A New Pathway for Protein Haptenation by β-Lactams

Article abstract of DOI:10.1002/chem.201702643

The covalent binding of β-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of β-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the β-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA).

Full text of DOI:10.1002/chem.201702643

A Journal of
Accepted Article
Title: A Novel Pathway of Protein Haptenation by ß-Lactams
Authors: M. Consuelo Jiménez, Raúl Pérez-Ruiz, Emilio Lence,
Inmaculada Andreu, Concepción González-Bello, Miguel A.
Miranda, and Daniel Limones-Herrero
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201702643
Link to VoR: http://dx.doi.org/10.1002/chem.201702643
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A Novel Pathway of Protein Haptenation by β-Lactams
Raúl Pérez-Ruíz,[a] Emilio Lence,[b] Inmaculada Andreu,[c] Daniel Limones-Herrero,[a] Concepción
González-Bello,[b] Miguel A. Miranda*[a] and M. Consuelo Jiménez*[a]
Abstract: The covalent binding of β-lactams to proteins upon
photochemical activation has been demonstrated using an
integrated approach that combines photochemical, proteomic
and computational studies, selecting human serum albumin
(HSA) as target protein and ezetimibe (1) as probe. The results
have revealed a novel protein haptenation pathway by this
family of drugs that is alternative to the known nucleophilic ring
opening of β-lactams by the free amino group of lysine residues.
Thus, photochemical ring splitting of the β-lactam ring following
a formal retro-Staudinger reaction gives a highly reactive ketene
intermediate that is trapped by the neighbouring lysine residues,
leading to an amide adduct. For the investigated 1/HSA system,
covalent modification of Lys414 and Lys525 residues, which are
located in sub-domains IIIA and IIIB, respectively, occurs. The
observed photobinding may constitute the key step in the
sequence of events leading to photoallergy. Docking and
Molecular Dynamics simulation studies provide an insight into
the molecular basis of the selectivity of 1 for these HSA sub-
domains and the covalent modification mechanism. The
computational studies also reveal a positive cooperative binding
of sub-domain IIIB that explains the experimentally observed
modification of Lys414, which is located in a hardly accessible
pocket (sub-domain IIIA).
of the most important achievements in the history of Medicine
(Chart 1). Also relevant are β-lactamase inhibitors, such as
clavams and carbapenems, which are used to block the most
prevalent cause of antibiotic resistance in bacteria, thus the
enzymatic inactivation of drugs by β-lactamases. In 2010, the
international consumption of β-lactams made up 65% of the
global antibiotics market.[1]
Currently marketed β-lactams are responsible for inducing a
variety of adverse reactions; allergy is probably the most
important and can be manifested as pruritus, erythema, urticaria,
dermatitis, fever, asthma or even anaphylactic shock.[2] The
most frequently reported allergy problems are due to penicillins,
with up to 10% of patients exhibiting hypersensitivity to these
drugs, followed by cephalosphorins.[3] Less prevalent but also
significant are the allergic processes due to carbapenems,
clavams and monobactams (Chart 1).[4],[5] These allergic
diseases are due to the interaction of β-lactam drugs with the
immune system that has been mainly explained by the hapten
hypothesis. It is based on the observation that small organic
molecules do not induce an immune response unless they are
covalently bound to a protein.[6]
Introduction
Drugs containing the β-lactam ring (azetidinones) are the first
choice for the treatment of a broad range of bacterial diseases.
That is the case of the widely prescribed β-lactam antiobiotics,
such as penicillins, cephalosporins and monobactams, which
have saved millions of lives and whose discovery has been one
[a]
Dr. R. Pérez-Ruiz, Dr. D. Limones-Herrero, Prof. M. A. Miranda,
Prof. M. C. Jiménez
Chart 1. Chemical structures of some families of β-lactam antibiotics and
inhibitors of antibiotic resistance that contain the β-lactam moiety.
