223

Recent biophysical studies on the interactions between leadand recombinant proteins and peptides that naturally bind zincor calcium have provided unparalleled insights into thebiological chemistry and molecular toxicology of lead. Thesestudies lay the foundation for the rational design of improvedmethods for detecting and treating lead poisoning.

AddressesDepartment of Chemistry, Northwestern University, 2145 Sheridan Road,Evanston, IL 60208-3113, USA; e-mail: h-godwin@northwestern.edu

Current Opinion in Chemical Biology 2001, 5:223–227

1367-5931/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsALAD δ-aminolevulinic acid dehydratase BLL blood lead levelCP-CCCC consensus zinc-binding domain with Cys4 zinc-binding

siteCP-CCHC consensus zinc-binding domain with Cys2HisCys zinc-

binding siteCP-CCHH consensus zinc-binding domain with naturally occurring

Cys2His2 zinc-binding siteHP2 human protamine 2PKC protein kinase C

IntroductionLead poisoning is the one of the most common pediatrichealth problems in the United States, affecting approxi-mately 890,000 children nationwide at any given time [1].The sources of this exposure are primarily leaded paint,which was not banned in the United States until 1978, andcontaminated soil. Recent studies suggest that much of theexisting soil contamination is probably a result of deposi-tion from exhaust from cars that used leaded gasoline, inaddition to leaded paint used on the exterior of buildings[2•]. Lead from these sources exists as or evolves into avariety of Pb2+ compounds, which are remarkably persis-tent in the environment. Unfortunately, these sources ofexposure are often expensive to remediate, and the politicssurrounding this issue are complex, suggesting that thelegacy of lead poisoning will continue to plague mankindfor many years to come [3,4].

Lead poisoning can afflict both children and adults, but thegreatest concern is for children, who experience symptomsat significantly lower blood lead levels (BLLs) than doadults [1]. In addition, children tend to develop permanentdevelopmental and neurological problems when exposedchronically to lead, whereas many of the symptoms experi-enced by adults are reversed when exposure is ceased.Although a broad range of epidemiological studies has beenconducted on lead poisoning, its molecular underpinningshave remained relatively obscure. However, recentadvances in biophysics and molecular biology have provid-ed the tools necessary to study the biological chemistry of

lead. These studies have helped to provide insights into thefollowing questions:

1. Out of all of the molecular targets that have been pro-posed for lead, which ones are physiologically relevant?

2. How does lead binding affect the structure and dynam-ics of target proteins?

3. Is lead binding to proteins under thermodynamic orkinetic control?

These studies and questions are the subject of this review,which focuses particularly on work from the past two years.

Molecular targets for leadSeveral classes of molecular targets have been proposed toaccount for the symptoms associated with lead poisoning.With few exceptions, these targets fall into two primarycategories: proteins that naturally bind calcium and pro-teins that naturally bind zinc [5–8,9••]. If these interactionsare to be physiologically relevant, lead must bind tightlyenough to the proposed target to occupy the site(s) underphysiologically relevant conditions. To ascertain whetherlead binds ‘tightly enough’, both the affinity of lead for theprotein and the concentration of free (or ‘bioavailable’)lead must be known. The affinity of lead for a given pro-tein is related to the disocciation constant for thelead–protein complex, . In the simple case, half of thepopulation of target protein (P) will have lead bound when

is equal to the concentration of ‘free’ (or ‘bioavail-able’) lead ([Pb]) in the cell. When lead is competing withanother metal ion (M, e.g. calcium or zinc) for a bindingsite, then the relative affinities and concentrations of thetwo metals must be considered, where:

How much bioavailable lead is present in cells?The amount of bioavailable (or ‘free’) lead has not beendetermined experimentally for most cell types, because ofthe lack of sensitive and selective fluorescent probes forlead [10•,11•]. A child is considered to have lead poisoningif he or she has a total BLL (as measured by atomic absorp-tion spectroscopy) greater than or equal to 10 µg/dl(0.5 µM or 100 parts per billion) [1]. By contrast, a typicalperson in the United States today who does not have leadpoisoning will have a BLL of ~2 µg/dl (0.1 µM or 20 partsper billion). It has been estimated that <5% of the totallead in blood is bound to plasma proteins [12] and that only0.01% of lead in plasma (~ picomolar lead) is bioavailable[13]. By contrast, the total concentrations of zinc and calci-um in human plasma are about 17 µM and 10–6 to 10–3 M,

