Cl(93é€l OCR Output

and MerA.

lf these studies are successful we will perform preliminary l99’"Hg—TDPAC studies on MerBin order to probe the origin of ultrasensitivity and metal selectivity.try in the i\/IerR protein and to elucidate the interaction of Hg2+/Cd2+—MerR with the DNAby time differential perturbed angular correlation (TDPAC) to identify their metal site geomeWe propose to utilize the nuclear quadrupole interaction (NQI) of l99'"Hg and m"‘Cd detectedmercuric ion coordination environment. respectively.stulated to arise from the unusual topology of the transcription complex and a rare trigonaltranscription. The molecular origin of the ultrasensitivity and selectivity phenomena are poother metal ions (eg. Cd2+) can bind to NlerR. Hg is uniquely effective at stimulating DNAsence of very low concentrations (1()‘8 Nl) of Hg2+. MerR activates transcription. Althoughdetoxification proteins. ln the absence of Hg2"'. NlerR inhibits DNA transcription. In the preThe XlerR protein. a mercury responsive genetic switch. controls expression of the other Hg

A protein MerR. the organomercurial lyase RlerB and the mercuric ion reductase 3lerA.detoxification proteins New (Rx- : T. P. .—\. B. D). The main components are the regulatoryduction of mercuric ion to elementary Hg°. All three activities are performed by the mercurymechanism involves three distinct steps: (i) uptake: (ii) cleavage of organomercurials: (iii) reHg2+ and organomercurials to non—toxic elemental Hgo. The most common bacterial resistancegroups. Xlany bacteria contain an elaborate detoxification system for a metabolic conversion ofKlercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol

Summary

Spokesman and (Yontactman: \\·`. Troger

HOI"l.`Hl`({ .l’[C({l·('(lf School. Boston. .l[..4.. ['..S'..4.

Depart‘men2‘ of Biological C/iernisfry and -lI0[ecuIar Pharnzaco/0gy

CT. Walsh

Snza/I Drug Discovery. Genetics Institute, Carn.bridge. -liI..—l.. [,.5..4.

ILM. Comess

Un i1rer.sz'tiit L eipzig, Ge rm an yInstitut fiiir E.1·per·irncntr:He Physi/t. I-lbfcilung IYM}:/erzrr Fest/vdrperp/1y.si}I·

W. Troger. T. Butz. C. Lippert. B. Ctortecka. P. Schmidt and Lv. Schmidt

. . . . E11zymat1c Mercury Detox1Hcat1on: The Regulatory Protem MerRQ -’ "`

QQEM

Cp PROPOSAL TO THE ISOLDE COMMITTEE} * (/O TNS}/P

ll Januarv 1995U5SC000O0199

ISC / P 69CERN/ISC* ‘“`*" llllllllllllllllllllllllllllllllllll

CIIRRI LIBRARIES, GENEVA AR RESEARCHEL ROPN

89] and which binds to the next region. t.he operator/promoter segment (OP). In the absence of OCR Outputwhich is a 144 amino acid polypeptide. which functions in the cell as a dimer [OHA 87. O HAThe mer operon (DNA. fig. 1]) begins with the regulatory gene MerR. [FOS T0]. the product of

Fig. 1: Schematic model of the Hg detoxification system of the plasmid pDI]1i355 [SIL