Departamento de Química/Instituto de Tecnología Química UPV-
CSIC
Universitat Politècnica de Valencia,
Camino de Vera s/n
46071 Valencia, Spain
Actually, β-lactams have been used to prove such hypothesis,
based on the high reactivity associated with the strained four
membered ring that facilitates covalent binding to proteins
through a nucleophilic attack by the amino groups.[7] In this
context, the most widely studied β-lactam is benzylpenicillin,
which is considered a reference model.[8] Opening of its four-
E-mail: mmiranda@qim.upv.es; mcjimene@qim.upv.es
Dr. E. Lence, Prof. C. González-Bello
Centro Singular de Investigación en Química Biolóxica e Materiais
Moleculares (CIQUS) and Departamento de Química Orgánica,
Universidade de Santiago de Compostela,
calle Jenaro de la Fuente s/n,
15782 Santiago de Compostela, Spain
Dr. I. Andreu
Instituto de Investigación Sanitaria La Fe,
Hospital Universitari i Politècnic La Fe,
Avenida de Fernando Abril Martorell 106,
46026 Valencia, Spain
[b]
[c]
membered ring by
a protein amino group leads to a
benzylpenicilloyl adduct through formation of an amide linkage.
Similar results have been reported for amoxicillin, the most
prescribed member of the family.[9] The main structural
difference between cephalosporins and penicillins is the nature
of the ring to which the β-lactam is fused, a 6-member
dihydrothiazine in the former and a 5-member thiazolidine in the
Supporting information for this article is given via a link at the end of
the document.
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latter. This feature modifies the electrophilic properties of the
carbonyl group and, therefore, its potential to react with
proteins.[10] Comparatively, little is known about drugs containing
a monocyclic β-lactam moiety, although the available chemical
data suggest that they should be much less reactive towards
amines and hence more reluctant to undergo covalent
attachment to proteins. For the above reasons, the covalent
binding of β-lactams to carrier proteins has been intensively
investigated, paying attention to detection and identification of
the products.
Chart 2. Chemical structure of ezetimibe 1.
Results and Discussion
Human serum albumin (HSA) is a carrier protein often employed
for model studies. It is the most abundant protein in human
plasma, complexing a wide variety of endogenous as well as
exogenous ligands.[11] Thus, HSA has been considered as the
main target protein in the haptenation by penicillins, and a
number of studies have focused on characterization of
penicilloyl-HSA adducts. Although the factors that determine
which amino acids become modified by β-lactams are not fully
established, lysine residues are clearly more reactive.[12]
Photochemistry of 1 in the absence of protein. Due to the
poor solubility of 1 in water, acetonitrile as well as a 9:1 (v/v)
mixture of acetonitrile and water were employed as solvents.
The results were similar, although the process was markedly
faster at shorter wavelengths. Irradiation of 1 was performed at

max = 300 and 254 nm, in a multilamp photoreactor. The course
of the reaction was followed by 1H-NMR spectroscopy. After
purification by column chromatography, imine 2[16] and lactone
3[17] were separated from the reaction crude as the sole
photoproducts (Scheme 1). The 1H- and 13C-NMR spectra of
isolated 2 matched with those of a commercially available
sample, while in the case of 3, they were compared with data
With this background, it appears relevant to explore whether
photochemical activation of β-lactams may enhance covalent
binding to proteins, a process that constitutes the key step in the
sequence of events leading to photoallergy. This is an important
issue, and the public health problem associated with
photobiological risk has been recognized by the major regulatory
entities (EMA, FDA, etc.), who have issued a set of guidelines
for compulsory photosafety testing of new active compounds.
reported in the literature.[17] The corresponding H and 13C-NMR
1
spectra are shown in the SI (Figures S1-S4).
The reaction mechanism is outlined in Scheme 1. The anilide
moiety would undergo N-CO bond splitting from the excited
state; subsequent C-C bond cleavage of the primary biradical I
would afford the corresponding retro-Staudinger products,
namely 2 and ketene II; the latter was not even detected, since it
rapidly evolves to the stable lactone 3, through an intramolecular
nucleophilic attack of the hydroxyl group to the ketene carbon.
