Pertactin beta-helix folding mechanism suggests common themes for the secretion and folding of autotransporter proteins

Many virulence factors secreted from pathogenic Gram-negative bacteria are autotransporter proteins. The final step of autotransporter secretion is C --> N-terminal threading of the passenger domain through the outer membrane (OM), mediated by a cotranslated C-terminal porin domain. The native structure is formed only after this final secretion step, which requires neither ATP nor a proton gradient. Sequence analysis reveals that, despite size, sequence, and functional diversity among autotransporter passenger domains, >97% are predicted to form parallel beta-helices, indicating this structural topology may be important for secretion. We report the folding behavior of pertactin, an autotransporter passenger domain from Bordetella pertussis. The pertactin beta-helix folds reversibly in isolation, but folding is much slower than expected based on size and native-state topology. Surprisingly, pertactin is not prone to aggregation during folding, even though folding is extremely slow. Interestingly, equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the C-terminal half of the protein. Examination of the pertactin crystal structure does not reveal any obvious reason for the enhanced stability of the C terminus. In vivo, slow folding would prevent premature folding of the passenger domain in the periplasm, before OM secretion. Moreover, the extra stability of the C-terminal rungs of the beta-helix might serve as a template for the formation of native protein during OM secretion; hence, vectorial folding of the beta-helix could contribute to the energy-independent translocation mechanism. Coupled with the sequence analysis, the results presented here suggest a general mechanism for autotransporter secretion.     

An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1

The cariogenic bacterium Streptococcus mutans uses adhesin P1 to adhere to tooth surfaces, extracellular matrix components, and other bacteria. A composite model of P1 based on partial crystal structures revealed an unusual complex architecture in which the protein forms an elongated hybrid alpha/polyproline type II helical stalk by folding back on itself to display a globular head at the apex and a globular C-terminal region at the base. The structure of P1’s N terminus and the nature of its critical interaction with the C-terminal region remained unknown, however. We have cocrystallized a stable complex of recombinant N- and C-terminal fragments and here describe a previously unidentified topological fold in which these widely discontinuous domains are intimately associated. The structure reveals that the N terminus forms a stabilizing scaffold by wrapping behind the base of P1’s elongated stalk and physically “locking” it into place. The structure is stabilized through a highly favorable ΔG_solvation on complex formation, along with extensive hydrogen bonding. We confirm the functional relevance of this intramolecular interaction using differential scanning calorimetry and circular dichroism to show that disruption of the proper spacing of residues 989–1001 impedes folding and diminishes stability of the full-length molecule, including the stalk. Our findings clarify previously unexplained functional and antigenic properties of P1.Streptococcus mutans is a recognized cause of human dental caries (cavities), the most common infectious disease worldwide (1). Identifying how S. mutans interacts with host components at the molecular level is essential to fully understand its virulence properties. The sucrose-independent adhesin P1 (AgI/II, antigen B, PAc) is localized on the surface of this oral pathogen, along with many other streptococci (2–7). In the oral cavity, S. mutans P1 interacts with the salivary agglutinin glycoprotein complex composed predominantly of scavenger receptor gp340/DMBT1 (2, 3, 5–10). Without a complete structural model, the mechanisms by which P1 binds to host components have not yet been fully characterized.P1’s primary structure (Fig. 1A) contains a 38-residue signal sequence, the heretofore uncharacterized N-terminal region, three alanine-rich repeats (A1–3), a central domain containing a so-called variable (V) region (11), three proline-rich repeats (P1–3), a C-terminal region consisting of three domains (C1–3), an LPxTG sortase-recognition motif, and wall- and membrane-spanning regions (12, 13). Recent partial X-ray crystal structure and velocity centrifugation studies of the intact protein unveiled a unique architecture in which the ∼185-kDa (1,561-aa) protein folds back on itself to form a ∼50-nm elongated hybrid helical stalk that separates two independent adherence domains, with a globular head at the apex and a globular C-terminal region at the base (13–15) (Fig. 1B).Open in a separate windowFig. 1.Schematic representation of the primary and modeled tertiary structure of P1. (A) Primary structure of P1 and location of polypeptides used in this study. (B) Proposed tertiary model of P1 based on velocity centrifugation and crystal structures of A3VP1 and C123 fragments (13). (C) Diagram showing the locations of the two engineered Cla1 sites (circled in red) that added isoleucine and aspartic acid residues to either side of the proline-rich region (20).The crystal structure of the third alanine-rich repeat through the first proline-rich repeat first revealed the unusual interaction between the A and P regions to form a hybrid alpha/polyproline type II helix (14). In this model, a globular β super sandwich domain sits at the apex of the molecule (15). At the other end of the hybrid helix, the three contiguous domains of the C-terminal region each adopt a DE-variant Ig-like (DEv-IgG) fold stabilized by isopeptide bonds (13, 16). Despite this recent progress, the structure of the ∼20-kDa N terminus of P1 remained unknown, however (Fig. 1B). We previously demonstrated that proper folding and function of P1 on the surface of S. mutans requires an interaction between N- and C-terminal segments (17), thus increasing the imperative to elucidate the structure of the N terminus in complex with its intramolecular binding partner.For this, we used the recombinant N-terminal (NA1) and C-terminal (P3C) P1 fragments (Fig. 1A), which have been shown to form a stable high-affinity and functionally active complex (17). The NA1/P3C protein complex was copurified and then cocrystallized for X-ray diffraction data collection to 2.0-Å resolution. We observed that the N terminus adopts a previously unidentified fold that serves as an intramolecular scaffold connecting it to C-terminal portions of the molecule, thus locking P1’s hybrid helical stalk into place. We validated this model experimentally using differential scanning calorimetry (DSC) and circular dichroism to demonstrate decreased thermal stability and altered secondary structure in a P1 mutant containing two extra amino acids within the region that normally reacts with the N-terminal intramolecular scaffold. Our X-ray crystallography model and stability measurements agree well with biophysical data characterizing the NA1/P3C complex (17) and provide mechanistic insight into why the N-terminal segment supports the proper folding, function, and stability of the full-length P1 protein. This information contributes to our ability to interpret data regarding protective and nonprotective immune responses and preventative therapies, and will inform future studies evaluating bacterial adhesion and biofilm formation by S. mutans and related organisms.     

Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.

Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.     

Genetic analysis of the folding pathway for the tail spike protein of phage P22.

Temperature-sensitive mutations in the gene encoding the trimeric tail spike protein of phage P22 interfere with protein maturation at 39 degrees C. We show here that temperature-sensitive mutations at many sites block the folding pathway prior to accumulation of the partially folded protrimer intermediate. Temperature-shift experiments indicate that at least some of the mutants accumulate an earlier intermediate in the folding pathway. Immunoprecipitation experiments suggest that the conformation of the isolated temperature-sensitive polypeptide chains is closer to that of the unfolded chain than to that of the mature spike formed at permissive temperature. The sites of these mutations probably represent amino acid sequences that play key roles during the folding of the tail spike polypeptide chain but are not important in the mature protein.     

Stable folding core in the folding transition state of an alpha-helical integral membrane protein

Defining the structural features of a transition state is important in understanding a folding reaction. Here, we use Φ-value and double mutant analyses to probe the folding transition state of the membrane protein bacteriorhodopsin. We focus on the final C-terminal helix, helix G, of this seven transmembrane helical protein. Φ-values could be derived for 12 amino acid residues in helix G, most of which have low or intermediate values, suggesting that native structure is disrupted at these amino acid positions in the transition state. Notably, a cluster of residues between E204 and M209 all have Φ-values close to zero. Disruption of helix G is further confirmed by a low Φ-value of 0.2 between residues T170 on helix F and S226 on helix G, suggesting the absence of a native hydrogen bond between helices F and G. Φ-values for paired mutations involved in four interhelical hydrogen bonds revealed that all but one of these bonds is absent in the transition state. The unstructured helix G contrasts with Φ-values along helix B that are generally high, implying native structure in helix B in the transition state. Thus helix B seems to constitute part of a stable folding nucleus while the consolidation of helix G is a relatively late folding event. Polarization of secondary structure correlates with sequence position, with a structured helix B near the N terminus contrasting with an unstructured C-terminal helix G.     

