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The biological chemistry of lead


Hilary Arnold Godwin
Recent biophysical studies on the interactions between lead lead. These studies have helped to provide insights into the
and recombinant proteins and peptides that naturally bind zinc following questions:
or calcium have provided unparalleled insights into the
biological chemistry and molecular toxicology of lead. These 1. Out of all of the molecular targets that have been pro-
studies lay the foundation for the rational design of improved posed for lead, which ones are physiologically relevant?
methods for detecting and treating lead poisoning.
2. How does lead binding affect the structure and dynam-
Addresses ics of target proteins?
Department of Chemistry, Northwestern University, 2145 Sheridan Road,
Evanston, IL 60208-3113, USA; e-mail: h-godwin@northwestern.edu 3. Is lead binding to proteins under thermodynamic or
Current Opinion in Chemical Biology 2001, 5:223–227 kinetic control?

1367-5931/01/$ — see front matter These studies and questions are the subject of this review,
© 2001 Elsevier Science Ltd. All rights reserved.
which focuses particularly on work from the past two years.
Abbreviations
ALAD δ-aminolevulinic acid dehydratase Molecular targets for lead
BLL blood lead level
CP-CCCC consensus zinc-binding domain with Cys4 zinc-binding
Several classes of molecular targets have been proposed to
site account for the symptoms associated with lead poisoning.
CP-CCHC consensus zinc-binding domain with Cys2HisCys zinc- With few exceptions, these targets fall into two primary
binding site categories: proteins that naturally bind calcium and pro-
CP-CCHH consensus zinc-binding domain with naturally occurring
Cys2His2 zinc-binding site
teins that naturally bind zinc [5–8,9••]. If these interactions
HP2 human protamine 2 are to be physiologically relevant, lead must bind tightly
PKC protein kinase C enough to the proposed target to occupy the site(s) under
physiologically relevant conditions. To ascertain whether
Introduction lead binds ‘tightly enough’, both the affinity of lead for the
Lead poisoning is the one of the most common pediatric protein and the concentration of free (or ‘bioavailable’)
health problems in the United States, affecting approxi- lead must be known. The affinity of lead for a given pro-
mately 890,000 children nationwide at any given time [1]. tein is related to the disocciation constant for the
The sources of this exposure are primarily leaded paint, lead–protein complex, KdPb . In the simple case, half of the
which was not banned in the United States until 1978, and population of target protein (P) will have lead bound when
contaminated soil. Recent studies suggest that much of the KdPb is equal to the concentration of ‘free’ (or ‘bioavail-
existing soil contamination is probably a result of deposi- able’) lead ([Pb]) in the cell. When lead is competing with
tion from exhaust from cars that used leaded gasoline, in another metal ion (M, e.g. calcium or zinc) for a binding
addition to leaded paint used on the exterior of buildings site, then the relative affinities and concentrations of the
[2•]. Lead from these sources exists as or evolves into a two metals must be considered, where:
variety of Pb2+ compounds, which are remarkably persis-
tent in the environment. Unfortunately, these sources of  PbP  KdM  Pb 
 MP  = K Pb ×  M 
exposure are often expensive to remediate, and the politics   d  
surrounding this issue are complex, suggesting that the
legacy of lead poisoning will continue to plague mankind How much bioavailable lead is present in cells?
for many years to come [3,4]. The amount of bioavailable (or ‘free’) lead has not been
determined experimentally for most cell types, because of
Lead poisoning can afflict both children and adults, but the the lack of sensitive and selective fluorescent probes for
greatest concern is for children, who experience symptoms lead [10•,11•]. A child is considered to have lead poisoning
at significantly lower blood lead levels (BLLs) than do if he or she has a total BLL (as measured by atomic absorp-
adults [1]. In addition, children tend to develop permanent tion spectroscopy) greater than or equal to 10 µg/dl
developmental and neurological problems when exposed (0.5 µM or 100 parts per billion) [1]. By contrast, a typical
chronically to lead, whereas many of the symptoms experi- person in the United States today who does not have lead
enced by adults are reversed when exposure is ceased. poisoning will have a BLL of ~2 µg/dl (0.1 µM or 20 parts
Although a broad range of epidemiological studies has been per billion). It has been estimated that <5% of the total
conducted on lead poisoning, its molecular underpinnings lead in blood is bound to plasma proteins [12] and that only
have remained relatively obscure. However, recent 0.01% of lead in plasma (~ picomolar lead) is bioavailable
advances in biophysics and molecular biology have provid- [13]. By contrast, the total concentrations of zinc and calci-
ed the tools necessary to study the biological chemistry of um in human plasma are about 17 µM and 10–6 to 10–3 M,
224 Bio-inorganic chemistry