In the present work, the photoinduced binding of β-lactam to
proteins has been investigated using an integrated approach
that combines photochemical, proteomic and docking and
Molecular Dynamics (MD) simulation studies, selecting HSA as
target protein and ezetimibe (1, Chart 2) as probe. This recently
marketed drug contains a monocyclic β-lactam core and its main
function is to decrease the plasma cholesterol levels, acting as
selective absorption inhibitor in the small intestine and reducing
in this way the amount of cholesterol normally available to liver
cells. This mode of action is complementary to that of statins,
and is recommended as a second line treatment.[13] Concerning
its reactivity, previous studies on degradation of 1 in aqueous
media describe slow rearrangement or hydrolysis of the four
membered ring.[14] Being a monocyclic β-lactam, 1 could in
principle undergo photochemical ring splitting to give reactive
intermediates, whereas it should react only sluggishly with
nucleophiles win the dark. Moreover, the photoreactivity of 1
should increase in the presence of electron donors, such as the
aromatic side chains of the Tyr or Trp units of HSA.[15] The
obtained results indicate that 1 is indeed photolabile, giving rise
to a reactive ketene intermediate that undergoes nucleophilic
attack by Lys414 and Lys525, which are located in sub-domains
IIIA and IIIB of HSA, respectively, leading to covalent adduct
formation. In addition, the docking and MD simulation studies
performed provide an insight into the molecular basis of the
selectivity of 1 for these sub-domains of HSA and the covalent
modification mechanism.
Scheme 1. Photoreactivity of 1 in acetonitrile.
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To gain deeper insight into the excited states involved in the
process, absorption, fluorescence and phosphorescence
spectroscopy measurements were carried out on 1. Thus, the
absorption spectrum of 1 in MeCN (Figure S5 in SI) displayed a
maximum at 285 nm and reached the UVB edge of the solar
radiation. The fluorescence spectrum in the same solvent
exhibited a maximum at 314 nm. From the intersection between
the normalized emission (black trace, Figure 1) and excitation
bands (red trace, Figure 1), a singlet energy of 97 kcal mol−1
was obtained. In the phosphorescence spectrum, recorded in
EtOH at 77 K (blue trace, Figure 1), the maximum was located
at 400 nm; the triplet energy obtained from this emission curve
was 80 kcal mol-1. Thus, since the reported value for bond
dissociation energy of the N-CO bond is ca. 82 kcal mol−1,[18]
thermodynamic requirements indicate that the initial N-CO bond
cleavage should preferentially occur from the singlet excited
state.
Experiments performed with 1 indicate that the drug possesses
higher affinity towards site II. Although the binding constant
value cannot be accurately determined due to the extremely low
solubility of 1 in PBS, 1 clearly does not displace DG from site II,
while IBU does. Interestingly, when DA is bound to site I, 1 binds
free site II, which produces an allosteric effect that provokes a
compression of site I and a parallel enhancement of DA
fluorescence. Similar examples of allosteric effects can be found
in the literature.[20] The trend is shown in Figure 2, a plot of I/I0 vs
equivalents of ligand added, constructed with data from the
fluorescence experiments that appear in Figures S6 and S7 in
the SI.
Figure 2. Variation of relative fluorescence intensity observed for probe-
albumin solutions (4 × 10-5 M, 1:1 molar ratio) at em= 480 nm for DA/HSA and
475 nm for DG/HSA, upon addition of WAR, IBU or 1.
Figure 1. Normalized fluorescence emission (black trace) and excitation (red
trace) spectra of 1 at 1 × 10-5 M concentration, in MeCN, at T = 295 K, under
air; phosphorescence emission (blue trace) of 1 at 2 × 10-5 M concentration, in
EtOH, at T = 77 K, under air.
From the above experiments, it can be inferred that the value of
the binding constant of 1 to site II is lower than that of IBU and
DG (that are in the range of 2-3 ×106).[21] Indeed, it is probably
one order of magnitude lower, since DG is not displaced even
after addition of four equivalents of 1. Concerning the
stoichiometry of the complex, Job Plot experiments could not be
performed, due to the poor solubility of 1 in PBS. However, it
appears to be close to 1:1 (may be with some contribution from
a 1:2 complex), since the fluorescence of DA in HSA increases
until addition of ca. 1.3 equivalents of 1, and then reaches a
plateau.