Single amino acid substitutions influencing the folding pathway of the phage P22 tail spike endorhamnosidase.

Temperature-sensitive mutations in the gene for the thermostable tail spike of phage P22 interyFere with the folding and subunit association pathway at the restrictive temperature but not with the activity or stability of the protein once matured. The local sites of these mutations and the mutant amino acid substitutions have been determined by DNA sequencing. Of 11 temperature-sensitive folding mutations, 3 were replacements of glycine residues by polar residues, and three were replacements of threonine residues by residues unable to form a side-chain H-bond. There were no proline replacements. Two of the temperature-sensitive sites in which threonine residues were replaced by isoleucine residues were homologous. These sequences probably maintain the correct local folding pathway at higher temperatures. The temperature-sensitive amino acid substitutions appear to destabilize a thermolabile intermediate in the wild-type folding pathway or to increase the rate of a competing off-pathway reaction.     

An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P.

Deposition of the beta-amyloid protein in senile plaques is a pathologic hallmark of Alzheimer disease (AD). Focal deposition of beta amyloid in the adult rat cerebral cortex caused profound neurodegenerative changes, including neuronal loss and degenerating neurons and neurites. Chronic induction of the Alz-50 antigen appeared in neurons around focal cortical deposits of beta amyloid. Immunoblot analysis showed that beta amyloid induced Alz-50-immunoreactive proteins in rat cerebral cortex that were very similar to the proteins induced in human cerebral cortex from patients with AD. The neuropeptide substance P prevented beta-amyloid-induced neuronal loss and expression of Alz-50 proteins when coadministered into the cerebral cortex. Systemic administration of substance P also provided protection against the effects of intracerebral beta amyloid. Thus, beta amyloid is a potent neurotoxin in the adult brain in vivo, and its effects can be blocked by substance P.     

A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.     

An essential intermediate in the folding of dihydrofolate reductase

The folding of Escherichia coli dihydrofolate reductase was examined at pH 7.8 and 15 degrees C by using stopped-flow fluorescence and absorbance spectroscopies. The formation of a highly fluorescent intermediate occurs with relaxation times ranging between 142 and 343 msec, whereas stopped-flow absorbance spectroscopy using methotrexate binding assays shows a distinct lag phase during these time frames for the native state. The lag in absorbance kinetics and the lack of fast-track folding events indicate that the formation of this ensemble of intermediates is an obligatory step in the folding reaction.     

Estimation of apparent tumor vascular permeability from multiphoton fluorescence microscopic images of P22 rat sarcomas in vivo

OBJECTIVE: To develop an image processing-based method to quantify the rate of extravasation of fluorescent contrast agents from tumor microvessels, and to investigate the effect of the tumor vascular disrupting agent combretastatin A-4-P (CA-4-P) on apparent tumor vascular permeability to 40 kDa fluorescein isothiocyanate (FITC) labeled dextran. METHODS: Extravasation of FITC-dextran was imaged in 3 dimensions over time within P22 sarcomas growing in dorsal skin flap "window chambers" in BDIX rats using multiphoton fluorescence microscopy. Image processing techniques were used to segment the data into intra- and extravascular regions or classes. Quantitative estimates of the tissue influx (vascular leakage) rate constant, K(i), were obtained from the time courses of the fluorescence intensities in the two classes. Apparent permeability, P, was calculated, assuming K(i) = PS/V, where S is vascular surface area in tumor volume V. RESULTS: Combining image processing and kinetic analysis algorithms with multiphoton fluorescence microscopy enabled quantification of the rate of tumor vascular leakage, averaged over a large number of vessels. Treatment with CA-4-P caused a significant increase in K(i) from 1.13 +/- 0.33 to 2.59 +/- 0.20 (s(- 1)x 10(- 4); mean +/- SEM), equivalent to an increase in P from 12.76 +/- 3.36 to 30.94 +/- 2.64 (cm s(-1)x 10(-7)). CONCLUSIONS: A methodology was developed that provided evidence for a CA-4-P-induced increase in tumor macromolecular vascular permeability, likely to be central to its anti-cancer activity.     

