Tài liệu Báo cáo khoa học: Thermodynamic characterization of interleukin-8 monomer binding to CXCR1 receptor N-terminal domain ppt

Thermodynamic characterization of interleukin-8 monomer
binding to CXCR1 receptor N-terminal domain
Harshica Fernando
1
, Gregg T. Nagle
2
and Krishna Rajarathnam
1
1 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA
2 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA
Chemokines constitute the largest family of proteins
that mediate leukocyte recruitment and trafficking
[1,2]. They show a remarkable range of receptor selec-
tion and function, with some binding a single receptor,
some binding multiple receptors, and some binding
one receptor with high affinity and others with low
affinity [3–7]. Chemokine receptors belong to the
superclass of G-protein-coupled receptors (GPCRs),
and structure–function studies show that all chemo-
kines bind their receptors using the same two-site
mechanism, which involves interaction between the lig-
and N-loop and the receptor N-terminal domain
(N-domain) residues and between ligand N-terminal
and receptor extracellular loop residues [5]. The largest
sequence difference among chemokines and their re-
ceptors is found in the N-loop and N-domain, respect-
ively, suggesting that these residues encode both the
specificity and promiscuity of interactions.
Interleukin-8 (IL-8, also known as CXCL8) and
related neutrophil-activating chemokines (such as
Keywords

interleukin-8; isothermal titration calorimetry;
monomer; N-terminal domain;
thermodynamics
Correspondence
K. Rajarathnam, 5.144 MRB, UTMB,
Galveston, TX 77555-1055, USA
Fax: +1 409 772 1790
Tel: +1 409 772 2238
E-mail:
(Received 25 August 2006, revised 2
November 2006, accepted 7 November
2006)
doi:10.1111/j.1742-4658.2006.05579.x
Chemokines elicit their function by binding receptors of the G-protein-cou-
pled receptor class, and the N-terminal domain (N-domain) of the receptor
is one of the two critical ligand-binding sites. In this study, the thermo-
dynamic basis for binding of the chemokine interleukin-8 (IL-8) to the
N-domain of its receptor CXCR1 was characterized using isothermal titra-
tion calorimetry. We have shown previously that only the monomer of
IL-8, and not the dimer, functions as a high-affinity ligand, so in this study
we used the IL-8(1–66) deletion mutant which exists as a monomer. Calori-
metry data indicate that the binding is enthalpically favored and entropical-
ly disfavored, and a negative heat capacity change indicates burial of
hydrophobic residues in the complex. A characteristic feature of chemokine
receptor N-domains is the large number of acidic residues, and experiments
using different buffers show no net proton transfer, indicating that the
CXCR1 N-domain acidic residues are not protonated in the binding pro-
cess. CXCR1 N-domain peptide is unstructured in the free form but adopts
a more defined structure in the bound form, and so binding is coupled to
induction of the structure of the N-domain. Measurements in the presence

of the osmolyte, trimethylamine N-oxide, which induces the structure of
unfolded proteins, show that formation of the coupled N-domain structure
involves only small DH and DS changes. These results together indicate
that the binding is driven by packing interactions in the complex that are
enthalpically favored, and are consistent with the observation that the
N-domain binds in an extended form and interacts with multiple IL-8
N-loop residues over a large surface area.
Abbreviations
ASA, accessible surface area; CXCR1, CXC chemokine receptor 1; GPCR, G-protein-coupled receptor; IL-8, interleukin-8; ITC, isothermal
titration calorimetry; N-domain, N-terminal domain; TMAO, trimethylamine N-oxide.
FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS 241
MGSA ⁄ CXCL1 and NAP-2 ⁄ CXCL2) all have the
characteristic N-terminal ELR residues, and bind and
activate CXCR1 and CXCR2 receptors. IL-8 binds
both receptors with high affinity, whereas all other lig-
ands bind CXCR2 with high affinity and CXCR1 with
low affinity [6,7]. Sequence analysis shows that the
N-terminal ELR residues are conserved, whereas the
N-loop residues are not, suggesting that the differences
in binding may be due to binding of N-loop residues
to the receptor N-domain.
The structure of IL-8 is known, and the structural
basis for its function has been well studied [8–17]. The
receptor structures are not known and are difficult to
obtain because of their membrane-embedded state.
IL-8 and all other chemokine receptors share some
unique properties compared with other members of
GPCR class A receptors. Chemokines (molecular mass
% 8 kDa) are unusually large for a GPCR class A lig-
and, as most are small molecules (< 1 kDa) with a