Dark binding of 1 to HSA. Binding experiments were performed
following an established methodology based on the
displacement of site-specific fluorescent probes by non-emitting
compounds, such as 1. For this purpose, dansylamide (DA) and
dansylglycine (DG) were taken as site I and site II markers,
respectively.[19]
Selective excitation at 350 nm of DA/HSA or DG/HSA
complexes gave rise to fluorescence emission centred at 480 or
475 nm, respectively. Figures S6 and S7 in the SI show the
emission changes observed upon addition of ibuprofen (IBP, site
II probe) or warfarin (WAR, site I probe) to the DG/HSA and
DA/HSA systems. As expected for a site II ligand, addition of
increasing amounts of IBP to the DG/HSA solution (at 1/1 molar
ratio) induced a decrease of its emission intensity. Thus, DG
was displaced out of the protein, and consequently a weaker
and red-shifted emission was observed. In the case of DA/HSA,
a similar behaviour was observed with WAR as displacer.
Proteomic analysis of the photobinding of 1 to HSA.
Formation of covalent photoadducts was investigated by
proteomic analysis; these experiments would indicate precisely
which (if any) amino acid(s) are covalently modified after
irradiation of 1 in the presence of HSA. Thus, the photoreactivity
of 1 within HSA was addressed by HPLC-nanoESI analysis. The
procedure consisted on trypsin digestion to cleave peptide
chains mainly at the carboxyl side of Lys or Arg residues (unless
they are neighbour to a Pro residue), followed by HPLC-MS/MS.
The process allowed obtaining information on the modified
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peptide sequence and characterizing the adduct. Full scan and
fragmentation data files were analysed by using the Mascot®
database search engine (Matrix Science, Boston, MA, USA) and
by entering variable modifications that take into account the
main possible residues (Lys) able to react with ketene II
obtained after cleavage of the β-lactam ring. Sequence
coverage was 94%. The results are shown in Figure 3, with the
modified peptides indicated in red.
takes place to afford the corresponding amide adducts (Figure
5).
The main result was identification of two adducts in
414KVPQVSTPTLVEVSR428 and 525KQTALVELVQ534 (Figure 3),
with an increment of 194 amu. The modification site of the two
peptide sequences was assessed by tandem mass experiments
on the trypsin digests. The ESI-MS/MS spectra and
fragmentation pattern are shown in Figures S8 and S9. The
MS/MS analysis of fragment ions 414KVPQVSTPTLVEVSR428
and 525KQTALVELVQ534 showed that the modified amino acids
are Lys414 and Lys525, respectively). Products resulting from
transimination of imine 2 were not detected, even if they were
specifically searched. However, their formation cannot be
completely ruled out, since once formed, they would probably be
hydrolysed under the trypsin digestion conditions. The lack of
covalent binding of benzaldehyde to proteins when analysed in a
similar manner has been described in a previous work.[22]
Figure 4. Crystal structure of HSA complexed with palmitic acid (PDB code
4BKE,[23] 2.35 Å). The three repeated domains IIII and each sub-domains
AB are highlighted with different colors and labeled. The complex contains
seven palmitic acid molecules, depicted in spheres. The side chain of Lys525
and Lys414, which are located in sub-domains IIIB (red) and IIIA (cyan),
respectively, are shown in sticks and labeled.
MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE ENFKALVLIA
FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT
VATLRETYGE MADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTA
FHDNEETFLK KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA
CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV ARLSQRFPKA
EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK
ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF
LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE
FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV
SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV LNQLCVLHEK TPVSDRVTKC
CTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL SEKERQIKKQ
TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV
AASQAALGL
Figure 5. Protein adduct resulting after irradiation of the HSA/1 protein
complex.
Figure 3. Amino acid sequence obtained after irradiation of HSA/1 protein
complex, with the non-matched amino acids in green. The modified peptides
are in red, and the altered amino acid residues are yellow-highlighted.
