Search

Background: Increasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties., Results: We report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats., Conclusions: The structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.

Unlabelled: In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site., Importance: Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.

Protein unfolding occurs at both low and high temperatures, although in most cases, only the high-temperature transition can be experimentally studied. A pressing question is how much the low- and high-temperature denatured states, although thermodynamically equivalent, are structurally and kinetically similar. We have combined experimental and computational approaches to compare the high- and low-temperature unfolded states of Yfh1, a natural protein that, at physiologic pH, undergoes cold and heat denaturation around 0 °C and 40 °C without the help of ad hoc destabilization. We observe that the two denatured states have similar but not identical residual secondary structures, different kinetics and compactness and a remarkably different degree of hydration. We use molecular dynamics simulations to rationalize the role of solvation and its effect on protein stability., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Calcium activation of the actin-modifying properties of gelsolin is sensitive to ATP. Here, we show that soaking calcium-free gelsolin crystals in ATP-containing media results in ATP occupying a site that spans the two pseudosymmetrical halves of the protein. ATP binding involves numerous polar and hydrophobic contacts and is identical for the two copies of gelsolin related by non-crystallographic symmetry within the crystal. The gamma-phosphate of ATP participates in several charge-charge interactions consistent with the preference of gelsolin for ATP, as a binding partner, over ADP. In addition, disruption of the ATP-binding site through Ca2+ activation of gelsolin reveals why ATP binds more tightly to the inactive molecule, and suggests how the binding of ATP may modulate the sensitivity of gelsolin to calcium ions. Similarities between the ATP and PIP2 interactions with the C-terminal half of gelsolin are evident from their overlapping binding sites and in that both molecules bind more tightly in the absence of calcium ions. We propose a model for how PIP2 may bind to calcium-free gelsolin based on the ATP-binding site.

Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ion-binding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb3+, results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ion-binding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange.

The actin filament-severing functionality of gelsolin resides in its N-terminal three domains (G1-G3). We have determined the structure of this fragment in complex with an actin monomer. The structure reveals the dramatic domain rearrangements that activate G1-G3, which include the replacement of interdomain interactions observed in the inactive, calcium-free protein by new contacts to actin, and by a novel G2-G3 interface. Together, these conformational changes are critical for actin filament severing, and we suggest that their absence leads to the disease Finnish-type familial amyloidosis. Furthermore, we propose that association with actin drives the calcium-independent activation of isolated G1-G3 during apoptosis, and that a similar mechanism operates to activate native gelsolin at micromolar levels of calcium. This is the first structure of a filament-binding protein bound to actin and it sets stringent, high-resolution limitations on the arrangement of actin protomers within the filament.

We present the 2.6 A resolution crystal structure of a complex formed between G-actin and gelsolin fragment Met25-Gln160 (G1+). The structure differs from those of other gelsolin domain 1 (G1) complexes in that an additional six amino acid residues from the crucial linker region into gelsolin domain 2 (G2) are visible and are attached securely to the surface of actin. The linker segment extends away from G1 up the face of actin in a direction that infers G2 will bind along the same long-pitch helical strand as the actin bound to G1. This is consistent with a mechanism whereby G2 attaches gelsolin to the side of a filament and then directs G1 toward a position where it would disrupt actin-actin contacts. Alignment of the sequence of the structurally important residues within the G1-G2 linker with those of WH2 (WASp homology domain 2) domain protein family members (e.g. WASp (Wiscott-Aldridge syndrome protein) and thymosin beta4) suggests that the opposing activities of filament assembly and disassembly may exploit a common patch on the surface of actin.

Gelsolin requires activation to carry out its severing and capping activities on F-actin. Here, we present the structure of the isolated C-terminal half of gelsolin (G4-G6) at 2.0 A resolution in the presence of Ca(2+) ions. This structure completes a triptych of the states of activation of G4-G6 that illuminates its role in the function of gelsolin. Activated G4-G6 displays an open conformation, with the actin-binding site on G4 fully exposed and all three type-2 Ca(2+) sites occupied. Neither actin nor the type-l Ca(2+), which normally is sandwiched between actin and G4, is required to achieve this conformation.

The structure of rabbit liver Hsp90 was reevaluated by limited trypsinolysis, N-terminal sequencing and determination of the site that is phosphorylated by casein kinase II. Limited proteolysis results in formation of four groups of large peptides with M(r) in the range of 26-41 kDa. Peptides with M(r) 39-41 kDa were represented by large N-terminal and central peptides starting at residue 283 of the alpha-isoform of Hsp90. All sites phosphorylated by casein kinase II were located in the large 39-41 kDa peptides. Peptides with M(r) 26-27 kDa were represented by short N-terminal and central peptides starting at Glu-400 of the alpha-isoform of Hsp90. The data of affinity chromatography and light scattering indicate that smooth muscle calponin interacts with Hsp90. The calponin binding sites are located in the large (37-41 kDa) N-terminal and in a short (26-27 kDa) central peptide starting at Glu-400 of the alpha-isoform of Hsp90. Phosphorylation by casein kinase II up to 2 mol of phosphate per mol of Hsp90 does not affect interaction of Hsp90 with calponin.