mcrP 9\" 2 H+

`_men,

Pcrblasm

hier Mcmbrano

Raductasc Cytoplasm"®

H v. ji [ ’/ v OHS Q _»' `~_ O /’i=Ao‘ no\\ /

Hg" ] ]Lyasc

at} l Y‘DNA \]o®0]*A€]l$G©,{`»©d( AL »O`$’>¢l©(`}@i

mcrF¢ OP merTmorP mcrA n·•crB merO

it will be reduced to Hgo [KIOO 90]. The function ofthe MerD protein is still unclear [BRO *6%].lyase) cleaves I·Ig—C` bonds and the resulting Hg2*‘ is given directly to the XIer_e\ protein whereduces Hg2"” to elementary Hg° [FOS T9. N.\I{ T9. MIS Sl]. The MerB protein (organomercurialHg"` through the membrane directly to the MerA protein (mercuric ion reductase] which reprotecting the cellular surface). brings it to the transport protein MerT. which transfers thediscussed only: the periplasmic scavanger protein MerP binds free Hg*+ immediately (thusproposal is devoted only to the MerR protein. the other detoxification enzymes are briefiyshows the organization of this genetic system of the plasmid pDI,'1358 [SIL S8]. Since thisresistance systems have been cloned and their DNA sequences determined [SIL 91]. Fig. Imechanisms to detoxify both. organomercurials and inorganic mercury salts. Eight mercuryWith this as backdrop, it is not surprising that several bacteria have developed resistancethiols and is much less toxic and volatilizes easily due to its high vapor pressure.sewage and sediments [RID TT]. In contrast. metallic mercury displays no propensity to bindnatural form is methyl mercury. arising from biomethylation of mercuric ions via methyl Big inmercury, merthiolate and mercurochrome have been used as disinfectants. The predominantcoal and petroleum products. Mercury compounds are used in industry as catalysts. Phenyl—sources of environmental contamination of mercury from human activities are from burningMercury compounds are leached from natural sources (weathering of rocks and soil). Majorfor Hg2"'-thiol chelates range from 10*38 M to 10‘*5 [CAS 80]).are highly toxic to living systems by virtue of tight binding to sulfur (at physiological pH. ILHSS(Cys) residues in proteins and glutathiones. Consequently. organomercurials and mercuric ionsCellular functions and enzymatic activities are often dependent on critical thiols such as cysteine

I. Motivation

tivity and metal selectivity of the regulatory protein. OCR Outputthe MerR protein may lead to a better understanding of the molecular origin of the ultrasensi—Hg2+ appears feasible. A comparison of the 199mHg NQI parameters with thOse of 1u'”Cd inUsing the NQl’s of the model compounds as fingerprints an unambiguous identification of themore yields information concerning structural distortions within a given coordination number.powerful tool to distinguish between two-, three-, and fourfold Hg2+—coordination, and furtherquadrupole interaction (NQI) of 199mHg via time differential perturbed angular correlation is aWith well-chosen model compounds we demonstrate that the measurement of the nuclearand four-fold [SHE 89b] coordinations are discussed.the Hg2+-coordination of MerR, two—fold [PEN 89], three-fold [RAL 90, COM 94a, HEL 90].the lack of nuclear magnetic resonance or crystallographic structural data in order to elucidatescopy [WAT 90], and X-ray absorption spectroscopy [NVRI 90. CLA 93] were applied due tomutagenesis [SHE 89b] of several residues. titration experiments [SHE89a]. UV/\i’is spectromost likely ligands of the bound Hg" [OHA ST. MIS 84]. Although random and site directedThe amino acid sequence of the NlerR protein is known: it contains 4 cysteins which are the

pression of the Mer proteins [Ans 92].complex induces a distortion of the DNA. thus allowing the transcription and ex

Fig. 2: The functioning of the regulatory protein MerR.. Only the DNA bound Hg2+-Mer}?

-3 3° underwound

ActivatorRggggorgHg-MerR

`lo

+ Hg(II)

-25 -10-35

not resistant against Cd2+, Zn2+, or Aulconcentrations much higher (>< 104) than Hg"' [RAL 90]. Cells containing the mer operon areCd"', Zn2+, and Au1+ substitute Hg2+ for the induction of transcription in vitro. however attranscriptionis activated by twisting the DNA [ANS 92, LIV 93].2 the supposed interaction of MerR and the Hg2+/MerR complex. respectively, is shown: theproteines Meri- (* = T, P, A, B, D) in response to the concentration of mercuric ions. In Hg.system. By this, the MerR regulatory protein controls the expression of the mercury resistancefunction to stimulate the transcription several 100 times, ”turning on” the mercuric resistance104 excess of exogenous thiols [SHE 89b] - the metal bridged dimer acts in a positive regulatoryare added - a concentration of 10‘8 M of Hg2+ is sufiicient [LUN 87] even in the presence of aHg2+ ions, the MerR represses the transcription of the next structural genes. When Hg2+ ions