rigid scaffold. In general, the sequence length of the
GPCR N-domain correlates with ligand size [18], and
chemokine receptors are a notable exception, as their
N-domains are unusually short (% 40 residues) com-
pared with the size of their ligands (% 70 residues).
Further, in contrast with most GPCR class A recep-
tors, chemokine receptor N-domains are also highly
acidic. Interestingly, the lowest sequence identity
between CXCR1 and CXCR2 lies in the N-domain
(% 50%), and the N-domains are also of different
length. Results from mutagenesis studies on both IL-8
and the CXCR1 receptor suggest that the binding
interactions between the IL-8 N-loop and receptor
N-domain residues cover an extended interface, and
can be described either by a model that involves mul-
tiple weak interactions or by a ‘hot spots’ model which
involves few strong interactions [12,19,20]. In principle,
both models allow the chemokine ⁄ receptor to fine-tune
and regulate binding affinity and ⁄ or ligand selectivity.
Currently, little is known about the relative enthalpic
(van der Waals, hydrogen-bonding, and electrostatic
interactions) and entropic (solvation ⁄ desolvation, loss
of conformational flexibility and dynamics) contribu-
tions to binding, and such knowledge is essential for
understanding the relationship between structure and
the thermodynamics of binding.
IL-8 binds the isolated N-domain with an affinity
similar to that for the N-domain in the intact receptor,
and so can be studied outside the context of the intact
receptor [21]. Such studies have already provided valu-

able insights into the molecular basis of ligand selectiv-
ity, ligand dimerization and binding affinity [22–24].
We have recently shown that the receptor N-domain
adopts a definite structure in the osmolyte, trimethyl-
amine N-oxide (TMAO), that promotes the folded
state of the protein, and that the binding affinity of
IL-8 for the N-domain is higher in osmolytes [24]. In
this study, we have characterized the thermodynamic
basis of IL-8 binding to the CXCR1 N-domain peptide
using isothermal titration calorimetry (ITC).
We have shown previously that only the monomer
of IL-8, and not the dimer, functions as a high-affinity
ligand for receptor binding [22,25], so in this study we
used the IL-8(1–66) deletion mutant which exists as a
monomer. ITC measures the heat released or absorbed
during a binding event, from which the free energy of
binding (DG), enthalpy (DH), entropy (DS), and stoi-
chiometry (n) are obtained in a straightforward man-
ner, and also provides DC
p
by measuring heat released
as a function of temperature [26]. To dissect coupling
between structure induction and binding, we also
measured binding in the presence of TMAO. As chemo-
kine receptor N-domains are acidic in nature, binding
experiments were also carried out using buffers with
different heats of ionization to determine whether bind-
ing is coupled to proton transfer. The data show that
the binding is enthalpically favored and entropically
disfavored, that coupled structure formation involves

only small enthalpy and entropy changes, and that
there is no net proton transfer. On the basis of the
structure of the complex and structure–function studies,
we propose that the favorable enthalpic contribution
arises from optimal packing interactions of apolar resi-
dues in the complex, and further propose that the ther-
modynamic basis of the binding of all chemokine
ligands to their receptor N-domains is similar to that
observed for the IL-8 ⁄ CXCR1 system, and the ability
to fine-tune the enthalpic and entropic components of
the binding to the N-domain plays a key role in modu-
lating affinity and ligand ⁄ receptor selectivity.
Results and Discussion
On the basis of structure–function data, a general two-
site mechanism of ligand–receptor interaction has been
proposed for all chemokines. Binding involves interac-
tions between the chemokine ligand N-loop and the
receptor N-domain, and ligand N-terminal and recep-
tor extracellular loop residues. N-domain peptides for
various chemokine receptors including CXCR1 have
been shown to bind to their cognate ligands, indicating
that studying isolated domains may give considerable
insight into the molecular basis of binding and func-
tion [11,21,27–31]. The IL-8 ⁄ CXCR1 pair is one of
the best studied, and for instance, studies using
CXCR1 N-domain peptide have shown that IL-8
dimer dissociation is essential for high-affinity binding,
Chemokine ligand–receptor interaction H. Fernando et al.
242 FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS
and that the CXCR1 N-domain plays a major role in

determining binding affinity and not in ligand selectiv-
ity [22–24].
Design and characterization of IL-8(1–66)
monomer
Previous studies using a ‘trapped’ monomer and native
protein that exists as both monomers and dimers have
shown that dimer dissociation is essential for high-
affinity binding to the receptor [22]. The trapped
L25NMe monomer contains a non-natural NMe-
amino acid as a dimer interface residue, and was
synthesized by solid-phase chemical synthesis [32].
Comparison of the trapped monomer and native dimer
structures shows that the last six C-terminal residues
(67–72) are unstructured in the monomer and struc-
tured in the dimer [8,10]. Therefore, we suspected that
deleting these residues would result in a monomer.
We had previously observed from ultracentrifugation
studies [33] that the IL-8(1–66) deletion mutant is a
monomer at micromolar concentrations, and we now
observe from NMR and ITC studies that it is a mono-
mer up to millimolar concentrations. The circular
dichroism (CD) spectrum of the IL-8(1–66) monomer
indicates that it is folded and shows a profile similar to
that observed for the native IL-8(1–72), both showing
characteristic minima at % 222 nm (Fig. 1). Higher
ellipticities and a pronounced minimum at % 208 nm
for the native protein are consistent with C-terminal
residues Trp57–Ser72 being structured and helical in
the dimer, whereas the monomer will have lower helical
content, as it is missing residues 67–72. An HSQC spec-

