Báo cáo khoa học: Human haptoglobin structure and function – a molecular modelling study pptx

Human haptoglobin structure and function – a molecular
modelling study
F. Polticelli
1
, A. Bocedi
1
, G. Minervini
1
and P. Ascenzi
1,2
1 Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, Italy
2 National Institute for Infectious Diseases I.R.C.C.S. ‘‘Lazzaro Spallanzani’’, Rome, Italy
Hemoglobin (Hb) is the most prominent protein in
blood. Hb transports O
2
in the circulatory system and
facilitates reactive oxygen and nitrogen species detoxifi-
cation [1–6]. Hb metabolism leads to the release of the
heme protein and of free heme into extracellular fluids,
with potentially severe consequences for health [7]. In
fact, extra-erythrocytic Hb undergoes renal filtration,
leading to renal iron loading if not bound to hapto-
globin (HPT) [8]. Hb release into plasma is a physio-
logical phenomenon associated with intravascular
hemolysis that occurs during the destruction of senes-
cent erythrocytes and enucleation of erythroblasts [7].
However, intravascular hemolysis becomes a severe
pathological complication when it is accelerated in
various autoimmune, infectious (such as malaria) and
inherited (such as sickle cell disease) disorders [1]. To
prevent Hb-mediated pathological events, Hb is com-

plexed to HPT for clearance by tissue macrophages [7].
In parallel to Hb : HPT complex formation, the free
heme is scavenged by hemopexin, which delivers it to
the liver [7].
HPT, the plasma protein with the highest binding
affinity for Hb (K
d
=10
)12
m), is mainly expressed in
the liver and belongs to the family of acute-phase pro-
teins, whose synthesis is induced by several cytokines
during the inflammatory processes [9]. HPT is syn-
thesized as a single chain and then cleaved into an
N-terminal light a-chain and a C-terminal heavy
Keywords
chaperone-like activity; covalent multimers;
haemoglobin; haptoglobin; homology
modelling
Correspondence
F. Polticelli, Department of Biology,
University Roma Tre, Viale Guglielmo
Marconi 446, I-00146 Rome, Italy
Fax: +39 06 57336321
Tel: +39 06 57336362
E-mail:
Database
Models data are available in the Protein
Model DataBase under the accession
numbers PM0075388 and PM0075389

(Received 8 July 2008, revised 11
September 2008, accepted 17 September
2008)
doi:10.1111/j.1742-4658.2008.06690.x
Hemoglobin is the most prominent protein in blood, transporting O
2
and
facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin
metabolism leads to the release of extra-erythrocytic hemoglobin, with
potentially severe consequences for health. Extra-erythrocytic hemoglobin
is complexed to haptoglobin for clearance by tissue macrophages. The
human gene for haptoglobin consists of three structural alleles: Hp1F,
Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only
one amino acid, whereas the Hp2 allele is the result of a fusion of the
Hp1F and Hp1S alleles, is present only in humans and gives rise to a
longer a-chain. Haptoglobin consists of a dimer of ab-chains covalently
linked by a disulphide bond between the Cys15 residue of each a-chain.
However, the presence of the Hp1 and Hp2 alleles in humans gives rise to
HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-
oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two
free Cys residues (Cys15 and Cys74) whose participation in intermolecular
disulphide bonds gives rise to higher-order covalent multimers. Here, the
complete modelling of both haptoglobin variants, together with their basic
quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is
reported. The structural details of the models, which represent the first
complete view of the molecular details of human haptoglobin variants, are
discussed in relation to the known haptoglobin function(s).
Abbreviations
C1R, complement protease C1R; CCP domain, complement control protein domain (also named the Sushi domain); Hb, hemoglobin; HPT,
haptoglobin; PDB, Protein Data Bank; SRCR domain, scavenger receptor cysteine-rich domain.