studies on 1umCd labelled Meri} protein appear feasible. OCR Outputextensive TDPAC studies on blue copper proteins [TRO 93, 94, DAN 95]. Thus, fingerprintthe Cd-NQI parameters for a lot of different coordination geometries a.re known based onNo 111'"Cd-TDPAC experiments with the MerR protein were performed until now. However,twin on the other monomer [COM 94b[,two binding geometries are not unexpected.involved in the Hg2+-coordination can be easily oxidized to form a homodisuliide linkage to itsdistorted trigonal or two-fold tilted Hg coordination. Because one of the cysteines probablya high asymmetry parameter. According to the vQ—r;—diagram, this can only be explained beThe fit (ii) also yields two high frequencies, however one site has a low and an the other sideand low asymmetry parameters, indicating a nearly linear S-Hg-S coordination for both sites.shown in the time spectrum (fig. 5, left, solid and dotted line). The fit yields high frequenciesapproximately equal populations were necessary to get a reasonable fit. Two possible fits areunsatisfactory statistical quality of the spectrum. lt turned out that two inequivalent sites withlabelling procedure only a very limited 199mHg-binding yield could be achieved resulting in anits Fourier transform are shown in fig. 5. Unfortunately, due to several problems during theprotein to proof the principal feasibility of this approach. The time spectrum obtained andUntil now, only one preliminary 199mHg TDPAC experiment was performed with the MerRcoordination unambiguously.coordinations are well separated from each other. This allows to identify an unknown Hg-Sdecreasing coordination number (see fig. 3, 4). Furthermore, the NQI parameters of differentcharge model of the first coordination sphere. Obviously, the NQI frequency increases withthe corresponding undistorted coordination geometries were estimated be means of a pointstretched two-fold sulfur coordination of Hg. In the UQ-17 diagram the NQI para.meters ofquadratic, Hg—thiophenol [CHR 84] for a distorted trigonal and Hg-cysteine [But 9lb[ for amercaptide [BUT 9l b] for a distorted tetrahedral, Hg-thiocrownether [SET 91) for a distortedsulfur coordination of mercury are summarized. The model compounds are Hg-tert-butylparameters obtained by a least squares lit (solid lines in the time spectra of fig. 3) and theIn fig, 3 the structures and the 199mHg TDPAC time spectra are shown; in fig. 4 the NQIinvestigated appropriate model compounds with known X-ray structures by 199mHg-TDPAC.

Hg is a powerful tool to distinguish between two-, three-, four-fold Hg-S coordination we199m

In order to demonstrate that the measurement of the nuclear quadrupole interaction (NQI) of

II. Actual Status of Research

compounds (bottom). OCR Output(top). Diagram of the NQI parameters uq and r; of the investigated Hg·S model

Fig. 4: Table of the NQI parameters to, ry, and VQ ofthe investigated Hg—S model compounds

thcpoimcbugemodcl.distortions of rh: first coordination sphere in Hg coomlimdonofthe NQ[p¤.¤mc:e1-s va mdn for dccxcasixig ccrnhcdnl, square plzmz, uigonalgnd linear

O Expcrimcnml dau Thcd.a.shcdli.nc.sdcpic:tl1c=11pcctcdchanges O Theoretical valuc tbrvq mdryfbr undismrwcd

V,. [GI-Izl0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

c o0.00l-<>——------»Hg_(SBu L

\ e0.102Hg(§_CyS)j

0.20

0.30_. Hg<16S4> Hg(SPh)1

0.40

0.50 planar060 F [C!iI'8.l1€d1'&l gqugye trjgonal linear

0-80¥ 0.70 r3—fo1d 2—fo1di4-fcld

0.90

1.00

F(S—Hg); 2.60 .5.0.Sl1(4) | 0.32T(8) { 0.TT5(T)AS—Hg-S: 92°. 162°[Hg(16S4)](C1O)4