×=

MPb

K

KMPPbP

Pbd

Md

PbdK

PbdK

The biological chemistry of leadHilary Arnold Godwin

respectively. The concentrations of bioavailable zinc andcalcium have been estimated to be 10–12 to 10–6 M and 10–8

to 10–6 M, respectively [13–16]. These estimates suggestthat, in general, lead must bind to proteins with a Kd of≤10–12 M and that lead must bind more tightly than thenative metal ion by at least one to three orders of magni-tude. Although it has been suggested that the localconcentration of lead may be higher in certain organelles(e.g. the nucleus) [17] or specific cell types (e.g. neurons)[12], these requirements serve as a reasonable minimalguideline until more detailed studies on lead biodistribu-tion become available.

Interactions between lead and zinc proteinsThe target for lead that has been studied most thoroughlyin vitro and in vivo is the zinc enzyme δ-aminolevulinicacid dehydratase (ALAD, also called porphobilinogen syn-thase) [8] (Figure 1). ALAD catalyzes the second reactionin the heme biosynthetic pathway, and inhibition of thisenzyme by lead explains (at least in part) the anemia oftenseen in adults and children with high BLLs (≥ 40 µg/dl)[8]. The log of the activity of ALAD in erythrocytesdecreases linearly with the individual’s BLL [18]; leadinhibits ALAD in vitro with an inhibition constant of0.07 pM (versus ) [19]. Why out of all thezinc enzymes in the body is ALAD the only one known tobe inhibited by lead? A recent crystal structure of yeastALAD offers insights into this ‘mystery’. Whereas mostzinc enzymes contain a zinc-binding site with a mixture ofhistidine, cysteine, and carboxylate residues, the yeast andmammalian forms of ALAD contain a unique catalytic

zinc-binding site with three cysteine residues [20,21•].When ALAD is co-crystallized with lead, lead binds prin-cipally to this three-cysteine site (Figure 1). Although thestructure of the lead protein is essentially identical to thatof the zinc form, incorporation of lead non-competitivelyinhibits substrate binding [20]. The suggestion by Erskineet al. [20] that lead prefers the Cys3 site in ALAD becausethis constitutes a tight binding site for lead is borne out byrecent model compound studies: lead binds more tightlythan zinc (about 500 to 1) to a novel tris-thiol ligand(tris(mercaptoarylimdazolyl)borate; Ar = Ph, Mes) but isconsiderably less Lewis-acidic in this environment than iszinc [22••]. These studies provoke several important ques-tions. Does lead bind tightly to other zinc proteins thatcontain three (or more) cysteines in their active sites?Could lead binding to any of these proteins account forsome of the other, more pressing, symptoms associatedwith lead poisoning?

Although no other catalytic zinc sites have been reportedthat contain three or more cysteine residues, there are sev-eral classes of proteins that contain structural zinc-bindingsites with three (Cys3His) or four (Cys4) cysteine residues.These proteins (e.g. retroviral nucleocapsid protein, withCys3His sites, and steroid receptors, with two tandem Cys4sites) act as transcription factors and regulate many of thedevelopmental processes associated with lead poisoning inchildren. Thus, the ability of lead to alter the activity ofthese proteins could account for the pressing developmen-tal problems associated with lead poisoning.

Until recently, the interactions between lead and tran-scription factors had not been probed directly, presumablybecause Pb2+ (electronic structure = [Xe]4f145d106s2) waswidely assumed to be spectrosopically silent. However,when lead binds to cysteine residues in proteins, itexhibits intense lead–thiolate charge-transfer bands in theultraviolet region of the electromagnetic spectrum[23,24,25••,26•] that can provide detailed and quantitativeinformation about lead–protein interactions [25••](Figure 2). Studies on a series of zinc-finger consensuspeptides with different metal-binding sites (consensuszinc-binding domain with naturally occurring Cys2His2zinc-binding site, CP-CCHH; consensus zinc-bindingdomain with Cys2HisCys zinc-binding site, CP-CCHC;and consensus zinc-binding domain with Cys4 zinc-bind-ing site, CP-CCCC) reveal that lead binds tightly tostructural zinc-binding sites ( ) andthat lead binds approximately two orders of magnitudemore tightly than zinc to the Cys4 site [25••]. Furthermore,competition experiments reveal that lead readily displaceszinc from the Cys4 site, which suggests that metal bindingis under thermodynamic, rather than kinetic, control. 1HNMR studies on a Cys2HisCys domain from HIV-nucleo-capsid protein and circular dichroism studies on both thisdomain and CP-CCCC reveal that even though lead bindstightly to these domains, it does not promote proper fold-ing of the site [25••]. These studies lay the foundation for