trum of the IL-8(1–66) monomer shows the characteris-
tic upfield (Phe17 and Val58) and downfield (Gln8 and
Lys20) shifted peaks previously observed in the native
dimer and the trapped monomer (Fig. 1). Chemical
shift and NOESY data analyses indicate that IL-8(1–
66) adopts a structure similar to that of the trapped
L25NMe monomer. We have also characterized the
dynamics of IL-8(1–66) from
15
N-T
1
, T
2
, and
1
H-
15
N
NOE relaxation measurements (unpublished observa-
tions). The correlation time (s
c
) of 5.2 ns calculated
from relaxation data is consistent with that expected
for a 7.7-kDa protein. Previous studies have shown that
the activity of IL-8(1–66) is similar to that of native
IL-8 [34]. We also observed that our recombinant
IL-8(1–66) is as active as native IL-8, and show below
that it binds with the same affinity as the trapped
L25NMe monomer to the CXCR1 N-domain peptide.
We used ITC to determine the enthalpy (DH),

entropy (DS), and the free energy (DG) of binding of
monomeric IL-8 to the receptor CXCR1 N-domain.
The binding isotherm of IL-8(1–66) and the trapped
L25NMe monomers to the CXCR1 N-domain are
shown in Fig. 2. The upper panels show the thermo-
grams, and the lower panels show the integrated heat
fitted to a standard binding isotherm. The negative
peaks indicate that the interaction is exothermic
(DH < 0); the data show excellent signal to noise
AB
Fig. 1. Characterization of the IL-8(1–66) monomer. (A) CD spectra of a 25-lM solution of the IL-8(1–66) monomer (solid line) and the native
IL-8(1–72) dimer(dash line) in 50 m
M sodium phosphate ⁄ 50 mM NaCl, pH 8.0 buffer. (B)
15
N-
1
H HSQC NMR spectrum of the IL-8(1–66)
monomer. The observed chemical shifts are similar to that observed for the trapped monomer, and some of the upfield and downfield
shifted peaks characteristic of a folded protein are labeled. The spectrum was acquired on a Varian Unity 750-MHz spectrometer in 50 m
M
acetate buffer, pH 5.5 at 25 °C.
H. Fernando et al. Chemokine ligand–receptor interaction
FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS 243
ratio, and could be adequately fitted to a single-site
binding model. Control titration of IL-8(1–66) in to a
buffer showed a weak exothermic peak, which further
confirms that IL-8(1–66) is a monomer (not shown), as
dimer dissociation is endothermic. The thermodynamic
parameters for the binding of the IL-8(1–66) monomer
to the N-domain peptide were observed to be similar

to those of the trapped L25NMe monomer [22].
For the IL-8(1–66) monomer, the binding constant
(K
D
) is 8.6 lm, binding is enthalpically favored
(DH )11.8 kcalÆmol
)1
) and entropically disfavored
(TDS )4.8 kcalÆmol
)1
). For the trapped monomer, the
thermodynamic parameters are DH )10.5 kcalÆmol
)1
,
TDS )3.4 kcalÆmol
)1
, and K
D
6.0 lm.
Enthalpy of binding
Enthalpic factors typically include van der Waals,
hydrogen-bonding, and electrostatic interactions. The
structure of a ligand–receptor N-domain complex is
essential to identify the pairwise interactions and to
describe how different interactions contribute to the
observed enthalpy. The only structure available is that
of IL-8 complexed to a chemically synthesized human
CXCR1 N-domain peptidomimetic [11]. The sequence
of the peptidomimetic is shown in Fig. 3 (labeled as
p1). It corresponds to residues 9–29 and contains a sin-

gle six-amino hexanoic acid linker (shown as lin) for
residues 15–19. Sequences of our rabbit CXCR1 34-
mer (residues 11–44) and the corresponding human
CXCR1(9–39) are also shown. Identical and conserved
residues are shaded grey and underlined, respectively.
The structure of the complex reveals that binding is
dominated by burial of apolar residues, involving van
der Waals interactions between IL-8 Tyr13, Phe17,
Phe21, Leu43 and receptor Pro21, Pro22, Tyr27, and
Pro29 residues (numbering corresponds to the human
sequence; Fig. 3). The structure also shows evidence of
less well-defined electrostatic interactions between IL-8
Lys15, Arg47, Lys11 and receptor N-domain Asp24,
Glu25, Asp26 residues. These observations show that
residues that mediate binding in the complex are quite
conserved between the human and rabbit sequences.
In addition to the structure, knowledge of how spe-
cific residues contribute to binding affinity is essential.
Proximity of residues in the structure does not always
mean that they are involved in favorable interactions,
and even if involved in favorable interactions, struc-
tures cannot provide the relative strengths of the indi-
vidual interactions; such information can be inferred
only from mutagenesis studies. Mutagenesis studies in
IL-8 have shown that both apolar (Ile10, Tyr13,
Phe17, Phe21) and charged residues (Lys11, Lys15 and
Lys20) are important [12,16,17,35,36]. However, inter-
pretation of the receptor mutagenesis studies has been
A
B

