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 m
Trang 1binding to CXCR1 receptor N-terminal domain
Harshica Fernando1, Gregg T Nagle2and Krishna Rajarathnam1
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 funcselec-tion, 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 lig-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: krrajara@utmb.edu
(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.
Trang 2MGSA⁄ 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 DCpby 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,
Trang 3and 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 15N-T1, T2, and 1H-15N NOE relaxation measurements (unpublished observa-tions) The correlation time (sc) 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
Fig 1 Characterization of the IL-8(1–66) monomer (A) CD spectra of a 25-l M 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 m M 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.
Trang 4ratio, 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
(KD) 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 KD6.0 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
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 C in 50 m M Hepes ⁄ 50 m M 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.
Trang 5less 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]:
DHITC¼ DHbindingþ nDHionization
where DHITC is the experimentally observed binding
enthalpy, DHbinding is the buffer-independent binding
enthalpy, DHionization 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 KDin three different buffers In all buffers, DHITC values were similar, within experimental error The independence of meas-ured DHITC from DHionization 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 (DCp) for IL-8(1–66) monomer binding to the CXCR1 N-domain
by measuring enthalpy (DH) at several temperatures ranging from 20 to 35C 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, DCp 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 DHionis the ionization enthalpy of the buffer.
KD (l M )
DHITC (kcalÆmol)1)
TDS (kcalÆmol)1)
DHion (kcalÆmol)1)
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 m M NaCl, pH 8.0 buf-fer, and the reported values are the mean of two experiments Temperature
K D (l M )
DH (kcalÆmol)1)
TDS (kcalÆmol)1)
Trang 6DH versus temperature, and the data indicate a slope
[DCp¼ d(DH) ⁄ dT] of)238 calÆmol)1ÆK)1 Correlating
experimentally measured DCp and structural changes
on binding and⁄ or folding has shown that positive
DCp involves burial of polar residues, and negative
DCp 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 DCp was calculated using the following
equa-tion:
DCp¼ 0:45DASAapolar 0:26 ASApolar
where DASAapolar and DASApolar are the changes in
ASA of the apolar and polar residues, respectively
[39] The structure-based calculations provide a DCpof
)407 calÆmol)1ÆK)1 and DASAapolar and DASApolar of
)1354 and )777 A˚2, indicating that more of the apolar
residues are buried on complex formation The
calcula-ted and experimental DCp values have the same sign but differ by 170 calÆmol)1ÆK)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 DCpis 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 DCp is smaller than the structure-based calculated DCpalso 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 m M
NaCl, pH 8.0 buffer The solid line represents the least-squares fit
of the experimental data.
Trang 7form (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 bindfold-ing 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 [DGfolding1.7 kcalÆmol)1 and a transition mid-point (C1⁄ 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 KD 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.
Trang 8escent-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
DCp, which is to be expected because of burial of
apolar residues [45] Our experimentally determined
DCp 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
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 C in 50 m M Hepes, 50 m M 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 m M NaCl, pH 8.0 buffer at 25 C, and the
reported values are the mean of two experiments.
[TMAO]
K D (l M )
DH (kcalÆmol)1)
TDS (kcalÆmol)1)
Trang 9the 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 A600of 0.5, and induced with 1 mm isopropyl
b-d-thiogalactopyranoside for 4 h at 37C 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 CaCl2,
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 25C 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)1Æcm)1, and for the N-domain peptide,
14962 m)1Æcm)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 (Kb), and binding enthalpy (DH)
Trang 10We 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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