thiol–disulfide
exchange between the N- and C-terminal redox centers, (ii) the type
II redox center evolved for efficient catalysis utilizing Cys instead
of Sec, and (iii) the type II redox center of PfTR is partly responsible
for substrate recognition of the cognate PfTrx substrate relative
to noncognate thioredoxins.
Phage P22 wild-type (WT) coat protein does not require GroEL/S to fold but temperature-sensitive–folding (tsf) coat proteins need the chaperone complex for correct folding. WT coat protein and all variants absolutely require P22 scaffolding protein, an assembly chaperone, to assemble into precursor structures termed procapsids. Previously, we showed that a global suppressor (su) substitution, T166I, which rescues several tsf coat protein variants, functioned by inducing GroEL/S. This led to an increased formation of tsf:T166I coat protein:GroEL complexes compared with the tsf parents. The increased concentration of complexes resulted in more assembly-competent coat proteins because of a shift in the chaperone-driven kinetic partitioning between aggregation-prone intermediates toward correct folding and assembly. We have now investigated the folding and assembly of coat protein variants that carry a different global su substitution, F170L. By monitoring levels of phage production in the presence of a dysfunctional GroEL we found that tsf:F170L proteins demonstrate a less stringent requirement for GroEL. Tsf:F170L proteins also did not cause induction of the chaperones. Circular dichroism and tryptophan fluorescence indicate that the native state of the tsf: F170L coat proteins is restored to WT-like values. In addition, native acrylamide gel electrophoresis shows a stabilized native state for tsf:F170L coat proteins. The F170L su substitution also increases procapsid production compared with their tsf parents. We propose that the F170L su substitution has a decreased requirement for the chaperones GroEL and GroES as a result of restoring the tsf coat proteins to a WT-like state. Our data also suggest that GroEL/S can be induced by increasing the population of unfolding intermediates.
Site-directed cysteine and disulfide chemistry is broadly useful in the analysis of protein structure and dynamics, and applications of this chemistry to the bacterial chemotaxis pathway have illustrated the kinds of information that can be generated. Notably, in many cases, cysteine and disulfide chemistry can be carried out in the native environment of the protein whether it be aqueous solution, a lipid bilayer, or a multiprotein complex. Moreover, the approach can tackle three types of problems crucial to a molecular understanding of a given protein: (1) it can map out 2° structure, 3° structure, and 4° structure; (2) it can analyze conformational changes and the structural basis of regulation by covalently trapping specific conformational or signaling states; and (3) it can uncover the spatial and temporal aspects of thermal fluctuations by detecting backbone and domain dynamics. The approach can provide structural information for many proteins inaccessible to high-resolution methods. Even when a high-resolution structure is available, the approach provides complementary information about regulatory mechanisms and thermal dynamics in the native environment. Finally, the approach can be applied to an entire protein, or to a specific domain or subdomain within the full-length protein, thereby facilitating a divide-and-conquer strategy in large systems or multiprotein complexes.
Rigorous application of the approach to a given protein, domain, or subdomain requires careful experimental design that adequately resolves the structural and dynamical information provided by the method. A full structural and dynamical analysis begins by scanning engineered cysteines throughout the region of interest. To determine 2° structure, the solvent exposure of each cysteine is determined by measuring its chemical reactivity, and the periodicity of exposure is analyzed. To probe 3° structure, 4° structure, and conformational regulation, pairs of cysteines are identified that rapidly form disulfide bonds and that retain function when induced to form a disulfide bond in the folded protein or complex. Finally, to map out thermal fluctuations in a protein of known structure, disulfide formation rates are measured between distal pairs of nonperturbing surface cysteines. This chapter details these methods and illustrates applications to two proteins from the bacterial chemotaxis pathway: the periplasmic galactose binding protein and the transmembrane aspartate receptor.
Vaccinia virus envelope protein A27 has multiple functions and is conserved in the Orthopoxvirus genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21–84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel α-helices and one unusual antiparallel α-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner in vitro in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly in vitro. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.