Fig. 2. Representative isothermal titration
calorimetric profiles of IL-8 (1–66) and
L25NMe IL-8 monomers binding to the
CXCR1 N-domain. The titrations were car-
ried out at 25 °Cin50m
M Hepes ⁄ 50 mM
NaCl, pH 8.0 buffer, and are shown in (A)
and (B), respectively. The upper panels rep-
resent the ITC thermograms, and the lower
panels represent the fitted binding iso-
therms.
Fig. 3. Sequence of the CXCR1 N-domains.
Chemokine ligand–receptor interaction H. Fernando et al.
244 FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS
less straightforward. A characteristic feature of the
N-domain is the preponderance of Asp ⁄ Glu residues,
so it is reasonable to assume that some of these are
involved in binding to the positively charged IL-8
Lys11, Lys15 and Lys20 residues. However, mutating
Asp ⁄ Glu residues in the CXCR1 receptor N-domain
did not seem to affect binding; in fact, mutagenesis
studies identified only four residues (Thr18, Pro21,
Pro22 and Tyr27) as being important [19,37]. All of
these residues except Thr18 were also identified as
being important from the NMR structural studies.
On the other hand, mutational studies using a
CXCR1 N-domain peptide showed that some of the
acidic residues, in addition to the apolar residues, are
involved in binding to neutrophil receptors [20].
Although the results from the mutational studies of

the receptor N-domain are inconclusive, they do indi-
cate that the binding involves interactions with mul-
tiple IL-8 N-loop residues over a large surface area.
On the basis of structure–function studies, residues
that could be involved in protonation ⁄ deprotonation-
coupled binding are the IL-8 N-loop residue His18 and
any of the Asp ⁄ Glu residues in the receptor N-domain.
The possibility that the Asp ⁄ Glu could be protonated
on binding was especially intriguing, considering that
only chemokine receptor sequences show the prepon-
derance of negatively charged residues. Thermody-
namic parameters measured by ITC can be influenced
by the choice of buffer if proton transfer accompanies
the binding process. In this case, the measured enthalpy
of binding is linearly related to the intrinsic ionization
enthalpy of the buffer. The relationship between the
ionization enthalpy of the buffer and the measured
enthalpy is given by the following equation [38]:
DH
ITC
¼ DH
binding
þ nDH
ionization
where DH
ITC
is the experimentally observed binding
enthalpy, DH
binding
is the buffer-independent binding

enthalpy, DH
ionization
is the ionization enthalpy of the
buffer, and n is the net number of protons transferred
during binding. To investigate whether there is a net
proton transfer on IL-8 binding to the receptor
N-domain, ITC measurements were carried out using
single-component buffers with different ionization en-
thalpies ranging from 1.0 to 11.5 kcalÆmol
)1
. Table 1
lists the values of DH , TDS, and K
D
in three different
buffers. In all buffers, DH
ITC
values were similar,
within experimental error. The independence of meas-
ured DH
ITC
from DH
ionization
indicates that binding is
not accompanied by net protonation ⁄ deprotonation
events.
Additional experiments such as mutagenesis and
calorimetry measurements on the CXCR1 N-domain
are necessary to provide a more definitive answer on
the role of negative charges in binding. It is also poss-
ible that the N-terminal acidic residues are necessary

for interactions with extracellular matrix constituents
and integrins, and that such interactions play a more
vital role in the leukocyte recruitment process and ⁄ or
in angiogenesis [39].
We determined the heat capacity (DC
p
) for IL-
8(1–66) monomer binding to the CXCR1 N-domain
by measuring enthalpy (DH) at several temperatures
ranging from 20 to 35 °C. Table 2 lists the thermo-
dynamic parameters, and the data show that at all
temperatures, the measured enthalpies are exothermic,
and that the interaction is energetically less favorable
at higher temperatures, as evidenced by the increased
values of the dissociation constants. Provided that the
temperature dependence of DH is linear over the tem-
perature range studied, DC
p
is obtained as the slope
of DH versus temperature. Figure 4 shows a plot of
Table 1. Thermodynamic parameters for binding of IL-8(1–66) to the CXCR1 N-domain in different buffers. Measurements were carried out
at 25 °C, and the reported values are the mean of two experiments. All buffers contained 50 m
M NaCl. DH
ITC
is the experimentally mea-
sured binding enthalpy, and DH
ion
is the ionization enthalpy of the buffer.
Buffer, pH 8.0 n
K

