James G. Bann

Periplasmic Peptidyl Prolyl cis-trans Isomerases Are Not Essential for Viability, but SurA Is Required for Pilus Biogenesis in Escherichia coli

In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.

Real-time and equilibrium 19F-NMR studies reveal the role of domain–domain interactions in the folding of the chaperone PapD

PapD is a periplasmic chaperone essential for P pilus formation in pyelonephritic strains of E. coli. It is composed of two domains, each of which contains a tryptophan residue (Trp-36 and Trp-128, in the N- and C-terminal domains, respectively). To explore the role of domain–domain interactions during folding, the protein was labeled with 6-fluorotryptophan for use in 19F-NMR experiments. 19F-NMR data collected as a function of urea concentration revealed the presence of a resonance caused by Trp-128 that was distinct from either the folded or unfolded resonances. The time course of refolding from urea was monitored by stopped-flow fluorescence, CD, and 19F-NMR, each method showing multiple kinetic phases. The 19F-NMR stopped-flow spectra, collected at 70 μM of protein with a fluorine cryoprobe, demonstrated that the intermediate was populated early in the folding process (<5 s). The slow disappearance of the intermediate and unfolded resonance occurred at the same rate as the appearance of the native resonances of both domains. The data are consistent with a model in which the C-terminal domain collapses rapidly to an intermediate, whereas the stabilization of the final structure is slow and requires folding of the N-terminal domain with concomitant readjustment of the C-terminal domain structure.

Biosynthetic incorporation of fluorohistidine into proteins in E. coli: a new probe of macromolecular structure.

Histidine is important for carrying out a number of protein functions. For instance, it can act as a general acid/base in enzymatic catalysis, it plays a role in ligating metal cations in metalloproteins, and stabilizes protein structure by metal binding, hydrogen bonding, or electrostatic interactions. One noteworthy example of histidine’s potential role in protein stability is observed in the pathogenesis of the anthrax toxin, where protonation of His side chains in one of the proteins leads to a large structural perturbation and subsequent toxicity. Given the unique role of this amino acid in such processes, the development of methods that probe the structural and mechanistic features of His in proteins would be extremely valuable. The biosynthetic incorporation of unnatural amino acids into proteins provides the experimentalist with a variety of methods that can probe protein structure and function. In particular, fluorinated amino acids can be used to achieve a relatively isosteric change (by replacing a single hydrogen with fluorine) that results in quite different electronic properties. Additionally, F NMR can be used to monitor changes in protein conformation in response to changes in the environment that are sometimes not detectable by other techniques. Although incorporating fluorine-labeled amino acids is not a new idea, access to an expanding tool box of fluorinated protein building blocks has promoted a renaissance in this area of research. Over 30 years ago, a photochemical Schiemann reaction was developed for synthesizing 2-fluorohistidine (2-FHis) and 4-fluorohistidine (4-FHis). To our knowledge this still represents the only fluorination procedure available for accessing these imidazole derivatives. The pKa of the side chain of both 2-FHis and 4-FHis has been measured previously, and is decreased from approximately 6.0–6.5 to 1 and 3, respectively. Because of this, these analogues provide a means for verifying the role of native His in pH-dependent processes. To this end, 4-FHis has been incorporated into the S peptide of ribonuclease, and into full-length ribonuclease A with chemical synthetic methods. Early experiments demonstrated that tritium-labeled 2-FHis could be incorporated into bacterial protein, and these analogues (2-FHis more so than 4-FHis) had an inhibitory effect on E. coli growth. However, in order to achieve high levels of incorporation for structural studies, it is typical to employ the use of bacterial auxotrophs. While the use of auxotrophs for the biosynthetic incorporation of novel His analogues into E. coli has been reported, a similar protocol for the incorporation of fluorohistidine derivatives has yet to be described. Herein, we provide unequivocal evidence for the incorporation of both 4-FHis and 2-FHis into a mutant form of the chaperone PapD by using an E. coli strain that is auxotrophic for His. PapD is the prototype for a wide variety of highly homologous chaperones that utilize the chaperone-usher pathway for the assembly of P-pili, and has been previously labeled with fluorophenylalanine and fluorotryptophan in protein-folding studies. The wild-type (WT) protein does not contain any His residues. Thus, site-specific labeling can be accomplished by the introduction of a single His residue by site-directed mutagenesis, and biosynthetic labeling can be performed according to previously described protocols. In this work, we used site-directed mutagenesis to introduce a single His residue at Arg200 in PapD. Among the chaperones that are homologous to PapD, the most similar is PmFD from Proteus mirabilis (47% identity), which possesses a His residue at position 200. Therefore, an R200H substitution was not expected to alter the structure and stability of PapD. To confirm this, urea denaturation studies were performed on PapD (R200H) and it was found to have a similar stability as that previously reported for WT PapD; PapD(WT): DG8=8.95 kcal [a] Dr. J. F. Eichler, Prof. Dr. J. G. Bann Department of Chemistry, Wichita State University Wichita, KS 67226 (USA) Fax: (+1)316-978-7373 E-mail : jim.bann@wichita.edu [b] Dr. J. C. Cramer, Dr. K. L. Kirk Laboratory of Bioorganic Chemistry National Institute of Diabetes & Digestive & Kidney Diseases National Institutes of Health Bethesda, MD 20892 (USA) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Anthrax toxin protective antigen—Insights into molecular switching from prepore to pore