Mature vaccinia virus has more than 20 envelope proteins, including the A27 protein, which has multiple functions in the virus life cycle. During virus entry, A27 mediates the attachment of mature vaccinia virus to cell surface heparan sulfate. A27 also tethers a viral fusion suppressor protein, A26, to mature virions. During virion morphogenesis, A27 mediates mature virus transport in infected cells. We used X-ray crystallography to determine the structure of tA27 protein, which forms a novel hexamer consisting of four parallel strands and two anti-parallel strands. Hexamerization depends on the coiled-coiled domain from L47 to L82 within each tA27 strand, and mutational analysis revealed that amino acid residues within the coiled-coiled domain are critical for tA27 self-assembly in vitro. We extended the importance of tA27 self-assembly into an in vivo system in which A27 protein dimer/trimer formation through the coiled-coiled domain is crucial to its biological activity, and revealed how A27 regulates virus-induced membrane fusion through its ability to form complexes with A26 protein. Since A27 is a critical target of neutralizing antibodies against pathogenic poxvirus infection in humans, our findings provide a structural basis for the development of anti-pox drugs.
Sod1 is an important antioxidant enzyme that becomes activated by its chaperone, Ccs1. The localization of Ccs1 to mitochondria is controlled by the oxidoreductase Mia40. The formation of a disulfide bond between Cys-27 and Cys-64 in Ccs1 is critical for import and stability but not for Ccs1 activity in the maturation of Sod1.
Superoxide dismutase 1 (Sod1) is an important antioxidative enzyme that converts superoxide anions to hydrogen peroxide and water. Active Sod1 is a homodimer containing one zinc ion, one copper ion, and one disulfide bond per subunit. Maturation of Sod1 depends on its copper chaperone (Ccs1). Sod1 and Ccs1 are dually localized proteins that reside in the cytosol and in the intermembrane space of mitochondria. The import of Ccs1 into mitochondria depends on the mitochondrial disulfide relay system. However, the exact mechanism of this import process has been unclear. In this study we detail the import and folding pathway of Ccs1 and characterize its interaction with the oxidoreductase of the mitochondrial disulfide relay Mia40. We identify cysteines at positions 27 and 64 in domain I of Ccs1 as critical for mitochondrial import and interaction with Mia40. On interaction with Mia40, these cysteines form a structural disulfide bond that stabilizes the overall fold of domain I. Although the cysteines are essential for the accumulation of functional Ccs1 in mitochondria, they are dispensable for the enzymatic activity of cytosolic Ccs1. We propose a model in which the Mia40-mediated oxidative folding of domain I controls the cellular distribution of Ccs1 and, consequently, active Sod1.
Protein disulfide isomerase (PDI) is a thiodisulfide oxidoreductase that catalyzes the formation, reduction and rearrangement of disulfide bonds in proteins of eukaryotes. The classical PDI has a signal peptide, two CXXCcontaining thioredoxin catalytic sites (a,a′), two noncatalytic thioredoxin fold domains (b,b′), an acidic domain (c) and a C-terminal endoplasmic reticulum (ER) retention signal. Although PDI resides in the ER where it mediates the folding of nascent polypeptides of the secretory pathway, we recently showed that PDI5 of Arabidopsis thaliana chaperones and inhibits cysteine proteases during trafficking to vacuoles prior to programmed cell death of the endothelium in developing seeds. Here we describe Arabidopsis PDI2, which shares a primary structure similar to that of classical PDI. Recombinant PDI2 is imported into ER-derived microsomes and complements the E. coli protein- folding mutant, dsbA. PDI2 interacted with proteins in both the ER and nucleus, including ER-resident protein folding chaperone, BiP1, and nuclear embryo transcription factor, MEE8. The PDI2-MEE8 interaction was confirmed to occur in vitro and in vivo. Transient expression of PDI2- GFP fusions in mesophyll protoplasts resulted in labeling of the ER, nucleus and vacuole. PDI2 is expressed in multiple tissues, with relatively high expression in seeds and root tips. Immunoelectron microscopy with GFP- and PDI2-specific antisera on transgenic seeds (PDI2-GFP) and wild type roots demonstrated that PDI2 was found in the secretory pathway (ER, Golgi, vacuole, cell wall) and the nuclei. Our results indicate that PDI2 mediates protein folding in the ER and has new functional roles in the nucleus.
chaperone; endoplasmic reticulum; nuclear factor; plant disulfide isomerase; protein folding