D
(lM)
DH
ITC
(kcalÆmol
)1
)
TDS
(kcalÆmol
)1
)
DH
ion
(kcalÆmol
)1
)
50 m
M phosphate 1.1 8.7 ± 0.3 )11.6 ± 0.1 )4.8 ± 0.1 1.0
50 m
M Hepes 1.05 8.6 ± 1 )11.8 ± 0.1 )4.8 ± 0.1 5.0
50 m
M Tris 1.1 7.5 ± 1.5 )12.3 ± 0.1 )5.2 ± 0.1 11.5
Table 2. Thermodynamic parameters for binding of IL-8(1–66) to
the CXCR1 N-domain as a function of temperature. All measure-
ments were carried out in 50 m
M Hepes ⁄ 50 mM NaCl, pH 8.0 buf-
fer, and the reported values are the mean of two experiments.
Temperature
(°C) n
K

D
(lM)
DH
(kcalÆmol
)1
)
TDS
(kcalÆmol
)1
)
20 1.0 5.2 ± 0.3 ) 10.6 ± 0.4 ) 3.5 ± 0.5
25 1.05 8.6 ± 1.0 ) 11.8 ± 0.1 ) 4.8 ± 0.1
30 1.05 12.9 ± 4.0 ) 13.2 ± 0.2 ) 6.5 ± 0.1
35 1.1 12.3 ± 0.4 ) 14.2 ± 0.1 ) 7.2 ± 0.2
H. Fernando et al. Chemokine ligand–receptor interaction
FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS 245
DH versus temperature, and the data indicate a slope
[DC
p
¼ d(DH) ⁄ dT]of)238 calÆmol
)1
ÆK
)1
. Correlating
experimentally measured DC
p
and structural changes
on binding and ⁄ or folding has shown that positive
DC
p

involves burial of polar residues, and negative
DC
p
involves burial of apolar residues, respectively
[40]. Thermodynamic parameters for ligand–protein
interactions can be related to changes in solvent-
accessible surface area (ASA) upon binding. Empirical
equations have been derived that allow comparison of
the experimental thermodynamic parameters and
structure-based calculated parameters based on sur-
face area parameterization. This approach is clearly
an approximation; according to this parameterization,
the changes in heat capacity arise from binding-
induced changes in the solvent polar and apolar
ASA. To evaluate the energetic contributions of the
binding interface, we made use of the NMR solution
structure of IL-8 complexed to the human CXCR1
receptor N-domain [11]. The thermodynamic parame-
ters were calculated for the complex with and without
the receptor N-domain using the vadar program
[41].
The DC
p
was calculated using the following equa-
tion:
DC
p
¼ 0:45DASA
apolar
À 0:26 ASA

polar
where DASA
apolar
and DASA
polar
are the changes in
ASA of the apolar and polar residues, respectively
[39]. The structure-based calculations provide a DC
p
of
)407 calÆmol
)1
ÆK
)1
and DASA
apolar
and DASA
polar
of
)1354 and )777 A
˚
2
, indicating that more of the apolar
residues are buried on complex formation. The calcula-
ted and experimental DC
p
values have the same sign
but differ by % 170 calÆmol
)1
ÆK

)1
. Despite the limita-
tions of the structure such as the N-domain peptidomi-
metic containing a linker for residues 15–19, the
agreement between calculated and experimental DC
p
is
quite good, suggesting that burial and packing of apo-
lar residues are the predominant determinants for
binding. The mutagenesis studies do indicate a role for
electrostatic interactions, and the observation that the
experimental DC
p
is smaller than the structure-based
calculated DC
p
also suggests that the structure may be
missing some of these native interactions. The struc-
ture of the complex was calculated on the basis of
intermolecular NOEs; NOEs between charged residues
are the most difficult to assign, especially if one of the
interacting partners (receptor N-domain) is not isotopi-
cally labeled.
Entropy of binding
Discussion of entropic factors in terms of structure is
less straightforward. As motional properties correlate
with entropy, a detailed knowledge of the conforma-
tional flexibility and dynamic motions before and after
binding of both the partners is essential to quantita-
tively discuss entropic changes in structural terms. It

has now become increasingly clear that the experiment-
ally determined structures are a snapshot of one of
many conformations that a protein can adopt, and
that proteins undergo a variety of fast and slow
motions [42]. For instance, NMR relaxation measure-
ments show that protein backbone atoms undergo fast
dynamics (nanosecond–picosecond time scale) about
the average structure, and, further, such dynamics con-
tribute significantly to the entropy of the protein [43].
It is generally thought that the conformational flexibil-
ity and dynamic motions are reduced on binding, and
so would be entropically disfavored. In contrast with
conventional thinking, NMR relaxation studies have
shown that the backbone dynamics in the bound form
are not always quenched and may remain the same or
actually increase [44]. Further, release of water on
binding, which is entropically favored, should also be
considered. If the binding interface were predomin-
antly hydrophobic, the release of ordered water from
interacting partners upon association could dominate
the binding process [45].
IL-8 is highly structured in the free form, and NMR
studies of the complex suggest that IL-8 does not
undergo structural changes on binding to the
N-domain [11]. On the other hand, our CD data show
that the CXCR1 N-domain is unstructured in the free
form, and relatively more structured in the bound
Fig. 4. Temperature dependence of the enthalpy of binding of IL-
8(1–66) monomer to CXCR1 N-domain in 50 m
M Hepes ⁄ 50 mM