5648 FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS
b-chain [10]. The two chains are covalently linked by
an intermolecular disulfide bond formed by Cys131
and Cys248 [11]. An Hb dimer binds to the HPT
heavy b-chain, and thus the HPT(ab dimer) : Hb stoi-
chiometry is 1 : 1 [12,13].
In plasma, stable HPT : Hb complexes are formed
and subsequently delivered to the reticulo-endothelial
system by receptor-mediated endocytosis. CD163, the
specific receptor for the HPT : Hb complex [14], is a
macrophage-differentiation antigen containing nine
copies of the scavenger receptor cysteine-rich (SRCR)
domain. Two variants of the SRCR domain proteins
(named class A and class B) have been identified in a
number of mosaic and transmembrane proteins [15].
CD163 belongs to group B of the SRCR domain pro-
teins, which are characterized by a short cytoplasmic
tail, a transmembrane segment and an extracellular
region consisting solely of the class B SRCR domains
[15,16]. CD163 is exclusively expressed by the mono-
cyte ⁄ macrophage lineage and its expression is induced
by inflammation [15].
Other than on macrophages, the existence of a
receptor for the HPT : Hb complex was demonstrated
also on hepatocytes and hepatoma cell lines. After
internalization into the liver parenchymal cells, organ-
elles containing the HPT : Hb complex distribute in
the microsome fraction where the complex dissociates
and the subunits are subsequently degraded [17–21].
The human gene for HPT, located on chromosome

l6q22, consists of three structural alleles: Hp1F, Hp1S
and Hp2 [22,23]. The products of the Hp1F and Hp1S
alleles differ by only one amino acid: Lys54 of the
Hp1S-chain is replaced by Glu in the Hp1F-chain [22].
The Hp2 allele, which probably originated by a nonho-
mologous crossing-over event, is the result of a fusion
of the Hp1F and Hp1S alleles, and is present only in
humans [22,23], although similar but independent
events have been very recently evidenced in other
mammals such as deer and cow [24,25]. The human
Hp2 allele gives rise to a longer chain (388 amino acids
as opposed to 329 in the chains originating from the
Hp1 alleles). The heavy b-chain of HPT displays a
fairly high homology to the catalytic domain of serine
proteases, although the residues His and Ser, partici-
pating in the catalytic triad, are not conserved [26]
(Fig. 1). On the other hand, it is interesting to note the
conservation of Asp193, orthologous to Asp194 of
serine proteases, which is involved in the conformational
A
B
Fig. 1. Amino acid sequence alignment
between HPT1 and C1R (A), and between
HPT2 and C1R (B). Conserved residues are
shaded in grey; Cys residues are highlighted
in yellow. Above the sequence alignment,
red, green and magenta bars indicate a- and
b-chains, and the Ile1–Leu2–Gly3–
Gly4 N-terminal sequence, respectively;
stars indicate residues orthologous to those

of the serine proteases catalytic triad; and
the black circle indicates Asp193, ortholo-
gous to the trypsin-like serine (pro)enzymes
Asp194.
F. Polticelli et al. Modelling haptoglobin structure
FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS 5649
change(s) taking place following proteolytic activation
of the zymogen, forming a salt bridge with the N-ter-
minal charged amino group. Remarkably, the homol-
ogy with trypsin-like enzymes extends also to the
N-terminal dipeptide (Ile1–Leu2 in HPT and Ile1–
Val2 ⁄ Leu2 ⁄ Ile2 in trypsin-like enzymes) and to the
Gly3–Gly4 hinge region [26] (Fig. 1). The quaternary
structure of HPT in organisms other than humans con-
sists of a dimer of ab-chains covalently linked by a
disulphide bond between the Cys15 residue of each
a-chain. However, in humans the presence of the Hp1
and Hp2 alleles gives rise to HPT1-1 dimers (cova-
lently linked by Cys15 residues), HPT1-2 hetero-oligo-
mers and HPT2-2 oligomers [11,22]. In fact, by the
effect of partial fusion of Hp1F and Hp1S alleles, the
HPT2 variant displays two free Cys residues (Cys15
and Cys74), whose participation in intermolecular
disulphide bonds gives rise to higher-order covalent
multimers [11].
Data regarding the molecular details of monomeric
HPT1 and HPT2 variants and oligomers are very
scarce. No 3D structure is available for any of the
human HPT variants, except for a molecular model
of the HPT1 variant built using a composite template

based on the homology between the HPT b-chain
and the serine protease fold, and the homology
between the HPT1 a-chain and the complement con-
trol proteins (CCP), or Sushi domain, of complement
C1S protease [26]. Additional data regarding the qua-
ternary structure of HPT variants are essentially those
deriving from a dated, albeit very careful, electron
microscopy analysis of both the Hb-free and
Hb-bound HPT1 and HPT2 variants [11,13]. In this
latter study it has been evidenced that HPT1 forms
covalent dimers made up of two ab-chains, while
HPT2 forms covalent trimers and higher-order oligo-
mers of ab-chains [11].
Recently, the crystal structure of the full-length
zymogen catalytic domain of the complement protease
C1R (C1R) has been determined [27]. This protein
displays a fairly high sequence identity to both HPT
variants (approximately 29%; Fig. 1) spanning the
entire length of both a- and b-chains. The availability
of a template that spans the entire length of both
HPT variants and provides the likely relative arrange-
ment of the two HPT chains prompted us to carry
out complete modelling of both HPT variants
together with their basic quaternary structures (i.e.
HPT1 dimer and HPT2 trimer). The structural details
of the models, which represent the first complete view
of the molecular details of human HPT variants, are
discussed in relation to the HPT physiological func-
tion(s).
Results and Discussion