M10to10 149 −−=PbdK

pM1.6=ZnmK

224 Bio-inorganic chemistry

Figure 1

Lead targets proteins that naturally bind calcium and zinc. Examples ofproteins that are targeted by lead include synaptotagmin, which actsas a calcium sensor in neurotransmission, and ALAD, the secondenzyme in the heme biosynthetic pathway. Each of these proteins wascrystallized with lead as a heavy atom derivative ([8,19,35,36]; SuttonRB, personal communication). These studies revealed that, despite itssize, lead (1.19 Å, blue sphere and circles) can substitute for calcium(0.99 Å, green spheres) in synaptotagmin ([35,36]; Sutton RB,personal communication) and zinc (0.74 Å, red spheres) in ALAD[8,19]. Protein coordinates were obtained from the Protein Data Bank(http://www.rcsb.org/pdb/).

Pb2+ (1.19 Å)Ca2+ (0.99 Å)

Synaptotagmin

Zn2+ (0.74 Å)

ALADCurrent Opinion in Chemical Biology

detailed molecular studies on the interactions betweenlead and larger proteins that contain structural zinc-bind-ing sites. Parallel studies have recently been reported forthe interactions of lead with the zinc protein human prota-mine 2 (HP2), which plays an important role inspermatogenesis; interactions between lead and HP2 mayaccount for increased infertility rates in men who are occu-pationally exposed to lead. Lead binds to thiol groups inHP2 and alters the structure of the protein [26•]. Theobservation that lead alters the structure of the zinc pro-teins to which it binds is not surprising given thedifferences in coordination preferences (6–8 for Pb2+ ver-sus 4 for Zn2+ and geometries (irregular for Pb2+ versustetrahedral for Zn2+) in structural zinc-binding sites for thetwo metals [27]. However, these results are extremelyimportant because they suggest that lead binding to struc-tural zinc-binding domains should disrupt theDNA-binding activity of the proteins and transcription fac-tors in which they are found.

The effects of lead on the DNA-binding activity ofCys2His2 zinc-finger proteins such as TFIIIA were recent-ly studied using DNase I protection assays. These studiesreveal that when sufficiently high levels of lead are present(5–20 µM), then TFIIIA does not bind DNA [28•] (see alsoUpdate). In addition, recent studies by Goldstein, Bressler,and co-workers [29•] reveal that lead alters expression ofimmediate early genes in PC12 cells, in a protein kinase C(PKC)-dependent fashion. Whether the alterations in geneexpression observed arise from interaction of Pb2+ with a

transcription factor upstream of PKC or solely by directactivation of PKC (see below) is not known. These studiespoint to the need for not only more detailed biophysicalstudies on a wide range of proposed targets, but also forcomprehensive, systemic studies on the mechanisms bywhich lead alters signal transduction in cells.

Interactions between lead and calcium proteinsIn addition to causing developmental problems, lead poison-ing results in pervasive neurological problems in bothchildren and adults [30]. Lead interferes with the ability ofcalcium to trigger exocytosis of neurotransmitters in neuronalcells [31], suggesting that lead might generally target proteinsinvolved in calcium-mediated signal transduction [6]. Thishypothesis was bolstered by the observation by Markovac andGoldstein [13] that picomolar concentrations of lead can acti-vate calcium-dependent PKC. By contrast, EF-hand calciumproteins, such as calmodulin, can be activated by lead, butonly at micromolar lead concentrations [32]. What is the sig-nificance of lead being able to activate PKC? Members of thePKC family regulate many cellular events, ranging from reg-ulation of cell growth to learning and memory [33].Calcium-dependent isoforms of PKC (α, β, and γ) contain aninteresting calcium-binding domain, termed a C2 domain[34]. The C2 domain of PKC contains a multinuclear calci-um-binding site on an exposed loop and binds phospholipidsin a calcium-dependent fashion. Lead promotes phospholipidbinding at lower concentrations than does calcium, suggestingthat lead binds to the calcium site of the C2 domain and thatit binds more tightly than does calcium [13].