4—fold. distorted quadratic

LS—HE·S: 90"

Hg(SBu‘)2 LS—Hg—S: 8T° F(S—Hg): 2.63 A [ 0.333(3l [ 0 0.353M)4—fold, distorted tetrahedral

LS—Hg—S: 13T.1°LS—Hg-S: 102.9** F(S—Hg): 2.45 X

1.201(3) | 0.31445) | 1.156(5)[Hg(SPh)3][NBu".,] LS—Hg-S: 120°3—fold, plana.r,distorted trigonal

F(S—Hg): 234 A1.360(16) | 0.149(1T) | 1.409(24)LS—Hg—S: 1T0°Hg(S—Cys)2

2—fold, stretched

OCR Outputcompound S—coo·rdinotion of Hg | to [Grad/sf | ly l UQ [GH;]

Due to the rather short half-life of the m"‘C<1— and 199"‘Hg—isotopes the collection point. the OCR Output

should be air~conditioned.

centrifuge and a successful labelling of the enzymes even on a hot summers day this laboratorycooling centrifuge and a cryobox is needed. To guarantee a perfect functioning of the coolingof the hoods of the Solid State Physics Laboratory is required. There. also some space for thetated/centrifuged, frozen or immobilized by adding sucrose. For the final preparation steps one

“""Cd— or ‘9""‘Hg solution with or without carrier for 5 to 230 minutes the enzymes are precipiconcentrations from pM to nM. After the incubation of the detoxification enzymes with the(CdC12. HgC12 dissolved in adequate buffer solutions) to the molten ice to increase the metalown target holder at the collection point. Occasionally, we subsequently add inactive carrierinto ice at 100 K (in order to guarantee the vacuum in the beamline). We will provide ourThe proposed experiments require the implantation of u1'"C`r1 and l°9"1Hg for 10 — 60 minutescooling unit is about 30 mz.called P.\(i'—canieras TBLLT S0}. The spare retpiired for the two P.\l `-·‘an1e1·as and the sample

A The 7-1 ecorrelation spectroscopy utilizes two G—detector Bal?;—TDP.\(.` spectrometers. the sotransported to ISOLDE on dry ice lz- —S0"(.`).spectroscopy in the divison of Nuclear Solid State Physics in Leipzig. The samples will besamples will be checked and prepared for the experiments at ISOLDE with l'\`/\'ls absorptionGenetics Institute and the Harvard Medical School in Boston. l`.S.A.. Furthermore. the proteinsupervision of Prof. (;'.T. \\t`alsh Rllil Dr. KAL Corness in the biocliemical laboratories of theThe isolation. purification and cliaracterisation of the Xlerfi protein will he done under the

III. Experimental Metliods

128(11 Grad/s. ry : 0. UQ = 1.3611) CH2.Grad/s. ry = 1. 1/Q : l).SZ1(2) GHZ: : 2. component: population 101 10171. ,.· =[ii) Dotted line: yl :0.Tl. 1. component: population 3116`1€Z. ..· = 1.38121.1; = 1.33(2) Grad/s. 1] : 0.10(4). 1zQ = 1.36lll GHZ.Grad/s. ry = 0.126). VQ = 1.49(5) GHZ: 2. component: population 43(S17.(i) Solid line: y2 :0.80. 1. component: population $(717. 0; : 1.43(21correspond to two possible least squares fits:regulamry protein )[erR. The solid and dotted lines in the time spec11·11111

Fig. 5: 199’"Hg~TDPAC time spectrum (left) and its Fourier transf01·r11 (righw 0i` the