Modelling of the ab ‘monomers’ of HPT1 and
HPT2
Figure 1 shows the sequence alignment of the two
HPT variants and C1R. C1R displays 29% sequence
identity to both HPT1 (spanning residues 15–328, cov-
ering both the HPT1 a- and b-chains) and HPT2
(spanning residues 15–387, covering both HPT2 a- and
b-chains) variants. The homology is widespread along
all the sequences, and almost all the Cys residues
involved in disulphide bonds in C1R are conserved in
both HPT variants. In detail, 14 Cys residues are pres-
ent in C1R, all of which are involved in disulphide
bonds (the Cys pairing being: 3–52, 32–65, 70–123,
100–141, 145–271, 314–333 and 344–374, numbered
according to the C1R crystal structure; protein data
bank (PDB) code: 1GPZ [27]). The Cys residue orthol-
ogous to C1R Cys123 is substituted by Leu in HPT1
(Fig. 1A) and Cys residues orthologous to C1R Cys52
and Cys123 are substituted by Gln and Leu, respec-
tively in HPT2 (Fig. 1B). The result of these substitu-
tions is that HPT1 Cys15 and HPT2 Cys15 and Cys74
are predicted not to be involved in disulphide bonds,
as indeed has been demonstrated experimentally [28],
whereas all other disulfide bonds are conserved in both
HPT variants.
The modelled 3D structures of HPT1 and HPT2 ab
‘monomers’ are shown in Fig. 2. Cys15 is located on
the tip of the N-terminal CCP domain in both HPT1
C1R HPT1 HPT2
Fig. 2. Schematic representation of the 3D structure of C1R (PDB

code: 1GPZ [27]) and of the modelled structures of HPT1 and
HPT2. HPT1 residue Cys15 and HPT2 residues Cys15 and Cys74
are shown in spacefill representation. This and the following figures
were produced using
CHIMERA [43].
Modelling haptoglobin structure F. Polticelli et al.
5650 FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS
and HPT2, whereas Cys74 is located in the region con-
necting the two CCP modules in HPT2, where the low
steric hindrance allows the formation of intermolecular
disulphide bonds (Fig. 2).
CD spectroscopy data, available only for the HPT1
variant [26], indicate an a-helix content ranging from
3.6 to 9.9% and a b-sheet content ranging from 32.9
to 40.9%. The HPT1 model presented here displays
9.5% a-helix content and 28.6% b-sheet content,
which is in fairly good agreement with the experimen-
tal data. In addition, both models display a good
stereochemical quality, as evaluated using procheck
[29]. In fact, G-values calculated using procheck were
)0.20 and )0.24 for HPT1 and HPT2, respectively,
well above the threshold of )0.5 for good quality mod-
els, and approximately 97% of residues in both models
were observed to lie in the allowed regions of the
Ramachandran plot. It is interesting to note that the
secondary structure content of the two CCP modules
of HPT2 was lower than that of the single CCP mod-
ule of HPT1. This could be a result of the fact that the
full-length zymogen catalytic domain of the comple-
ment protease C1R is a better template for HPT1 than