Figure 1 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 the
structure of the lead protein is essentially identical to that
Ca2+ (0.99 Å) Pb2+ (1.19 Å) Zn2+ (0.74 Å) of the zinc form, incorporation of lead non-competitively
inhibits substrate binding [20]. The suggestion by Erskine
et al. [20] that lead prefers the Cys3 site in ALAD because
this constitutes a tight binding site for lead is borne out by
recent model compound studies: lead binds more tightly
than zinc (about 500 to 1) to a novel tris-thiol ligand
(tris(mercaptoarylimdazolyl)borate; Ar = Ph, Mes) but is
considerably less Lewis-acidic in this environment than is
zinc [22••]. These studies provoke several important ques-
Synaptotagmin ALAD tions. Does lead bind tightly to other zinc proteins that
Current Opinion in Chemical Biology contain three (or more) cysteines in their active sites?
Could lead binding to any of these proteins account for
Lead targets proteins that naturally bind calcium and zinc. Examples of some of the other, more pressing, symptoms associated
proteins that are targeted by lead include synaptotagmin, which acts with lead poisoning?
as a calcium sensor in neurotransmission, and ALAD, the second
enzyme in the heme biosynthetic pathway. Each of these proteins was
crystallized with lead as a heavy atom derivative ([8,19,35,36]; Sutton Although no other catalytic zinc sites have been reported
RB, personal communication). These studies revealed that, despite its that contain three or more cysteine residues, there are sev-
size, lead (1.19 Å, blue sphere and circles) can substitute for calcium eral classes of proteins that contain structural zinc-binding
(0.99 Å, green spheres) in synaptotagmin ([35,36]; Sutton RB, sites with three (Cys3His) or four (Cys4) cysteine residues.
personal communication) and zinc (0.74 Å, red spheres) in ALAD
[8,19]. Protein coordinates were obtained from the Protein Data Bank These proteins (e.g. retroviral nucleocapsid protein, with
(http://www.rcsb.org/pdb/). Cys3His sites, and steroid receptors, with two tandem Cys4
sites) act as transcription factors and regulate many of the
developmental processes associated with lead poisoning in
respectively. The concentrations of bioavailable zinc and children. Thus, the ability of lead to alter the activity of
calcium have been estimated to be 10–12 to 10–6 M and 10–8 these proteins could account for the pressing developmen-
to 10–6 M, respectively [13–16]. These estimates suggest tal problems associated with lead poisoning.
that, in general, lead must bind to proteins with a Kd of
≤10–12 M and that lead must bind more tightly than the Until recently, the interactions between lead and tran-
native metal ion by at least one to three orders of magni- scription factors had not been probed directly, presumably
tude. Although it has been suggested that the local because Pb2+ (electronic structure = [Xe]4f145d106s2) was
concentration of lead may be higher in certain organelles widely assumed to be spectrosopically silent. However,
(e.g. the nucleus) [17] or specific cell types (e.g. neurons) when lead binds to cysteine residues in proteins, it
[12], these requirements serve as a reasonable minimal exhibits intense lead–thiolate charge-transfer bands in the
guideline until more detailed studies on lead biodistribu- ultraviolet region of the electromagnetic spectrum
tion become available. [23,24,25••,26•] that can provide detailed and quantitative
information about lead–protein interactions [25••]
Interactions between lead and zinc proteins (Figure 2). Studies on a series of zinc-finger consensus
The target for lead that has been studied most thoroughly peptides with different metal-binding sites (consensus
in vitro and in vivo is the zinc enzyme δ-aminolevulinic zinc-binding domain with naturally occurring Cys2His2
acid dehydratase (ALAD, also called porphobilinogen syn- zinc-binding site, CP-CCHH; consensus zinc-binding
thase) [8] (Figure 1). ALAD catalyzes the second reaction domain with Cys2HisCys zinc-binding site, CP-CCHC;
in the heme biosynthetic pathway, and inhibition of this and consensus zinc-binding domain with Cys4 zinc-bind-
enzyme by lead explains (at least in part) the anemia often ing site, CP-CCCC) reveal that lead binds tightly to
seen in adults and children with high BLLs (≥ 40 µg/dl) structural zinc-binding sites ( KdPb = 10 −9 to 10 −14 M ) and
[8]. The log of the activity of ALAD in erythrocytes that lead binds approximately two orders of magnitude
decreases linearly with the individual’s BLL [18]; lead more tightly than zinc to the Cys4 site [25••]. Furthermore,
inhibits ALAD in vitro with an inhibition constant of competition experiments reveal that lead readily displaces
Zn
0.07 pM (versus K m = 1.6 pM ) [19]. Why out of all the zinc from the Cys4 site, which suggests that metal binding
zinc enzymes in the body is ALAD the only one known to is under thermodynamic, rather than kinetic, control. 1H
be inhibited by lead? A recent crystal structure of yeast NMR studies on a Cys2HisCys domain from HIV-nucleo-
ALAD offers insights into this ‘mystery’. Whereas most capsid protein and circular dichroism studies on both this
zinc enzymes contain a zinc-binding site with a mixture of domain and CP-CCCC reveal that even though lead binds
histidine, cysteine, and carboxylate residues, the yeast and tightly to these domains, it does not promote proper fold-
mammalian forms of ALAD contain a unique catalytic ing of the site [25••]. These studies lay the foundation for
The biological chemistry of lead Godwin 225