The protective antigen is a key component of the anthrax toxin, as it allows entry of the enzymatic components edema factor and lethal factor into the host cell, through the formation of a membrane spanning pore. This event is absolutely critical for the pathogenesis of anthrax, and although we have yet to understand the mechanism of pore formation, recent developments have provided key insights into how this process may occur. Based on the available data, a model is proposed for the kinetic steps for protective antigen conversion from prepore to pore. In this model, the driving force for pore formation is the formation of the phi (ϕ)‐clamp, a region that forms a leak‐free seal around the translocating polypeptide. Formation of the ϕ‐clamp elicits movements within the prepore that provide steric freedom for the subsequent conformational changes required to form the membrane spanning pore.

Evidence That Histidine Protonation of Receptor-Bound Anthrax Protective Antigen Is a Trigger for Pore Formation

The protective antigen (PA) component of the anthrax toxin forms pores within the low pH environment of host endosomes through mechanisms that are poorly understood. It has been proposed that pore formation is dependent on histidine protonation. In previous work, we biosynthetically incorporated 2-fluorohistidine (2-FHis), an isosteric analogue of histidine with a significantly reduced pK(a) ( approximately 1), into PA and showed that the pH-dependent conversion from the soluble prepore to a pore was unchanged. However, we also observed that 2-FHisPA was nonfunctional in the ability to mediate cytotoxicity of CHO-K1 cells by LF(N)-DTA and was defective in translocation through planar lipid bilayers. Here, we show that the defect in cytotoxicity is due to both a defect in translocation and, when bound to the host cellular receptor, an inability to undergo low pH-induced pore formation. Combining X-ray crystallography with hydrogen-deuterium (H-D) exchange mass spectrometry, our studies lead to a model in which hydrogen bonds to the histidine ring are strengthened by receptor binding. The combination of both fluorination and receptor binding is sufficient to block low pH-induced pore formation.

Comparison of the Structural Stability and Dynamic Properties of Recombinant Anthrax Protective Antigen and its 2-Fluorohistidine-Labeled Analogue

Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigens conformational stability and dynamic properties were investigated by various biophysical techniques. Temperature/pH stability profiles of rPA and 2-FHis rPA were analyzed by the empirical phase diagram (EPD) approach, and physical stability differences between them were identified. Results showed that rPA and 2-FHis rPA had similar stability at pH 7-8. With decreasing solution pH, however, 2-FHis rPA was found to be more stable. Dynamic sensitive measurements of the two proteins at pH 5 found that 2-FHis rPA was more dynamic and/or differentially hydrated under acidic pH conditions. The biophysical characterization and stability data provide information useful for the potential development of 2-FHis rPA as a more stable rPA vaccine candidate.

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH‐dependent pore formation. We present here the 1.95 Å crystal structure of domain 4, which adopts a fold that is identical to that observed in the full‐length protein. We have also investigated the structural properties of the isolated domain 4 as a function of pH, as well as the pH‐dependence on binding to the von Willebrand factor A domain of capillary morphogenesis protein 2 (CMG2). Our results provide evidence that the isolated domain 4 maintains structure and interactions with CMG2 at pH 5, a pH that is known to cause release of the receptor on conversion of the heptameric prepore (PA63)7 to a membrane‐spanning pore. Our results suggest that receptor release is not driven solely by a pH‐induced unfolding of domain 4.

3S-Fluoroproline as a probe to monitor proline isomerization during protein folding by 19F-NMR

Variable-temperature inversion transfer NMR is used to determine the kinetic and thermodynamic parameters of cis-trans isomerization of N-Ac-(3R) and (3S)-fluoroproline-OMe.

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional 13C‐edited 1H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.

pH effects on binding between the anthrax protective antigen and the host cellular receptor CMG2

The anthrax protective antigen (PA) binds to the host cellular receptor capillary morphogenesis protein 2 (CMG2) with high affinity. To gain a better understanding of how pH may affect binding to the receptor, we have investigated the kinetics of binding as a function of pH to the full‐length monomeric PA and to two variants: a 2‐fluorohistidine‐labeled PA (2‐FHisPA), which is ∼1 pH unit more stable to variations in pH than WT, and an ∼1 pH unit less stable variant in which Trp346 in the domain 2β3‐2β4 loop is substituted with a Phe (W346F). We show using stopped‐flow fluorescence that the binding rate increases as the pH is lowered for all proteins, with little influence on the rate of dissociation. In addition, we have crystallized PA and the two variants and examine the influence of pH on structure. In contrast to previous X‐ray studies, the domain 2β3‐2β4 loop undergoes little change in structure from pH ∼8 to 5.5 for the WT protein, but for the 2‐FHis labeled and W346F mutant there are changes in structure consistent with previous X‐ray studies. In accord with pH stability studies, we find that the average B‐factor values increase by ∼20–30% for all three proteins at low pH. Our results suggest that for the full‐length PA, low pH increases the binding affinity, likely through a change in structure that favors a more “bound‐like” conformation.