for HPT2 in the CCP modules protein region. In fact,
considering only this region, HPT1 displays 31% iden-
tity with C1R, whereas HPT2 displays 25% identity.
The HPT1 model shown in Fig. 2 differs from that
reported by Ettrich and coworkers [26] in the location
of the Cys15 residue. In the model reported by Ettrich
and coworkers [26], Cys15 appears to be located in the
middle of the b strands of the CCP module, near the
region connecting the a and b HPT1 chains, whereas
in the models presented here Cys15 is located on the
tip of the N-terminal CCP domain. This latter location
is consistent with the low steric hindrance required for
the formation of inter-chain disulphide bonds in HPT1
and with the location of the orthologous Cys residue
found in C1S (PDB code: 1ELV [30]). Furthermore,
the terminal location of Cys15 in both HPT variants
results in HPT1 covalent dimers and HPT2 covalent
trimers whose dimensions are in very good agreement
with those obtained by electron microscopy measure-
ments (see below) [11,13].
Modelling of the quaternary structure of HPT1
and HPT2
Based on the electron microscopy data [11,13], the
HPT1 quaternary structure consists of a dimer cova-
lently linked by a disulphide bond between Cys15 resi-
dues of each a-chain. Accordingly, the molecular model
of dimeric HPT1 is formed through a tip-to-tip
arrangement of two ab ‘monomers’, giving rise to a
bilobated structure in which the two heavy b-chains are
separated by two a-chains in a linear arrangement

(Fig. 3). The minimum and maximum distance between
the two heavy b-chains in the modelled HPT1 dimer
are approximately 60 and 130 A
˚
, respectively, which
compare quite well with distances obtained by electron
microscopy measurements (50 and 124 A
˚
, respectively
[13]). Moreover, the heavy b-chain diameter determined
here is approximately 35 A
˚
compared with the value of
37 A
˚
measured by electron microscopy [13].
At variance with dimeric HPT1, the HPT2 variant
forms higher-order multimers, the minimum number
of subunits involved being three [11,22]. The simplest
‘closed symmetric’ arrangement of HPT2 ab ‘mono-
mers’ that can give rise to covalently linked HPT2
trimers is the one shown in Fig. 3B, in which Cys15 of
each monomer forms a disulphide bond with Cys74 of
a neighbour monomer. The modelled trimer is fully
compatible with both the symmetric arrangement of
the heavy b-chains and the formation of a triangle-
shaped connecting region observed in electron micros-
copy studies [11]. In addition, the center-to-center
distance between two heavy b-chains in the modelled
HPT1

HPT2
Fig. 3. Quaternary structure model of dimeric HPT1 and trimeric
HPT2. Interchain disulphide bonds are shown in spacefill represen-
tation.
F. Polticelli et al. Modelling haptoglobin structure
FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS 5651
HPT2 trimer is approximately 120 A
˚
, a value which
compares well with that estimated by electron micros-
copy (approximately 129 A
˚
) [11].
An interesting feature of the ‘closed symmetrical’
modelled trimer, which sheds light on the possible
mechanism of formation of HPT2 trimeric species, is
the presence of several intermolecular electrostatic
interactions taking place at the interface between the
tip of the N-terminal CCP module of one ab ‘mono-
mer’ (where Cys15 is located) and the region connect-
ing the two CCP modules of another ab ‘monomer’
(where Cys74 is located). In detail, positively charged
Lys17 and Lys64 of one monomer are located in the
vicinity of negatively charged Glu79 and Asp45,
respectively, of a second monomer (Fig. 4). These resi-
dues provide a sort of ‘electrostatic docking’ site,
which can facilitate the proper relative orientation of
two monomers to form the Cys15–Cys74 disulphide
bond and give rise to trimers, tetramers and higher-
order multimers (Fig. 5).

Hb binding and chaperone-like activity of HPT
Selective proteolysis studies have demonstrated that
the Hb-binding site lies in the region surrounding resi-
dues 9 and 10, and the 128–137 loop of the HPT
b-chain, while the HPT a-chain is not involved in Hb
binding [31]. In agreement, in our model the b-chain
N-terminal residues partially overlap with the 128–137
loop region (Fig. 6). An interesting feature of the HPT
b-chain is that its N-terminus is highly homologous to
that of serine proteases [26] and that residue Asp194,
which in the latter class of enzymes binds the N-termi-
nal ammonium group following proteolytic activation
of the zymogen, is conserved in HPT as Asp193. Thus,
it is probable that also in HPT the N-terminal region
binds in the protein cavity on the bottom of which
Asp193 lies, thus leading to structuring of this protein
region to form the Hb-binding site (see below).
A large hydrophobic region is adjacent to the
Hb-binding site (Fig. 6). This region, the largest hydro-
phobic solvent-exposed area on the HPT b-chain, has
been hypothesized to be responsible for the chaperone-
like activity of HPT [26], the property of HPT to
prevent thermally induced aggregation of proteins
Fig. 4. Schematic representation of the electrostatic interactions
taking place between HPT2 monomers at the closed trimer inter-
face. For clarity only residues at the interface between monomers
A and C are labeled. Interchain disulphide bonds are shown in
spacefill representation.
Fig. 5. Schematic representation of the main quaternary structure
arrangements of HPT1 and HPT2. The spheres represent HPT