In an effort to understand in atomic detail the covalent
modification mechanism and to gain insight into the molecular
basis selectivity for sub-domains IIIA and IIIB, molecular docking
and MD simulation studies were carried out, which is described
below. The binding modes of 1 in sub-domains IIIA and IIIB of
HSA were first studied by molecular docking using the program
GOLD[24] version 5.2 and the available enzyme coordinates of
the crystallographically determined HSA in complex with palmitic
acid (PDB code 4BKE,[25] 2.35 Å). This structure was chosen
because palmitic acid, in addition to domains I and II, also binds
in sub-domains IIIA and IIIB (Figure 4). The positions of the
palmitic acid molecules in each of the identified sub-domains
were used to define the recognition site and the radius was set
to 10 Å. Docking studies were independently carried out in sub-
domains IIIA and IIIB. Moreover, in order to assess the reliability
of the postulated binding, MD simulation studies were then
Computational studies. Docking and MD simulations. The
results of our proteomic studies revealed that irradiation of the
HSA/1 protein complex at 1:1 molar ratio causes the chemical
modification in a similar proportion of Lys414 and Lys525, which
are located in sub-domains IIIA and IIIB, respectively (Figure 4).
The structure of HSA consists in three homologous helical
domains, namely I-III, each of which is divided into two sub-
domains, A and B.[23] In addition, the experimentally observed
additional mass of 194 of the lysine residues suggests that in
both cases nucleophilic addition of their corresponding -amino
groups to the generated ketene intermediate after UV irradiation
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conducted with the highest score solutions obtained by docking
for each sub-domain IIIA and IIIB. The monomer of the HSA-
during 76% of the simulation with an average distance of 2.7 Å.
Another relevant interactions that contribute to the binding
specificity of 1 to sub-domain IIIB would be the numerous apolar
contacts within the pocket. Specifically, it would involve residues
Phe507, Phe509, Ala528, Leu529, Leu532, Leu547, Phe551,
Ala552, Leu547, and Met548 (Figure 6B).
IIIA/1 and HSA-IIIB/1 protein complexes in
a truncated
octahedron of water molecules obtained with the molecular
mechanics force field AMBER[26] was employed. To get insight
into the covalent addition mechanism, these simulation studies
were carried out considering the two possible protonation states
of Lys414 and Lys525. The results of these studies are
discussed below.
Three main reasons led us to consider that Lys525 would be
protonated in the HSA-IIIB/1 complex: (1) the pKa of Lys525,
calculated using the H++ Web server,[27] is 9.6; (2) comparison of
the simulation studies with the two possible protonation states of
Lys525 revealed that the ammonium group of the protonated
Lys525 was located closer to the carbonyl group of 1 than the
corresponding free amino group in the neutral Lys525; and (3)
during the simulation, the protonated Lys525 establishes an
electrostatic interaction with the carboxylate group of Glu518
(Figure S11A). The latter residue would be therefore well located
to deprotonate Lys525 to afford the required nucleophile for the
covalent modification reaction. Comparison of the position of the
ligand during the simulation (Figures S12AS12B) and the
analysis of the variation of the relative distance between the
atoms involved in the aforementioned polar interactions during
the whole simulation revealed that the ligand is well fixed in this
pocket and its conformation properly controlled by these
residues (Figures S13AS13C). In particular, the hydrogen
bonding interaction between Lys525 and the secondary hydroxyl
group in 1 is stable during 89% of the simulation with an
average distance of 2.8 Å. Moreover, the hydrogen bond of the
phenol group of 1 with the carboxylate of Asp549 is observed
during 76% of the simulation with an average distance of 2.7 Å.
Another relevant interactions that contribute to the binding
specificity of 1 to sub-domain IIIB would be the numerous apolar
contacts within the pocket. Specifically, it would involve residues
Phe507, Phe509, Ala528, Leu529, Leu532, Leu547, Phe551,
Ala552, Leu547, and Met548 (Figure 6B).
Binding of 1 and covalent addition to sub-domain IIIB  The
results from 100 ns dynamic simulation showed that the
proposed binding for 1 in sub-domain IIIB obtained by docking
was reliable as this complex proved to be stable during the
simulation. Analysis of the root-mean-square deviation (rmsd)
for the whole protein backbone (C, C, N and O atoms)
calculated in the complex obtained from MD simulations studies
revealed that it varies between 1.53.4 Å (average of 2.2 Å) and
is relatively low for sub-domain IIIB (0.81.6 Å and an average of
1.2 Å) (Figure S10). These studies revealed that 1 would be
anchored to sub-domain IIIB of HSA via four polar interactions
involving the lactam carbonyl group and the two hydroxyl groups
(Figure 6). Specifically, the ligand would establish: (a) an
electrostatic interaction between its lactam carbonyl and the
protonated ɛ-amino group of Lys525; (b) two hydrogen bonds
between its secondary hydroxyl group and the main carbonyl
group of Lys525 and the amide side chain of Asn405; and (c) a
hydrogen bond between its phenol group and the carboxylate of
Asp549.