NaCl, pH 8.0 buffer. The solid line represents the least-squares fit
of the experimental data.
Chemokine ligand–receptor interaction H. Fernando et al.
246 FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS
form (Fig. 5). The N-domain peptide in the free form
shows a minimum at 199 nm, which is characteristic of
random coil structure, and on binding, the minimum is
shifted to % 203 nm and also shows a new peak at
% 220 nm. We measured the CD spectra of the bound
N-domain at three different stoichiometries, and
observed the spectrum of the bound N-domain to be
essentially the same (data not shown). The CD spectra
rule out helical structure (absence of characteristic
double-well minima at 208 and 222 nm), suggesting
that the N-domain binds in an extended fashion. These
observations are also consistent with the previous
NMR structural studies, which show that the receptor
N-domain binds in an extended form to a cleft formed
by the IL-8 N-loop residues [11].
The ITC data show that the binding is entropically
disfavored, and also the relatively smaller change in
entropy could be interpreted as entropic factors play-
ing only a marginal role in complex formation. How-
ever, this is not necessarily true, as the change in
individual entropic factors, such as release of water
or change in backbone dynamics, may be significant,
and the overall change cancels out the individual con-
tributions. Folding of N-domain on binding to IL-8
would be entropically disfavored, as the N-domain is
unstructured in the free form and structured in the

bound form. Knowledge of the dynamic characteris-
tics of both IL-8 and N-domain before and after
binding and whether binding is accompanied by
release or retention of water molecules is lacking, and
is also essential to provide a more quantitative des-
cription for the role of entropy in binding. Future
structural and dynamic studies of the complex should
provide such an answer.
Binding and folding
We have discussed the calorimetry data so far simply
in terms of binding, and have not explicitly considered
contribution of enthalpy and entropy from folding of
the N-domain. Mechanistically, binding could be des-
cribed by a model in which the N-domain adopts a
structure only on binding, or by an ensemble model in
which the free N-domain exists in multiple freely inter-
converting substates, one of which corresponds to a
folded state that is binding-competent. In the former
model, binding precedes folding, and in the latter, fold-
ing precedes binding. Although these two models are
mechanistically not equivalent, they are thermodynam-
ically equivalent. Therefore, it is possible, in principle,
to dissect the thermodynamics of folding and binding.
We have shown previously that the CXCR1 N-domain
is structured in the osmolyte, TMAO, and that IL-8
binds to the N-domain with higher affinity in TMAO
[22]. In that study, a CXCR1 N-domain modified with
a fluorescent tag was used; fluorescence spectroscopy
was used to show that the N-domain becomes struc-
tured in the presence of TMAO [DG

folding
1.7 kcalÆmol
)1
and a transition mid-point (C
1 ⁄ 2
) 1.6 m TMAO].
Therefore, to dissect the contribution between binding
and folding of the N-domain, we carried out binding
experiments in the presence of TMAO. Our rationale
was that the N-domain is structured in TMAO, so the
measured thermodynamics should be predominantly
due to binding. It is now well established that organic
osmolytes such as TMAO promote structure of parti-
ally and natively unfolded proteins and impart biologi-
cal activity to these proteins, and so serve as excellent
tools for studying the thermodynamic basis of protein
folding [46].
The ITC thermograms for 1 m and 2 m TMAO are
shown in Fig. 6, and the thermodynamic parameters
are listed in Table 3. We could not carry out the bind-
ing at higher TMAO concentrations because of limited
solubility of TMAO and the 34-mer. Our previous
studies have shown that a significant fraction of the
N-domain peptide should be folded in 2 m TMAO
[24]. The data indicate that in 2 m TMAO, binding is
tighter (lower K
D
values), and that both the enthalpy
and entropy values are higher. The approximately
threefold increase in binding affinity is comparable to