b-chains while ellipses represent the CCP modules of HPT
a-chains. Intermolecular disulphide bonds are represented by dou-
ble lines and indicated by arrows. The bottom panel highlights the
fact that HPT2 exists also in oligomerization states larger than 3
that can give rise to both closed (no free Cys residues) and opened
(free Cys residues) oligomers.
Modelling haptoglobin structure F. Polticelli et al.
5652 FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS
[26,32]. Titration experiments have also demonstrated
that the chaperone-like activity of HPT decreases by
up to 50% upon addition of Hb, until a 1 : 1
HPT : Hb ratio is reached [26]. Our model is con-
sistent with these data, in that binding of the Hb
ab-dimer (approximately 50 A
˚
in diameter) to the 128–
137 loop region of HPT would, at least partially, cover
up the solvent-exposed hydrophobic surface of HPT.
The comparative analysis of the primary and tertiary
structures of trypsin-like serine proteases and HPT
suggests that HPT chaperone-like activity may be
modulated by the formation of the intramolecular salt
bridge between the Ile1 N-terminus and Asp193. Note
that the endogenous and exogenous Ile–Leu-like dipep-
tides have been demonstrated to activate trypsinogen
by forming a salt bridge with the Asp194 residue
[33,34], hortologous to HPT Asp193. Interestingly, the
pH-dependent chaperone-like activity of HPT increases
with pH in the range 5.5–7.5 with an apparent
pK  6.5 [32]. To test the hypothesis that HPT activa-

tion could be related to binding of the Ile1–Leu2
N-terminal tail to Asp193, the pK
a
values of HPT
b-chain and trypsinogen-ionizable residues were calcu-
lated using the propka software [35]. The HPT Asp193
pK
a
value was 6.2; this value correlates well with the
experimentally determined midpoint of the pH depen-
dence of HPT chaperone-like activity ( 6.5). Interest-
ingly, the calculated pK
a
value of trypsinogen Asp194
resulted to be 6.2 as well.
The CD163-binding region
Recently, using recombinant HPT ⁄ HPT-related protein
chimeras complexed to Hb, it has been shown that
only the HPT b-chain is involved in binding of the
HPT : Hb complex by the CD163 receptor [36]. In
particular, the loop encompassing residues Val157–
Thr162 of the HPT b-chain has been demonstrated to
be essential for receptor binding [36]. The 157–162
loop is located near the Hb-binding loop in our HPT
model (Fig. 6), in agreement with the observation that
the epitope recognized by CD163 is formed by residues
contributed by both HPT and Hb. In fact, CD163
binds the HPT : Hb complex with an affinity at least
two orders of magnitude higher than HPT and Hb
alone [36].

Conclusions
The best-characterized function of HPT is that of
binding free Hb and promoting its endocytosis and
subsequent intracellular degradation through the
formation of high-affinity complexes with the CD163
scavenger receptor on macrophages. In this way HPT
reduces the loss of free Hb through glomerular filtra-
tion and promotes the recycling of iron. In addition,
heme and iron released from free Hb generate reactive
oxygen species leading to tissue injury, as demon-
strated in vivo in HPT knockout mice. Thus, HPT,
promoting immediate clearance of free Hb, acts as an
antioxidant agent [37].
An additional activity of HPT is related to its ability
to suppress heat-induced and oxidative stress-induced
unfolding and precipitation of a number of proteins,
thus reducing the toxic effects caused by aggregation
of misfolded extracellular proteins. Taken together,
these findings indicate that HPT plays a significant role
in re-establishing homeostasis after local or systemic
A
B
Fig. 6. Schematic representation of the 3D
structure of the HPT b-chain (A). The 128–
137 residue region, involved in Hb binding,
is coloured in blue. Residues building up the
adjacent large hydrophobic region are
shown in stick representation. The red
arrow indicates the cavity on the bottom of
which Asp193 is located. The green arrow