Three main reasons led us to consider that Lys525 would be
protonated in the HSA-IIIB/1 complex: (1) the pKa of Lys525,
calculated using the H++ Web server,[27] is 9.6; (2) comparison of
the simulation studies with the two possible protonation states of
Lys525 revealed that the ammonium group of the protonated
Lys525 was located closer to the carbonyl group of 1 than the
corresponding free amino group in the neutral Lys525; and (3)
during the simulation, the protonated Lys525 establishes an
electrostatic interaction with the carboxylate group of Glu518
(Figure S11A). The latter residue would be therefore well located
to deprotonate Lys525 to afford the required nucleophile for the
covalent modification reaction. Comparison of the position of the
ligand during the simulation (Figures S12AS12B) and the
analysis of the variation of the relative distance between the
atoms involved in the aforementioned polar interactions during
the whole simulation revealed that the ligand is well fixed in this
pocket and its conformation properly controlled by these
residues (Figures S13AS13C). In particular, the hydrogen
bonding interaction between Lys525 and the secondary hydroxyl
group in 1 is stable during 89% of the simulation with an
average distance of 2.8 Å. Moreover, the hydrogen bond of the
phenol group of 1 with the carboxylate of Asp549 is observed
In an effort to obtain further details of the covalent modification
of Lys525 by 1, the HSA/ketene protein complex and amide
adduct were then explored by MD simulation studies. The
results suggested that the capture of the ketene intermediate by
nucleophilic attack of Lys525 must be very fast since this
intermediate moves away quickly from the Lys525 vicinity. This
was already observed during the minimization step, which is
previous to the dynamic simulation. As shown in Figure 6C, the
position of the resulting amide would be frozen by two hydrogen
bonds involving the hydroxyl and NH groups of the amide adduct.
In addition, the introduced chain in the ɛ-amino group of Lys525
would be embedded in the aforementioned apolar pocket.
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Figure 6. Proposed binding mode of 1 in sub-domains IIIB (AC) and IIIA (DE) obtained by docking and MD simulation studies. (A) Overall view of the HSA-IIIB/1 complex. The
main backbone of 1 is shown as yellow spheres while sub-domains IIIB and IIIA are highlighted in red and blue, respectively. (B) Interactions of 1 with sub-domain IIIB of HSA
(gray). A snapshot after 80 ns is shown. (C) Detailed view of the HSA-IIIB/1 adduct. The binding mode of the covalently modified Lys525 (green) through the formation of an
amide bond (yellow) is shown. Snapshot taken after 80 ns. (D) Overall view of the HSA-IIIA/1 complex. (E) Interactions of 1 with sub-domain IIIA of HSA. A snapshot after 100 ns
is shown. (F) Detailed view of the HSA-IIIA/1 adduct. The binding mode of the covalently modified Lys414 (violet) through the formation of an amide bond (yellow) is shown.
Hydrogen bonding and electrostatic interactions between the ligand and HSA are shown as blue dashed lines. Relevant side chain residues are shown and labelled.
Binding of 1 and covalent addition to sub-domain IIIA  Our
computational studies showed that 1 would be fixed to sub-
domain IIIA by three hydrogen bonds (Figures 6D and 6E). Two
of them are direct strong contacts, one between the carbonyl
group in 1 and the hydroxyl group of Ser489, and another one
between the hydroxyl group in 1 and the main carbonyl group of
Phe488. The analysis of the variation of the relative distance
between the atoms involved in the aforementioned interactions
revealed that they are very stable as they are present during
89% of the simulation with an average distance of 2.8 Å
(Figures S13DS13E). The third hydrogen bond, which would be
mediated by a water molecule, would involve the phenol group
in 1 and the main carbonyl group of Val456 (Figure 6E). On the
contrary to Lys525, Lys414 seems not to be contributing to the
binding of 1. Moreover, as for sub-domain IIIB, Lys414 would
also be establishing an electrostatic interaction with Glu492
(Figure S11B) and 1 would have numerous aliphatic interactions
with the apolar residues within the pocket. Specifically, residues
Leu387, Ile388, Tyr411, Val415, Leu430, Val433, Leu453,
Val456, Leu460, Val418 and Phe488 would be engaged.
