ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
SL11. The Role of Histidine-rich Proteins in Helicobacter pylori
H. Sun, S. Cun, R. Ge and Y. Zeng
Department of Chemistry, The University o Hong Kong, Pokfulam Road, Hong Kong
e-mail: hsun@hku.hk
Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative spiral-shaped bacterium which causes
chronic inflammation of the stomach and peptic ulcer formation in humans [1]. Bismuth compounds such as
colloidal bismuth subcitrate (De-Nol ® ) and ranitidine bismuth citrate (Pylorid ® ) have been widely used for the
treatment of H. pylori infection together with antibiotics [2,3]. Proteins and enzymes have been thought to be the
target(s) of bismuth in vivo. Our comparative proteomic data of H. pylori cells before and after treatment with
colloidal bismuth subcitrate showed that eight proteins are significantly up- or down-regulated. Using
immobilized-metal affinity chromatography (Bi- and Ni-IMAC), we isolated and subsequently identified seven
bismuth-binding proteins from H. pylori extracts [4]. One of the binding proteins, the Heat-Shock Protein A
(HspA), was then overexpressed and purified to confirm its metal-binding properties. Recombinant H. pylori
HspA was found to bind both Ni 2+ and Bi 3+ at its C-terminal histidine- and cysteine-rich domain of the proteins.
Binding of bismuth to the protein is much stronger than nickel. Importantly, Bi 3+ induces the structural changes
of the protein from its native form (heptamer) to a dimer [5]. When cultured in Ni 2+ -supplemented M9 minimal
media, E. coli BL21(DE3) expressing the wild-type HspA or C-terminal deletion mutant clearly indicated a role
that the C-terminus might protect cells from higher concentration of external Ni 2+ . In contrast, an opposite
phenomenon was observed when the same E. coli hosts were grown in Bi 3+ -supplemented media. The histidine-
and cysteine-rich domain may play a critical role in nickel homeostasis and bismuth susceptibility in vivo.
Binding of the metallodrug to other histidine-rich proteins was also characterized [6]. Our preliminary
bioinformatic search has found that there are actually any histidine-rich proteins and motifs in microorganisms
[7]. Hpn (28 His residues out of 60 aa) is a small cytoplasmic protein in H. pylori and is present as a multimer
with 20-mer being the predominant species in solution and binds to five Ni 2+ and four Bi 3+ per monomer
moderately (Kd of 7.1 and 11.1 µM respectively) [6]. Although in vitro, it binds to Cu 2+ stronger than Ni 2+ and
Bi 3+ , the in vivo protection by the protein is in the order of Ni 2+ > Bi 3+ > Cu 2+ [4]. Hpn may therefore serve to
buffer intracellular Ni 2+ in much the similar way to that the small and cysteine-rich protein, metallothionein
interacts with Zn 2+ /Cu + .
Acknowledgement: This work was supported by Research Grants Council of Hong Kong (HKU7039/04P,
HKU7043/06P, HKU1/07C), National Science Foundation of China and the University of Hong Kong!
References:
[1] B.J. Marshall, J.R. Warren, Lancet 1984, i, 1311.
[2] S. Suerbaum, P. Michetti, New Engl. J. Med. 2003, 347, 1175.
[3] (a) H. Sun, L. Zhang, K.Y. Szeto, Met. Ions Biol. Syst. 2004, 41, 333; (b) R.G. Ge, H. Sun, Acc. Chem. Res.
2007, 40, 267.
[4] R. Ge, X. Sun, Q. Gu, R.M. Watt, B.C.Y.;Wong, H.H.X. Xia, J. Huang, Q. He, H. Sun J. Biol. Inorg. Chem.
2007, 12, 831.
[5] S.J. Cun, H. Li, R. Ge, M.C. Lin, H. Sun, J. Biol. Chem. 2008, 283, 15142.
[6] (a) R. Ge, Y. Zhang, X. Sun, R.M. Watt, Q.Y. He, J. Huang, D.E. Wilcox, H. Sun, J. Am. Chem. Soc. 2006,
128, 11330; (b) R. Ge, R.M. Watt, X. Sun, J.A. Tanner, Q.Y. He, J. Huang, H. Sun, Biochem. J. 2006, 393,
285.
[7] J.F. Tomb et al (1997) Nature 388, 539-547.
_____________________________________________________________________
63