the approximately fivefold increase observed in our
previous fluorescence studies, which is quite good,
considering the intrinsic differences between the fluor-
Fig. 5. CD spectra of free (solid line) and bound (dash line) CXCR1
receptor N-domain. The spectrum of the bound form was obtained
by subtracting the spectrum of a 25-l
M solution of free IL-8(1–66)
monomer from the spectrum of an equimolar mixture (25 l
M each)
of IL-8(1–66) and receptor N-domain in 50 m
M sodium phos-
phate ⁄ 50 m
M NaCl, pH 8.0 buffer.
H. Fernando et al. Chemokine ligand–receptor interaction
FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS 247
escent-tagged and unlabeled N-domain, and that the
binding was measured using two different techniques.
The ITC measurements suggest that the folding
is energetically and enthalpically disfavored (DG
0.8 kcalÆmol
)1
, DH 3.7 kcalÆmol
)1
) and entropically
favored (TDS 2.9 kcalÆmol
)1
). Essentially the same
enthalpic and entropic factors (such as van der
Waals interactions and loss of conformational flexi-
bility) that govern binding also govern folding and

induction of structure [26,47]. The most important
observation is that these thermodynamic changes on
folding are small compared with binding. The
measured heat capacity also has contributions from
folding and binding. Most protein folding and struc-
ture-induction events are accompanied by a negative
DC
p
, which is to be expected because of burial of
apolar residues [45]. Our experimentally determined
DC
p
is negative, which, however, most likely reflects
the binding process, as the apolar residues of the
N-domain are buried predominantly because of
intermolecular interactions (binding) and not because
of intramolecular interactions (folding). Small chan-
ges in the thermodynamic parameters on folding also
suggest that the folded form is only slightly more
stable.
Conclusion
The thermodynamic basis of IL-8 binding to its
receptor CXCR1 N-domain has been characterized
using ITC. This report describes how different
enthalpic and entropic factors could mediate chemo-
kine binding to its receptor N-domain, and is one of
the few calorimetric studies that describes thermo-
dynamics of a GPCR class receptor. The major con-
clusion from this study is that the binding is
enthalpically favored and is mediated by optimal

packing interactions of apolar residues and to a les-
ser extent also by electrostatic and hydrogen-bonding
interactions. Future high-resolution structure deter-
mination of the complex and thermodynamic meas-
urements of both IL-8 and CXCR1 N-domain
mutants should provide a more quantitative relation-
ship between enthalpy and entropy and different
binding interactions, and be able to distinguish
between a model that involves multiple weak interac-
tions and a hot-spots model that involves a few key
interactions providing most of the binding energy.
We propose that all chemokine receptor N-domains
interact with their ligands using principles observed
for IL-8 ⁄ CXCR1 system, and that binding affinity
and receptor selectivity are mediated by modulating
A
B
C
Fig. 6. Representative isothermal titration calorimetric profiles of IL-8 (1–66) monomer binding to CXCR1 N-domain in TMAO. The titrations
were carried out at 25 °Cin50m
M Hepes, 50 mM NaCl, pH 8.0 buffer, and the data for 0, 1, and 2 M TMAO are shown in panels A, B, and
C, respectively. The upper panels represent the ITC thermograms, and the lower panels represent the fitted binding isotherms.
Table 3. Thermodynamic parameters for binding of IL-8(1–66) to
the CXCR1 N-domain in TMAO. All measurements were carried out
in 50 m
M Hepes ⁄ 50 mM NaCl, pH 8.0 buffer at 25 °C, and the
reported values are the mean of two experiments.
[TMAO]
(
M) n

K
D
(lM)
DH
(kcalÆmol
)1
)
TDS
(kcalÆmol
)1
)
0 1.05 8.6 ± 1 )11.8 ± 0.1 )4.8 ± 0.1
1 1.1 3.4 ± 0.6 )12.6 ± 0.1 )5.1 ± 0.1
2 1.0 2.7 ± 0.3 )15.5 ± 0.7 )7.7 ± 0.1
Chemokine ligand–receptor interaction H. Fernando et al.
248 FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS
the enthalpic and entropic components of the bind-
ing to the receptor N-domain.
Experimental procedures
Cloning, expression, and purification
of IL-8(1–66) monomer
The IL-8(1–66) construct was generated by introducing a
stop codon after residue 66 in the wild-type IL-8(1–72)
cloned in the pet32Xa vector at the LIC site. PCR amplifica-
tion was carried out using the upstream primer 5¢-GAGA
AGTTTTTGTAAGGCTCTAACTCTCCTCTG-3¢ and the
downstream primer 5¢-AGAGTTAGAGCCTTACAAAAA
CTTCTCCACAAC-3¢ with the QuickChange Site-Directed
Mutagenesis kit (Stratagene Inc., La Jolla, CA, USA). The
IL-8(1–66) monomer was expressed and purified using a pro-