Endoscopic ultrasound guided 22 gauge core needle biopsy for the diagnosis of Autoimmune pancreatitis

Background

It is difficult to obtain adequate tissue sample for diagnosing autoimmune pancreatitis (AIP) with the help of traditional EUS-guided FNA. As per ICDC guidelines, EUS-guided FNA is not recommended for diagnosing AIP(1). We herein present a report of 2 cases of using a new flexible 22 gauge (G) core biopsy needle (SharkCore, Medtronic, Sunnydale, Calif) for diagnosing AIP.

Role of group-conserved residues in the helical core of beta2-adrenergic receptor

G protein-coupled receptors (GPCRs) belonging to class A contain several highly conserved (>90%) amino acids in their transmembrane helices. Results of mutational studies of these highly conserved residues suggest a common mechanism for locking GPCRs in an inactive conformation and for their subsequent activation upon ligand binding. Recently, a second set of sites in the transmembrane helices has been identified in which amino acids with small side chains, such as Gly, Ala, Ser, Thr, and Cys, are highly conserved (>90%) when considered as a group. These group-conserved residues have not been recognized as having essential structural or functional roles. To determine the role of group-conserved residues in the beta(2)-adrenergic receptor (beta(2)-AR), amino acid replacements guided by molecular modeling were carried out at key positions in transmembrane helices H2-H4. The most significant changes in receptor expression and activity were observed upon replacement of the amino acids Ser-161 and Ser-165 in H4. Substitution at these sites by larger residues lowered the expression and activity of the receptor but did not affect specific binding to the antagonist ligand dihydroalprenolol. A second site mutation, V114A, rescued the low expression of the S165V mutant. Substitution of other group-conserved residues in H2-H4 by larger amino acids lowered receptor activity in the order Ala-128, Ala-76, Ser-120, and Ala-78. Together these data provide comprehensive analysis of group-conserved residues in a class A GPCR and allow insights into the roles of these residues in GPCR structure and function.     

A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo

Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted.     

Single versus parallel pathways of protein folding and fractional formation of structure in the transition state.

Protein engineering and kinetic experiments indicate that some regions of proteins have partially formed structure in the transition state for protein folding. A crucial question is whether there is a genuine single transition state that has interactions that are weakened in those regions or there are parallel pathways involving many transition states, some with the interactions fully formed and others with the structural elements fully unfolded. We describe a kinetic test to distinguish between these possibilities. The kinetics rule out those mechanisms that involve a mixture of fully formed or fully unfolded structures for regions of the barley chymotrypsin inhibitor 2 and barnase, and so those regions are genuinely only partially folded in the transition state. The implications for modeling of protein folding pathways are discussed.     

An in vivo model for evaluation of the postantibiotic effect

A new experimental model to evaluate the postantibiotic effect (PAE) in vivo was developed using subcutaneously implanted tissue cages in rabbits with normal host defence mechanisms. The rabbits received benzylpenicillin i.v. in a dose giving a free penicillin concentration of 10 X MIC in the tissue cage fluid (TCF). A log-phase suspension of group A streptococci was injected into the tissue cages exposing them to penicillin in vivo. After 2 h bacterial samples were withdrawn, treated with penicillinase and transferred to 2 tissue cages in untreated rabbits. Simultaneously, unexposed streptococci were implanted in 2 other cages in the same animals. By repeated sampling of TCF, growth curves of the streptococci exposed to penicillin and the controls could be compared and a PAE of 1.6-2.4 h demonstrated. The PAE was of the same magnitude as that found in vitro. The model has several advantages for the demonstration of PAE in vivo: repeated samplings are easy to perform percutaneously, the effect of subinhibitory antibiotic concentrations are avoided, interindividual variations are eliminated since each animal is its own control, and the experiments can be performed in animals with undisturbed host defence mechanisms.     

