Structural and functional aspects of the multifaceted SlyD in Helicobacter pylori

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.