Zeit [ns] Frcqucnz [M1ad/s]0 4 8 12 16 20 5000 10000 15000

0.0-0.02

0.00

0.022.0

an 0.04

UC, mia (NH.),SO. gellllx*99mHg-MMR-Proteinws r W, M5 (~.i§,s¤, §2.%1

¤99¤·H -M R·P z` "4.0

other groups. OCR Outputin the 1986 Users Guide (.1. Kluge ed.). and therefore we would like t.o share the beam withWe can utilize the beam about 10 — 60 minutes every 3 hours only, assuming the yields listed

2 >< 13 shifts *99mHg (Pb plasma source)2 >< 13 shifts ‘“’"Cd (Sn plasma source)

'We ask for a total of 52 shifts within one vear to be used as follows:

V. Beam Time Request

lead to a new ISOLDE proposal dedicated to these proteins.the XlerB protein (organomercurial lyase). lf successful. these investigations will

l\*) First exploratory 199'"Hg studies on the NlerA protein (mercuric ion reductase) andstant, origin of the ultrasensitivity.MerR-DNA binding studies: influence on the coordination geometry. binding con

selectivitv of NlerR.

(iii) Comparative coordination studies with 111mCd and 199mHg to elucidate the metalconcentrations of different reducing agents to stabilize the thiols for Hg/Cd binding`.’avoid or to diminish the bridging of cystein thiolgroups. VVhat are the minimal

(ii) Tests of several reducing agents (eg., 2-mercaptoethanol. ascorbate) in order tobenzoate) [SHB 89b]; in situ monitoring with UV/\°is and / or TDPAC.tein with DTNB(5.5`—dithiobis(2—nitrobenzoic acid)) or pHMB(p-(hydroxi—mercury)metry of Hg2+ in the MerR protein: extensive titration studies of the pro

(i) Investigations of the influence of bridged cystein thiols on the coordination geo

IV. Proposed Experiments

Koordinationsstudien mit 199'"Hg”, Bu 594/9-1.This work is supported by the DFG—grant ”Enzymatische Quecksilber—Detoxifikation: Hg;would very much appreciate the very efficient support of Dr. Doris Forkel—Wirth.We need some manpower (womanpower) for the beam setting and nuclear spectroscopy andA room in the light building to store equipment between the beam times is also required.no inflammable, explosive chemicals.There are no safety risks: all chemicals used are non—toxic due to the very low concentrations:

of ISOLDE.

other, i.e. the best collection points for us would be close to the stairs in the experimental hallPAC—camera.s and the Solid State Physics Laboratory have t0 be as close as possible to eacli

A. M. Chakrabarty, and S.E. Kornguth, Eds., Kluwer, Boston 1991. 265-186 OCR Outputmediation, in ”Bi0technology: Bridging Research and Applications”,D.Kame1y,

[SIL 91] S. Silver, Bacterial heavy metal resistance systems and possibility of bi0re—[SIL 88] S. Silver and TK. Misra. Ann. Rev. Microbiol. Q(1988)717—743

6145

[SHE 89b] L.M. Shewchuk, G.L. Verdine. H. Nash and C.T. Wlalsh. BiochemistryQ§_(1989)6140—[SHE 89a] LM. Shewchuk. G.L. Yerdine. and C.T. Walsh. Biochemistry §(1989)2331—2339

1991 )3652—3656

[SET 91] WN. Setzer, Y. Tang. GHI. Grant. and D.G. VanDerveer. [norg. Chem.[RID TT] W.P. Ridley, L..l. Dizikes. and .l.M. Wiood: Science 1_9_7(1977)329·332

3850 und Adv. Inorg. Biochem. §(1990)1—31

D.M. Ralston and T.\`. O`Hall0rau. Proc. Natl. .—1cml. Sci. I’..S..—1. ;7(l990)3840e[RAL 90]@(1989)11Te11S

[PEN 89] .].E. Penner»Hahn. H.T. Tsang. T.\¤’. O`Halloran. and .I. Wlright. Physica B