An in vivo study of muscle phosphate metabolism in Becker's dystrophy by 31P NMR spectroscopy

The forearm flexor muscles of five patients with Becker's dystrophy were examined by the painless and noninvasive technique of high resolution phosphorus nuclear magnetic resonance spectroscopy. In the mildly affected cases, the ratios of the signals of phosphocreatine to ATP and to inorganic phosphate were normal but they were reduced in the patients with advanced disease. Absolute quantitation under the conditions of the study was not feasible, but it was probable that whereas in advanced Becker's dystrophy the intramyocellular concentration of phosphocreatine was reduced, that of ATP was unchanged. The intramyocellular pH was normal in three of the four patients in whom this could be measured and an additional unidentified signal between those of phosphocreatine and inorganic phosphate was recorded in two patients. This study emphasizes some metabolic similarities between Becker's and Duchenne type muscular dystrophy and suggests that nuclear magnetic resonance spectroscopy may be a useful and objective technique with which to investigate the biochemistry of these and other muscle diseases.     

The Role of the Coat Protein A-Domain in P22 Bacteriophage Maturation

Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.     

ATP depletion in macrophages in the core of advanced rabbit atherosclerotic plaques in vivo

The cores of rabbit plaques in vivo are hypoxic, suggesting that ATP depletion due to an insufficient supply of oxygen and nutrients could contribute to macrophage death in atherosclerotic plaques. During hypoxia, however, macrophages maintain ATP levels by anaerobic glycolysis. To directly assess ATP and glucose metabolites in plaques in vivo, we used bioluminescence imaging to map the concentrations of ATP, glucose, glycogen, and lactate in normal and atherosclerotic rabbit aortas in vivo. Hypoxia was assessed with NITP (7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline). Normal aortas and plaques <500 microm thick were not hypoxic and had homogenous concentrations of energy metabolites. In plaques >500 microm thick, however, the cores were characterized by ATP depletion, low concentrations of glucose and glycogen, and a high concentration of lactate. A majority of ATP-depleted macrophages within the core were viable but severely hypoxic and glucose depleted. Hyperoxia in vitro reversed the ATP depletion in macrophages in viable areas of the core. Our findings suggest that ATP depletion contributes to the death of macrophages in atherosclerotic lesions and to the formation of a necrotic core.     

Evidence suggesting that insulin-like growth factor-I is necessary for the trophic effects of insulin on cartilage growth in vivo.

The possibility that insulin-stimulated cartilage growth in hypophysectomized rats involves local production of insulin-like growth factor I (IGF-I) was investigated. Osmotic minipumps with attached catheters were used to infuse insulin into the right hindlimb of rats via the common iliac artery for 7 days starting 14 days after pituitary removal. The left, noninfused limb served as an internal control. Doses of insulin ranging from 0.25 to 50 mU/day caused significant increases in the tibial epiphyseal plate width (TEPW) of the infused limbs. The optimal dose of 1.25 mU/day increased the TEPW by 51 microns. Immunohistochemical analysis with an antiserum to human IGF-I showed that the growth response to the optimal dose was accompanied by the accumulation of IGF-I in the chondrocytes in the tibial plate. Infusion of insulin at a dose of 10 mU/day increased the TEPW by 18.6 +/- 3.0 microns. When the same dose of insulin was infused with the antiserum to human IGF-I, the growth response was completely nullified. These results indicate that cartilage cells in hypophysectomized rats are highly sensitive to the direct growth-promoting action of insulin. Furthermore, the growth effect appears to be mediated by or dependent on local production of IGF-I.