As for sub-domain IIIB, the results from our MD simulations with
the ketene intermediate suggested that this compound would
also react rapidly with Lys414. Moreover, the resulting amide
adduct would establish two hydrogen bonds involving the
hydroxyl and the NH amide groups and the main carbonyl
groups of Phe488 and Leu491, respectively (Figure 6F). In
addition, the covalently modified Lys414 would be well
embedded in the apolar pocket involving the aforementioned
residues.
Remarkably, the analysis of the rmsd of the whole protein
backbone for the HSA-IIIA/1 complex revealed that it is
significantly larger than for the HSA-IIIB/1 one with values
between 1.55.1 Å (average of 4.0 Å) (Figure S10). Although the
rmsd values involving sub-domain IIIB are low (0.81.6 Å and an
average of 1.2 Å), it seems that the HSA-IIIB/1 complex would
be more stable than the HSA-IIIA/1 one. Comparison of the two
protein complexes clearly reveals that when 1 binds to sub-
domain IIIB it causes a large conformational change in this sub-
domain of up to 16 Å (Figure 7A). Similar findings were
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obtained for MD simulation studies carried out with the
corresponding palmitic acid protein complexes (Figures 7B and
7C).
Curry et al.[27] Thus, comparison of the crystal structures of
HSA/myristic acid protein complex (PDB code 1BJ5,[28] 2.5 Å)
with the wild-type HSA (PDB code 1AO6,[29] 2.5 Å) proved to
have a rmsd value after superposition of 479 -carbon atoms of
2.90 Å (Figure S14). Therefore, taking into account: (i) the
similar proportion obtained for the covalent modification of
Lys414 and Lys525 in the proteomic studies, (ii) the greater
accessibility of sub-domain IIIB than IIIA and (iii) the higher
stability of the HSA-IIIB/1 complex than the HSA-IIIA/1 one, we
considered that the formation of a ternary complex rather than a
mixture of binary complexes would be reasonable; this is
evaluated below.
Thus, when the ligand binds to sub-domain IIIB it causes a large
opening of this protein region, whereas the binding to sub-
domain IIIA leads to the opposite effect. This seems to be due to
the interaction of the ligand in the interface between the two sub-
domains (Figure 6A) that causes a remarkable increase of the
protein volume in this region and a dramatic reduction of the
flexibility of sub-domain IIIB. The relevant conformational
changes that HSA can undergo on binding several ligands,
specifically in domains I and III, was also previously reported by
Figure 7. (A) Comparison of the HSA-IIIB/1 (red) and HSA-IIIA/1 (green) complexes. Ligands are not shown for clarity. Note the large conformational change that undergoes sub-
domain IIIB when binding to 1. (B) Comparison of the HSA-IIIA/palmitic acid binary complex after minimization and prior simulation (grey) and after 100 ns of dynamic
simulation (pink). (C) Comparison of the HSA-IIIB/palmitic acid binary complex after minimization and prior simulation (grey) and after 100 ns of dynamic simulation (orange).
Note how the binding of palmitic acid to sub-domain IIIB causes the opening of this sub-domain of up to 11.4 Å, whereas the binding to sub-domain IIIA yields the opposite
effect. (D) Overall view of the proposed ternary HSA/1 complex obtained by MD simulation studies. Sub-domains IIIA and IIIB are highlighted in blue and red, respectively,
whereas the molecules of 1 are shown using yellow spheres. A snapshot after 60 ns is shown. (E) RMSD plots for the protein backbone (Cα, C, N, and O atoms) calculated in the
three complexes [HSA-IIIA/1 (green), HSA-IIIB/1 (red) and HSA-IIIBA/1+1 (black)] obtained from MD simulations studies.
