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. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/188237 |
| Date | January 2012 |
| Creators | Cheng, Tianfan., 程天凡. |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B48199199 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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