indicates the CD163-binding loop. Panel B
shows the molecular surface of the HPT
b-chain shown in the same orientation as
panel A. Surface areas generated by hydro-
phobic and polar residues are coloured in
green and grey, respectively.
F. Polticelli et al. Modelling haptoglobin structure
FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS 5653
infection by virtue of its various anti-inflammatory
activities [32].
Particularly intriguing is the hypothesis that HPT
may undergo a conformational re-arrangement follow-
ing proteolytic maturation of the protein, similarly to
the proteolytic activation mechanism observed in
serine proteases. The structural model presented in this
work supports this hypothesis. In fact, binding of the
Ile1–Leu2 N-terminal tail of the HPT b-chain to the
Asp193 pocket would lead to a structural re-arrange-
ment of both the Hb-binding region and of the adja-
cent hydrophobic surface area available for binding
misfolded proteins. In addition, pK
a
calculations indi-
cate that Asp193, as a result of its poorly solvent-
accessible location on the HPT surface, is characterized
by an altered pK
a
value (= 6.2) which correlates well
with the pH dependence of the chaperone-like activity
of HPT (midpoint  6.5 [32]). This could easily be

explained by the ability of Asp193 to drive the confor-
mational rearrangement of HPT only in the deproto-
nated form, with the ability to form a salt bridge with
the N-terminal ammonium group of the protein, as
reported for serine (pro)enzyme activation [33,34]. The
common architecture of the HPT b-chain and of tryp-
sin-like (pro)enzymes, together with similar structural
and electrostatic properties at the root of the activa-
tion mechanism, may represent a case of divergent
evolution. Indeed, the HPT b-chain appears to have
evolved from a common ancestor by mutation of only
two out of three catalytic residues (His fi Lys and
Ser fi Ala) while preserving the overall fold and prob-
ably the activation mechanism [26,33,34].
In conclusion, in this work we present the first com-
plete description of the molecular details of HPT1 and
HPT2 monomers and multimers and show that the
information deriving from the calculated models can
be useful to explain some of the properties of the
multifunctional human HPT variants.
Materials and methods
The molecular models of HPT1 and HPT2 were built using
the crystal structure of C1R as the template (PDB code:
1GPZ [27]). In detail, protein sequences displaying signifi-
cant similarity with HPT variants were retrieved through
three PSI-Blast [38] iterations against the nonredundant
protein database using the sequences coded GI 1212947
and GI 4826762 for HPT1 and HPT2 variants, respectively,
as a bait. The 18 N-terminal amino acids of both HPT1
and HPT2 sequences, which represent a signal peptide, were

previously removed. PSI-Blast E-values of C1R were
2 · 10
)109
and 1 · 10
)126
for HPT1 and HPT2 variants,
respectively. Amino acid sequence alignment between the
template C1R and the two HPT variants was then obtained
through multiple sequence alignment of the PSI-Blast hits
using clustalw [39]. This procedure yielded the alignments
shown in Fig. 1.
The molecular models of the two HPT1 and HPT2 vari-
ants were built using the program NEST [40], a fast model-
building program that applies an ‘artificial evolution’
algorithm to construct a model from a given template and
alignment. The NEST option tune 2 was used to refine
the alignment, avoiding the unlikely occurrence of insertions
and deletions within template secondary-structure elements.
The HPT1 covalent dimer and the HPT2 covalent trimer
were constructed using the molecular graphics package
Discovery Studio visualizer 1.7 (Accelrys Software Inc.,
San Diego, CA, USA) and the rototranslation matrices
given in Table S1. Constraints considered for HPT1 dimer
construction were its binary symmetry and the formation of
the Cys15–Cys15 intermolecular disulphide bond [11,13].
Analogously, constraints used for HPT2-2 trimer construc-
tion were its ternary symmetry and the formation of the
Cys15–Cys74 intermolecular disulphide bonds [11,13].
Monomer and multimer models obtained using the
above-described procedure were stereochemically regular-

ized through energy minimization using the CHARMM
macromolecular mechanics package [41], c33b1 version,
and the CHARMM27 parameters and force field [42]. The
stereochemical quality of the models was evaluated using
procheck [29].
The molecular surface of the HPT b-chain was calculated
and visualized using the program chimera [43]. The HPT
b-chain and bovine trypsinogen (PDB code 1TGN [44])
ionizable residue pK
a
values were calculated using propka
[35].
Acknowledgements
This work was supported by a grant from the Italian
Ministry of University and Research.
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Supporting information
The following supplementary material is available:
Table S1. Rototranslation matrices used to generate
HPT1-1 dimer and HPT2-2 trimer.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
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sponding author for the article.
Modelling haptoglobin structure F. Polticelli et al.
5656 FEBS Journal 275 (2008) 5648–5656 ª 2008 The Authors Journal compilation ª 2008 FEBS