The biological chemistry of lead Godwin 225

Figure 2

Lead binding to structural zinc-bindingdomains can be determined directly andquantitatively by monitoring lead–thiolatecharge-transfer bands in the ultraviolet [25•• ].By conducting competition experiments withzinc, the relative affinities of lead and zincwere determined for a series of consensuszinc finger peptides with different metal-binding residues. These studies revealed thatlead binds more tightly than zinc to Cys4sites and that lead can displace zinc fromthese sites on a physiologically relevanttimescale [25•• ]. Adapted from reference[25•• ] with permission.

S N

NSM

(KbPb)

(KbZn)

Wavelength (nm)

250 300 350 4000

2000

4000

6000

8000

10,000

12,000

14,000

16,000

Mol

ar a

bsor

ptiv

ity (

M–1

cm

–1)

Pb(CP-CCHH)

Pb(CP-CCHC)

Pb(CP-CCCC)

Pb2+ in bis-Tris

0.04 43= 0.1

~ <<S N

S SM

S

S S

SM

Is the effect of lead on C2-containing proteins a general one?Interestingly, two C2 domains are also found in synaptotag-min (syt), the protein that senses calcium influx at nervetermini and mediates calcium-triggered release of neuro-transmitters. The structure of the first C2 domain from syt(Figure 1) was solved using lead as a heavy-atom derivative,and the lead was found to occupy one of the calcium-bind-ing sites ([35,36]; Sutton RB, personal communication).Recent studies by Bouton et al. [37••] reveal that lead bindstightly to syt and that nanomolar concentrations of lead pro-mote binding of synaptotagmin to phospholipid micelles.However, lead interferes with the ability of synaptotagminto bind to its protein partner syntaxin; this inhibition offersa possible molecular explanation for the ability of lead tointerfere with calcium-triggered neurotransmitter release.

Conclusions and remaining questionsOver the past five years, a quantum leap has been made inour understanding of the molecular mechanism of lead poi-soning. Detailed biophysical studies have revealed thatlead binds tightly to both zinc and calcium sites in proteinsand alters their activity. However, lead binds to the ‘best’(cysteine-rich) zinc sites many orders of magnitude moretightly than to the ‘best’ (C2 domain) calcium sites. Thistempts the chemist to say that effects of lead on zinc pro-teins are ‘more important’ than those of lead on calciumproteins. However, the biology of lead is more complex:the multitude of symptoms associated with lead poisoningsuggest that no single target (or class of targets) will explainall of lead’s effects. Additionally, until more is known aboutthe distribution and speciation of lead within the body, wecannot definitively know which proteins are targeted andwhen. Questions that remain to be addressed include, butare not limited to:

1. How is lead taken up into cells?

2. What is the speciation of lead within different cell types andhow is this speciation affected by normal cellular homeostasis?

3. Does lead target Cys4 zinc sites (e.g. in steroid receptors)in vivo?

4. How can lead bind tightly to the calcium sites of synap-totagmin and yet prevent binding to syntaxin?

5. Are other C2 domain proteins affected by lead?

Interestingly, many of the targets that have been explored todate on a molecular level are involved in signal transductionpathways. The implications of these effects are immense:the alteration of one protein molecule by lead could beamplified throughout the subsequent pathway. New toolssuch as DNA microrrays [38] and proteomics [39] are pro-viding the means to test these molecular hypotheses bystudying the effects of lead (and other toxins) on signalingpathways. In addition, recent developments in our under-standing of the mechanism of metal ion transport

[40–44,45•,46•] lay the foundation for understanding howtoxic metals are taken up and distributed about the body.Finally, new developments in fluorescent sensors for metalions offer the promise that we will be able to correlate thesesystematic changes with changes in lead concentrations andspeciation [10•,11•].

UpdateRecent work has demonstrated not only that Pb2+-TFIIIAdoes not bind to DNA, but also that addition of Pb2+ to the Zn2+-TFIIIA-DNA adduct causes dissociation of the protein from DNA [47].

AcknowledgementsThe original experimental work described herein that was conducted byHAG and co-workers was supported by the Burroughs Wellcome Fund (NewInvestigator Award in Toxicology to HAG) and the National Institutes ofHealth (R01 GM58183). HAG is a recipient of a Camille and Henry DreyfusNew Faculty Award, a National Science Foundation CAREER Award, aCamille Dreyfus Teacher-Scholar Award, and a Sloan Research Fellowship.

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• of special interest••of outstanding interest

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The biological chemistry of lead Godwin 227