tocol similar to that used for wild-type human IL-8 [23].
Briefly, transformed Escherichia coli BL21DE3pLysS cells
were grown in Luria–Bertani medium in the presence of
ampicillin to a A
600
of 0.5, and induced with 1 mm isopropyl
b-d-thiogalactopyranoside for 4 h at 37 °C. The pelleted cells
were solubilized in lysis buffer (500 mm NaCl, 20 mm
Tris ⁄ HCl, 5 mm benzamidine, 5 mm imidazole, pH 8.0), and
then subjected to four freeze–thaw cycles and sonication. The
protein-containing supernatant was loaded on to a Ni ⁄ nitril-
otriacetate column and eluted with the same buffer as above,
except containing 250 mm imidazole. Fractions containing
the protein were pooled and dialyzed against the cleavage
buffer (20 mm Tris ⁄ HCl, 50 mm NaCl, 2 mm CaCl
2
,
pH 7.4). The dialyzed protein was cleaved with Factor Xa,
and then purified by RP-HPLC using a gradient of acetonit-
rile in 0.1% heptafluorobutyric acid. The fractions contain-
ing protein were pooled, lyophilized, and stored at )20 °C
until further use. Both MS and analytical HPLC show that
the recombinant IL-8(1–66) is pure with no evidence of
impurities. The mass was verified using MALDI TOF MS.
Synthesis of the CXCR1 N-domain peptide
The rabbit CXCR1 34-mer (LWTWFEDEFANATGMPP
VEKDYSPSLVVTQTLNK) used in this study is the same
as that was used in all of our previous studies [22–25], and
was synthesized at the Biomedical Research Center, Van-
couver, Canada. The peptides were purified by RP-HPLC

and eluted with a gradient of acetonitrile in 0.1% trifluoro-
acetic acid, and the mass was confirmed MALDI TOF MS.
The sequence corresponds to residues 11–44, and is missing
the first 10 residues, which have been shown not to be
essential for binding [20]. At the time we initiated our
calorimetric and biophysical studies, we synthesized both
human and rabbit peptides, and observed that the rabbit
CXCR1 34-mer was easy to synthesize, better behaved, and
showed good heat signature.
CD studies
All CD spectra were collected on a Jasco J-720 spectropola-
rimeter at 25 °C in a 50 mm sodium phosphate ⁄ 50 mm
NaCl, pH 8.0 buffer. Samples of the receptor N-domain,
IL-8(1–66) monomer, and native IL-8 dimer were exten-
sively dialyzed against the buffer and then filtered before
determination of the protein concentration. Spectra were
recorded from 260 to 195 nm with a scan rate of 10 nmÆ
min
)1
in a 0.1-cm-path-length cuvette. Scans of the buffer
alone were averaged and subtracted from the averaged
spectrum of each sample. Spectra of the bound receptor
N-domain were obtained by measuring the spectra of the
equimolar mixture of the receptor N-domain and IL-8(1–
66), and subtracting the spectra of the free IL-8(1–66) at
the same concentration. Raw ellipticities were plotted,
because molar ellipticities cannot be accurately determined
in the case of protein complexes.
ITC
The ITC experiments were performed using the VP-ITC

system at 298 K as described previously [48]. The proteins
and the CXCR1 34-mer peptide were extensively dialyzed
against the appropriate buffer, centrifuged, filtered and
degassed just before the start of the experiment. Protein
and peptide concentrations were measured using
UV absorbance spectroscopy, and the absorption coeffi-
cients were determined by amino-acid analysis: for IL-8(1–
66), 7044 m
)1
Æcm
)1
, and for the N-domain peptide,
14962 m
)1
Æcm
)1
. Protein concentrations used for the titra-
tion ranged from 0.5 to 0.8 mm for the IL-8(1–66) mono-
mer, and 0.04–0.07 mm for the CXCR1 N-domain
peptide. For ITC experiments carried out in the presence
of TMAO, the samples were dialyzed in the appropriate
buffer, and aliquots of TMAO were added from a 4-m
stock solution. The 1.42-mL sample cell and the injector
were first washed with the dialysis buffer before the
CXCR1 34-mer and the IL-8(1–66) monomer were intro-
duced into the sample cell and injector, respectively. One
to five injections of 3 lL followed by 20–25 of 9 lL were
made, with a 6-min equilibration period between injec-
tions. The reference cell was filled with distilled water.
Control experiments such as protein and TMAO titration

into buffer alone were performed to evaluate the heats of
dilution, and subtracted from the experimental titration
results. The heat of dilution of IL-8(1–66) was small and
exothermic, providing further evidence that it is a mono-
mer, as dimer dissociation is endothermic. The heats of
dilution of the peptide and the buffer were small com-
pared with the heat of reaction. Data were fitted using a
nonlinear least squares routine using a single-site binding
model in Origin for ITC version 5.0 (Microcal), varying
the stoichiometry (n), binding constant (K
b
), and binding
enthalpy (DH°).
H. Fernando et al. Chemokine ligand–receptor interaction
FEBS Journal 274 (2007) 241–251 ª 2006 The Authors Journal compilation ª 2006 FEBS 249
Acknowledgements
We thank Dr Bolen for access to instrumentation, Drs
Bolen, Ro
¨
sgen, and Konkel for critical reading of the
manuscript, and Ms Eapen for technical assistance.
This work was supported by grants from the National
Institutes of Health and the American Heart Associ-
ation (to KR).
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