The Three-dimensional Structure Of The Extracellular Adhesion Domain Of HpaA From Helicobacter Pylori

The adherence of Helicobacter pylori?H.pylori?to gastric epithelial cells,which contributed to the initial colonization and persistence of the bacteria in human stomach,was associated with a number of outer membrane proteins?OMPs?such as HpaA,BabA/B,AlpA/B,etc.H.pylori adhesin A?HpaA?has been detected on the surface and the flagellar sheath of H.pylori,and it is a member of neuraminyllactose-binding hemagglutinin precursor?NLBH?.NLBH is found exclusively in Helicobacter and bind to sialic acid containing moiety of molecules exposed on the gastric epithelium.However,the basis of binding specificity to sialoglycoconjugates is still poor understood due to the lack of detailed information for NLBH.Thus,neither the mechanism of adhesion or colonization of H.pylori mediated by HpaA is well identified,nor the receptors on gastric epithelial cells accounted for the attachment.Furthermore,as HpaA is demonstrated a promising immunogen for use in a vaccine against H.pylori,the reveal of its three-dimensional structure may provide insight into epitopes involved in protective immunity and aid in vaccine design.The objective of this study was to express,purify,crystallize and determine the crystal structure of HpaA.The protein contains an N-terminal trans-membrane domain which anchors HpaA to the outer membrane of H.pylori and a predicted highly flexible loop region linked the N-terminal and C-terminal extracellular adhesion region.Here,the hp0797 gene segment encoding the C-terminal soluble domain of HpaA was amplified from H.pylori 26695 genomic DNA and cloned into the pET22b?+?vector.Three recombinant constructions without the predicted disorder region of HpaA were tried to increase the chance of crystallization,and the expressed proteins were designated rHpaA36,rHpaA31,and rHpaA53,respectively.Each rHpaA with a C-terminal His tag was expressed as a soluble species in the E.coli BL21?DE3?host strain with a high yield and purified to ⁹5% with a dimeric form in solution.After initial crystallization screening and optimization,rHpaA36 and rHpaA31 appeared elusive and stubborn,while single crystals of rHpaA53 were suitable for a high-resolution X-ray diffraction analysis.The purified rHpaA53 crystallized in two different crystal forms using lithium sulfate or ammonium sulfate as the precipitant.The former crystal with approximate dimensions of 800 × 280 × 60 ?m diffracted X-ray to 2.03 ? resolution,analysis of the data indicated that the crystal was hexagonal,belonged to space group P65,with one molecule in the crystallographic asymmetric unit and unit-cell parameters a=b=95.33,c = 54.81 ?,? = 120°.The latter crystal with approximate dimensions of 70 × 70 × 30 ?m diffracted X-ray to 2.60 ? resolution,orthorhombic,space group P22₁2₁,with eight molecules in the crystallographic asymmetric unit and unit-cell parameters a = 106.31,b = 121.008,c = 190.56 ?.Both sets of diffraction data were processed and structures were solved by the molecular-replaement method.In conclusion,we determined the crystal structure of the HpaA extracellular adhesion domain and established structural basis for understanding the adhesion fuction of HpaA as well as the NLBH family.The structure will also be instrumental for the development of new strategies to prevent and treat H.pylori infections by inducing strong immune responses or through the inhibition of attachment to the gastric mucosa.