Crystal structure of c5321 : a protective antigen present in uropathogenic Escherichia coli strains displaying an SLR fold

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 crystalcontacts 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

Towards a crystallographic structure for the N-terminal half of gelsolin bound to actin

Gelsolin belongs to a family of proteins that participates in the reorganization of cytoskeletal actin, a shared requirement of such processes as cell movement, cell division and apoptosis. Its functions include actin filament nucleation, severing and capping. Gelsolin consists of six structurally analogous domains (G1-G6) that, in the Ca2+-free, inactive state, are packed in such way that none of the actin-binding sites are accessible. Binding of Ca2 + ions to gelsolin causes large changes in the relative positions and orientations of the six domains, resulting in exposure of several actin-binding surfaces, and subsequent binding and severing of actin filaments. Filament end-binding sites have been identified previously in domains Gl and G4, while a distinct filament side-binding site has been attributed to G2. This thesis describes protein preparation and crystallization experiments that led to the structure at 3 A resolution of the N-terminal half of gelsolin (G1-G3) bound to one actin molecule in the presence of Ca2 + ions. The structure reveals for the first time the details of how G2 and G3 interact with the same actin to which Gl is attached. As expected, the changes in the relative orientation of domains within the N-terminal half of gelsolin on activation are large. Previously unidentified contacts between G3 and actin are observed. The existence of a Ca2 + in the type-2 binding site in activated G3, which was inferred by sequence comparison within gelsolin domains, is confirmed. In addition, docking the structure into existing molecular models for an actin filament permits proposal of a self-consistent mechanism for how intact gelsolin is activated, binds to the side of an actin filament, severs and then caps one of the newly cut filament ends.Science, Faculty ofChemistry, Department ofGraduat

Structural studies of the regulation of gelsolin by small ligands

Gelsolin regulates the dynamic rearrangement of the actin cytoskeleton by severing actin filaments (F-actin) and capping the newly generated barbed filament ends. Calcium ions and phosphatidylinositol 4,5-bisphosphate (PIP₂), in turn, control these activities. Ca²⁺ -binding within gelsolin primes the protein for binding actin filaments and, during the process, causes drastic rearrangement of its six domains (G1-G6). The significance of specific Ca²⁺ -binding events in this activation process is slowly emerging. A structural basis for the ability of PIP₂ to inhibit ab initio interactions of gelsolin with actin filaments, as well as to induce dissociation of gelsolin already bound to F-actin, remains largely unknown. The reported binding of ATP to gelsolin has been suggested to modulate gelsolin-actin interactions. Association of gelsolin with both of these phosphate-rich molecules is sensitive to the presence of calcium ions. In the projects described in this thesis, I report results of X-ray crystallographic and computational molecular docking experiments to investigate aspects of the regulation of gelsolin by ATP, PIP₂ and calcium ions. Successful introduction of ATP into crystals of inactive gelsolin identify for the first time detailed features of its molecular interactions with gelsolin. Computations confirm the binding of ATP to the observed site to be strong and specific. Demonstration that the ATP-binding site spans both the N- and C-terminal halves of the protein explains the decreased affinity of gelsolin for this ligand in the presence of calcium ions, which induce separation of the halves as part of the activation process. Computational docking experiments suggest residual affinity of activated gelsolin for ATP to be retained at the G2-G3 interface with actin. We propose a model for PIP₂ -binding in the same surface-exposed pocket in gelsolin that is associated with binding ATP phosphates. The model concurs with both the previously reported binding of PIP₂ in this vicinity on gelsolin and the higher affinity of gelsolin for PIP₂ than for ATP. The model, together with the structure of the G1-G3/actin complex, provide insight into the roles of putative PIP₂ -binding sites in both the N- and C-terminal halves of gelsolin. Lastly, exchangeability of metal ions in crystals of G1-G3/actin reflects the transient nature of Ca²⁺ -binding in G2 and helps to explain the loss of local structural stability in a gelsolin mutant that experiences enhanced susceptibility to proteolysis.Science, Faculty ofChemistry, Department ofGraduat

Localization of calponin binding sites in the structure of 90 kDa heat shock protein (Hsp90)

AbstractThe 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 Mr in the range of 26–41 kDa. Peptides with Mr 39–41 kDa were represented by large N-terminal and central peptides starting at residue 283 of the α-isoform of Hsp90. All sites phosphorylated by casein kinase II were located in the large 39–41 kDa peptides. Peptides with Mr 26–27 kDa were represented by short N-terminal and central peptides starting at Glu-400 of the α-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 α-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

Crystal structure of c5321 : a protective antigen present in uropathogenic Escherichia coli strains displaying an SLR fold

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 crystalcontacts 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