Figure 2

Lead binding to structural zinc-binding


domains can be determined directly and 16,000
quantitatively by monitoring lead–thiolate

Molar absorptivity (M–1 cm–1)


14,000
charge-transfer bands in the ultraviolet [25••]. Pb(CP-CCHH)
By conducting competition experiments with 12,000
zinc, the relative affinities of lead and zinc Pb(CP-CCHC)
were determined for a series of consensus 10,000
zinc finger peptides with different metal- Pb(CP-CCCC)
8000
binding residues. These studies revealed that
lead binds more tightly than zinc to Cys4 6000 Pb2+ in bis-Tris
sites and that lead can displace zinc from
these sites on a physiologically relevant 4000
timescale [25••]. Adapted from reference
2000
[25••] with permission.
0
250 300 350 400
Wavelength (nm)

S N
~ S N
<< S S
M M M
S N S S S S

(KbPb)
= 0.1 0.04 43
(KbZn)

detailed molecular studies on the interactions between transcription factor upstream of PKC or solely by direct
lead and larger proteins that contain structural zinc-bind- activation of PKC (see below) is not known. These studies
ing sites. Parallel studies have recently been reported for point to the need for not only more detailed biophysical
the interactions of lead with the zinc protein human prota- studies on a wide range of proposed targets, but also for
mine 2 (HP2), which plays an important role in comprehensive, systemic studies on the mechanisms by
spermatogenesis; interactions between lead and HP2 may which lead alters signal transduction in cells.
account for increased infertility rates in men who are occu-
pationally exposed to lead. Lead binds to thiol groups in Interactions between lead and calcium proteins
HP2 and alters the structure of the protein [26•]. The In addition to causing developmental problems, lead poison-
observation that lead alters the structure of the zinc pro- ing results in pervasive neurological problems in both
teins to which it binds is not surprising given the children and adults [30]. Lead interferes with the ability of
differences in coordination preferences (6–8 for Pb2+ ver- calcium to trigger exocytosis of neurotransmitters in neuronal
sus 4 for Zn2+ and geometries (irregular for Pb2+ versus cells [31], suggesting that lead might generally target proteins
tetrahedral for Zn2+) in structural zinc-binding sites for the involved in calcium-mediated signal transduction [6]. This
two metals [27]. However, these results are extremely hypothesis was bolstered by the observation by Markovac and
important because they suggest that lead binding to struc- Goldstein [13] that picomolar concentrations of lead can acti-
tural zinc-binding domains should disrupt the vate calcium-dependent PKC. By contrast, EF-hand calcium
DNA-binding activity of the proteins and transcription fac- proteins, such as calmodulin, can be activated by lead, but
tors in which they are found. only at micromolar lead concentrations [32]. What is the sig-
nificance of lead being able to activate PKC? Members of the
The effects of lead on the DNA-binding activity of PKC family regulate many cellular events, ranging from reg-