Dekker. NY 1989. Vol.25. 105v1—L0

[OHA 89] T.\’. O°Halloran in "Metal Ions in Biological Systems". ed. by H. Sigel. MarcelT.V. O’Hall0ran and C.T. Walsh. Science &§(1987)211[OHA 87]H. Nakahara, S. Silver. T. Miki. and R.H. Rownd. J. Bacteriol. l_J&(1979)161—166[NAK T9]Acc. Chem. Res. jZj(1990)301-308

M..I. Moore. M.D. Distephano. I..D. Zydowski. R.T. (ilumniings. and C. T. Walsh.[R100 90]

Natl. Acad. Sci. VSA S1|19S1I5973—5979

T.K. Misra. N.L. Brown. D. Fritzinger. R. Pridmore. W`. Barnes. and S.Silv<-r. Proc.[MIS 84]P.I.und and N.Brown. (}'E.\’E Qi 1987) 207[LUN ST]@(1993)2631—2639

LW'. Lee, V. Livrelli. S.—.l. Park. RA. Totis and .\.O. Summers. J. Biol. Chem.

V. Livrelli. I.W. Lee. and A.O. Summers. J. Biol. Chem. @(1993]2623—2631 und[LIV 9:3].l.D. Helman. B.T. Ballard. and C.T. Wlalsh. Scieizce g(1990)946·9—1S[HEL 90]T..I. Foster and H. Nakahara. .]. Bacteriol. _L1_Q(1979)301—305[FOS T9]serschmidt. J. Biol. Chem. QQ(1995)573—580W'. Tréger, G.W. Canters. C.W.G. Hoitink. G. Karlsson. O. Hansson. and A. Mes·E. Danielsen. R. Bauer. L. Hemmingsen. M.—L. Andersen. M..l.Bjerrum. T. Butz.[DAN 95]private communication[COM 94b]Q(1994)4175—4186

K.M. Comess. L.M. Sliewcluik. K. Ivanetich. (i`.T. Wialsh. Biocheiniisfry[COM 94a]the Arnerican Chemical Soc. &(1993)—151K. Clark, L. Utschig, T.\·’. O`Ha1l0ran. and .I.Penner—Ha1in. Abstracts of Papers of[CLA 93]C. Christou, K. Folting, and .l.C. Huliman. Polyhedron §(1984)1247—1253[CHR 84].l.S. Casas, M.M. Jones. .]. [norg. .Vucl. Chem. ;l_Q(1980)99—102[CAS 80]T. Butz, W. Tréger, O. Nuyken. Z. Naturfoinsch. @(1991)217[BUT 9lb] _T. Butz, Th. V61k1, and O. Nuyken. J. Organomet. Chem. 1;1;9(1991)217[BUT 91a,]T. Butz, S. Saibene, Th. Fraenzke. and M. Wveber. ;Vuc. Inst. ;1Ieth. ;&(1989)117[BUT 89]Gen. Genet. gQ_2(1986)143—151

N.L. Brown, T.K. Misra. .1.N. Winnie. A. Schmidt. M. Seifli. and S. Silver. .110/ec.[BRO 86]A.Z. Ansari. M.L. Chael. T.V. O`Ha1loran. Nature QJj(1992)S7—89[ANS 92]

References

Chem. $00. lQ(1990)2~13~l—2435J.G. `\/Vright, Him—Ta.i Tsang, J.E. Per1m=:r—Hahn. and T.V. O7H&TTOFH.I1. J. Am.[WRI 90]T.V. O’Ha1l0ra.n, J. Am. Chem. Soc. }_TQ(1990)2824—2826S.P. Wvatton, J.G. Wright. F.M. MacD0nell. .].W. Bryson, M. Sabat. and[WAT 90]W. Trégcr, PhD—Thesis. Physikdepa.rtment. TU Miinchen[TRO 94]Huber, G.VV. Canters. and T. den Blaauwcn. Hyp. Int. @(1993)1133—l14lVV. Tréger, T. Butz. E. Damielsen. R. Bauer. .\. NT€‘SS€l`S(`h1I]j(T[. L'. Thoeues. R.[TRO 9:3]