Huang, JD

Sun, HZ

Tanner, JA

Watt, RM

Hong Kong Medical Journal

HKU Scholars Hub

Title Determination of the functions of the putative metal-bindingdomain of the SCV helicase.Author(s) Huang, JD; Sun, HZ; Tanner, JA; Watt, RMCitation Hong Kong Medical Journal, 2009, v. 15 suppl. 6, p. 15-16Issued Date 2009URL http://hdl.handle.net/10722/124519Rights Creative Commons: Attribution 3.0 Hong Kong LicenseHong Kong Med J Vol 15 No 5 Supplement 6 October 2009      15RESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASESDepartment of Biochemistry, The University of Hong KongJD Huang, JA TannerDepartment of Chemistry, The University of Hong KongHZ SunOral Biosciences, Faculty of Dentistry, The University of Hong Kong RM WattRFCID project number: 02040192Principal applicant and corresponding author:Dr Jiandong HuangDepartment of Biochemistry, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, ChinaTel: (852) 2819 2810Fax: (852) 2855 1254E-mail: jdhuang@hkucc.hku.hkJD Huang 黃建東HZ Sun 孫紅哲JA TannerRM Watt Determination of the functions of the putative metal-binding domain of the SCV helicaseKey Messages1. The inhibition of SARS coronavirus (SCV) helicase by bismuth compounds is mediated via the abilities of bismuth ions to displace (essentially required) zinc ions that are bound to cysteine residues located within the metal-binding domain.2. Several novel bismuth compounds targeting SCV helicase have been synthesised. These compounds can inhibit SCV helicase activity and viral growth and potentially target other viruses.Hong Kong Med J 2009;15(Suppl 6):S15-6IntroductionSevere acute respiratory syndrome (SARS) was first recognised in late 2002 and was shown to be caused by the SARS coronavirus (SCV).1,2 We have reported the purification and characterisation of the SCV helicase,3 and identified a number of lead compounds able to inhibit SCV helicase activities and viral growth.4,5 One of these contained bismuth—ranitidine bismuth citrate (RBC). To reveal the mechanisms underlying RBC action, we investigated the interaction between zinc, bismuth, and the helicase protein. Mutational analysis was used to pinpoint the key amino acid residues necessary for the helicase function. Several novel bismuth compounds with SCV inhibiting activity were synthesised. This study was conducted from February 2005 to January 2007.Characterisation of the Zn2+- SARS coronavirus helicase interactionThe N-terminal of the SCV helicase consisted of a cysteine rich domain: the metal-binding domain (MBD). Sequence alignment suggested that this entailed Zn2+ binding. To determine how helicase binds to Zn2+ ions, we used 4-(2-pyridylazo) resorcinol / p-hydroxymercuriphenylsulfonic acid assay to demonstrate that zinc did indeed bind to the MBD of SCV helicase via the cysteine residues (Fig 1).  To study the effect on the structure of MBD upon zinc binding, we measured the circular dichroism of MBD (Fig 2). The zinc-bound form of MBD showed the characteristics expected of high helix content. However, addition of excess ethylenediaminetetraacetic acid to remove the zinc ions led to a decrease in ultraviolet light absorption, suggesting that the protein changed to a random structure in the absence of Zn2+. Mutational analysis of the SARS coronavirus helicase metal-binding domainTo define the precise role of individual amino acids within the SCV helicase MBD, we individually mutated several key cysteine residues within the MBD. The mutant proteins were then studied by measuring their ATPase and unwinding activities. All the mutant proteins lost their activities.Determination of the mechanism of ranitidine bismuth citrate–mediated inhibition of SARS coronavirus helicase activityBi3+ is known to have a high affinity for thiolate sulphur.6 We therefore measured the ultraviolet light absorbance spectrum of the SCV helicase, both in the absence and presence of RBC. The cysteine-rich MBD was the target of the bismuth ion. Addition of Bi3+ to the Zn2+-bound form of MBD led to an increase in ultraviolet light absorbance, indicative of replacement of Zn2+ by Bi3+.7Synthesis of bismuth complexes against SARS coronavirusBismuth compounds effectively inhibited SCV growth in cell culture. The Huang et al16      Hong Kong Med J Vol 15 No 5 Supplement 6 October 2009mechanism was through replacement of zinc bound to the MBD. We then designed and synthesised a series of bismuth complexes, including complexes of: bismuth porphyrin, bismuth macrocyclin, bismuth 12-crown-4, bismuth bipyridine, bismuth phenanthroline, bismuth nitrilotriacetate, bismuth ethylenediaminetetraacetic acid, and bismuth acetohydroxamate.3 The newly synthesised compounds were tested for their activity against SARS helicase. The two bismuth porphyrin complexes and RBC exhibited the the most significant inhibition activities as revealed by in vitro experiments.8ConclusionWe determined the interaction between zinc, bismuth, and the helicase protein, enabling us to construct mechanistic models for bismuth-mediated inhibition of the SCV helicase functions. The zinc ions bound to the MBD of SCV helicase were replaced by bismuth ions upon addition of RBC and other bismuth compounds, resulting in dysfunction of the helicase. These compounds may also be used to inhibit other viruses.AcknowledgementsThis study was supported by the Research Fund for the 1.501.351.201.050.900.750.600.450.300.150.00440 460 480 500 520 540 560 580 600Zinc released by p-hydroxymercuri-phenylsulfonic acidZinc released by bismuthFig 1. Zinc that binds to the metal-binding domain can be replaced by bismuth2.50.0-2.5190 200 210 220 230 240 250MBD and ethylenediaminetetraacetic acidMBD and zincFig 2. Circular dichroism of metal-binding domain (MBD) with and without zinc bindingControl of Infectious Diseases (#02040192), Food and Health Bureau, Hong Kong SAR Government. We thank all the research workers for their hard work.References1. Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003;348:1953-66.2. Peiris JS, Lai ST, Poon LL, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003;361:1319-25.3. Tanner JA, Watt RM, Chai YB, et al. The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5’ to 3’ viral helicases. J Biol Chem 2003;278:39578-82.4. Kao RY, Tsui WH, Lee TS, et al. Identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics. Chem Biol 2004;11:1293-9.5. Tanner JA, Zheng BJ, Zhou J, et al. The adamantane-derived bananins are potent inhibitors of the helicase activities and replication of SARS coronavirus. Chem Biol 2005;12:303-11.6. Rao N, Feldman S. Disposition of bismuth in the rat. II. Pharmacokinetics and biliary excretion. Pharm Res 1990;7:237-41.7. Yang N, Tanner JA, Zheng BJ, et al. Bismuth complexes inhibit the SARS coronavirus. Angew Chem Int Ed Engl 2007;46:6464-8.8. Yang N, Tanner JA, Wang Z, et al. Inhibition of SARS coronavirus helicase by bismuth complexes. Chem Commun (Camb) 2007;42:4413-5.Wavelength (nm)AbsorbanceWavelength (nm)θ (deg cm2  dmol-1)

Determination of the functions of the putative metal-binding domain of the SCV helicase.

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Determination of the functions of the putative metal-binding domain of the SCV helicase.

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