Cys2His2 zinc-finger proteins such as TFIIIA were recent- ulation of cell growth to learning and memory [33].
ly studied using DNase I protection assays. These studies Calcium-dependent isoforms of PKC (α, β, and γ) contain an
reveal that when sufficiently high levels of lead are present interesting calcium-binding domain, termed a C2 domain
(5–20 µM), then TFIIIA does not bind DNA [28•] (see also [34]. The C2 domain of PKC contains a multinuclear calci-
Update). In addition, recent studies by Goldstein, Bressler, um-binding site on an exposed loop and binds phospholipids
and co-workers [29•] reveal that lead alters expression of in a calcium-dependent fashion. Lead promotes phospholipid
immediate early genes in PC12 cells, in a protein kinase C binding at lower concentrations than does calcium, suggesting
(PKC)-dependent fashion. Whether the alterations in gene that lead binds to the calcium site of the C2 domain and that
expression observed arise from interaction of Pb2+ with a it binds more tightly than does calcium [13].
226 Bio-inorganic chemistry

Is the effect of lead on C2-containing proteins a general one? [40–44,45•,46•] lay the foundation for understanding how
Interestingly, two C2 domains are also found in synaptotag- toxic metals are taken up and distributed about the body.
min (syt), the protein that senses calcium influx at nerve Finally, new developments in fluorescent sensors for metal
termini and mediates calcium-triggered release of neuro- ions offer the promise that we will be able to correlate these
transmitters. The structure of the first C2 domain from syt systematic changes with changes in lead concentrations and
(Figure 1) was solved using lead as a heavy-atom derivative, speciation [10•,11•].
and the lead was found to occupy one of the calcium-bind-
ing sites ([35,36]; Sutton RB, personal communication). Update
Recent studies by Bouton et al. [37••] reveal that lead binds Recent work has demonstrated not only that Pb2+-TFIIIA
tightly to syt and that nanomolar concentrations of lead pro- does not bind to DNA, but also that addition of Pb2+ to
mote binding of synaptotagmin to phospholipid micelles. the Zn2+-TFIIIA-DNA adduct causes dissociation of the
However, lead interferes with the ability of synaptotagmin protein from DNA [47].
to bind to its protein partner syntaxin; this inhibition offers
a possible molecular explanation for the ability of lead to Acknowledgements
interfere with calcium-triggered neurotransmitter release. The original experimental work described herein that was conducted by
HAG and co-workers was supported by the Burroughs Wellcome Fund (New
Investigator Award in Toxicology to HAG) and the National Institutes of
Conclusions and remaining questions Health (R01 GM58183). HAG is a recipient of a Camille and Henry Dreyfus
Over the past five years, a quantum leap has been made in New Faculty Award, a National Science Foundation CAREER Award, a
Camille Dreyfus Teacher-Scholar Award, and a Sloan Research Fellowship.
our understanding of the molecular mechanism of lead poi-
soning. Detailed biophysical studies have revealed that
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