Structural and Functional Aspects of the Multifaceted Slyd in Helicobacter Pylori

This dissertation, "Structural and Functional Aspects of the Multifaceted SlyD in Helicobacter Pylori" by Tianfan, Cheng, 程天凡, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: As a ubiquitous protein-folding helper in bacterial cytosol, SlyD is a peptidylprolyl isomerase (PPIase) of the FK506-binding protein (FKBP) family. It has two important functional domains, the IF (insert-in-flap) domain with chaperone activity and the FKBP domain with PPIase activity. It also possesses a histidine- and cysteine-rich C-terminal metal-binding domain, which binds to selected divalent metal ions (e.g. Ni2+, Zn2+) and is critical for participation in metal trafficking for metalloenzymes. SlyD from Helicobacter pylori was investigated both structurally and functionally by a variety of biophysical, biochemical and molecular biology techniques. HpSlyD was cloned, expressed and purified. It binds to Ni2+ and Zn2+ with dissociation constants (Kd) of 2.74 and 3.79 μM, respectively. Both Ni2+ and Zn2+ can competitively bind to HpSlyD. The C-terminus was demonstrated to convey nickel resistance in vivo. It also binds to Bi3+ with Kd of 4.4 10-24 M. Furthermore, Zn2+, Cu2+ and Bi3+ can induce the dimerization or oligomerization of HpSlyD. The solution structure of the C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC) was determined by NMR, which demonstrates that HpSlyDΔC folds into two well-separated, orientation-independent domains. Both the FKBP and IF domains fold into a structure consisting of a four-stranded antiparallel β-sheet and an α-helix. Binding of Ni2+ instead of Zn2+ induced the conformational changes in FKBP domain, where the active sites are positioned, suggesting a regulatory role of nickel on the function of HpSlyD. It was also confirmed that HpSlyD can associate with the Tat (twin-arginine translocation) signal peptide from small subunit of [NiFe] hydrogenase (HydA), an accessory protein HpHypB for [NiFe] hydrogenase mainly by the IF domain. Surprisingly HpSlyD was found to form a complex with HpUreE, a urease chaperone, indicative of the "cross-talk" between [NiFe] hydrogenase and urease. The possible mechanism of HpSlyD for the cooperation with HpHypB was also explored. In the presence of different metal ions, HpSlyD was shown to regulate the GTPase activity of HpHypB, implicating the possible metal transfer induced by HpSlyD. It was suggested that HpSlyD modulates the nickel insertion of [NiFe] hydrogenase by controlling the GTPase activity of HpHypB. In this thesis, the SlyD protein from H. pylori was shown as an important regulator for the activation of both [NiFe] hydrogenase and urease. DOI: 10.5353/th_b4819919 Subjects: Helicobacter pylori - Molecular aspects Protein binding