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Ternary HSA/1 complex. Taking into account that: (i) depending
result of the interaction of the ligand with sub-domain IIIB
enhance the incorporation of a second ligand molecule to the
more hidden pocket, sub-domain IIIA. Thus, HSA seems
capable of transporting two 1 molecules in domain III due to a
“positive cooperative binding” of sub-domain IIIB. This effect
explains the unexpected and efficient experimentally observed
covalent modification of Lys414, which is located in a highly
hidden pocket of HSA.
on whether
1
binds to the sub-domain IIIA or IIIB
conformationally different complexes may be formed, and (ii) in
order to assess the affinity of 1 to sub-domain IIIA or IIIB, two
possible pathways should be evaluated for the formation of the
ternary complex (Scheme 2). Thus, we explored the
incorporation of 1 to the free sub-domain IIIB in the HSA-IIIA/1
protein complex to afford the HSA-IIIAB/1+1 one (A) and the
opposite situation to give complex B. The binding free energies
of 1 in each sub-domain of complexes A and B were calculated
using the MM/PBSA[30] approach in explicit water (generalized
Born, GB) as implemented in Amber (Table 1).
Hence, once the ketene intermediate is generated by irradiation
of the HSA/1 protein complex, covalent attachment would
probably occur by nucleophilic attack of Lys525 and Lys414,
which would be triggered by Glu518 and Glu492, respectively,
acting as the general base (Scheme S1). The MD simulation
studies carried out with the HSA/ketene intermediate in both
sub-domains suggest that the ketene reacts rapidly with both
lysine residues.
Conclusions
The results obtained in the present work constitute an
unambiguous proof supporting that, in addition to the known
nucleophilic ring opening of β-lactams by the free amino
group of lysine residues, there can be an alternative protein
haptenation pathway by this family of drugs. It consists on
the photochemical ring splitting of the β-lactam ring
following a formal retro-Staudinger reaction, to give a highly
reactive ketene intermediate that is attacked by the
neighbouring lysines. In the case of ezetimibe, a β-lactam
drug with anti-cholesterol activity, selective photobinding to
Lys414 and Lys525 of HSA is demonstrated by proteomic
analysis, in agreement with the expectations from docking
and MD simulation studies. The latter studies also show
that the specificity of ezetimibe for sub-domains IIIA and
IIIB is due to the formation of several strong hydrogen
bonding interactions with residues: (i) Phe488, Ser489 and
Val456 (for sub-domain IIIA) and (ii) Asp549, Asn405 and
Lys525 (sub-domain IIIB) as well as numerous lipophilic
interactions with apolar residues within those pockets. The
nucleophilic attack of Lys525 and Lys414 to the ketene
intermediate would be triggered by Glu518 and Glu492,
respectively, acting as the general base. These
computational studies also reveal that the unexpected and
efficient covalent modification of Lys414, which is located in
a quite hidden pocket (sub-domain IIIA), would be possible
due to a positive cooperative binding of sub-domain IIIB.
Thus, the interaction of the ligand with sub-domain IIIB
seems to cause a large opening of the protein that would
enhance the binding of a second ligand molecule in this
less accessible internal region. Work is currently in
progress, to investigate the possible generality of the novel
haptenation route in the widely employed penicillins and
cephalosporins antibiotics.
Scheme 2. Possible formation of the protein ternary complex.
Table 1. Calculated Binding Free Energies using MM/PBSAa
Ligand
Complex
Energyb
1
HSA-IIIA/1
43.6  0.2c
1
HSA-IIIB/1
44.3  0.2c
PALd
PAL
1
HSA-IIIA/PAL
HSA-IIIB/PAL
HSA-IIIAB/1+1 (A)
43.9  0.5c
50.2  0.2c
43.9  0.2c (sub-domain IIIA)
16.5  0.2c (sub-domain IIIB)
41.0  0.2c (sub-domain IIIA)
44.0  0.2c (sub-domain IIIB)
1
HSA-IIIBA/1+1 (B)
aEnergy units = kcal mol-1; bonly the last 80 ns of the whole simulation were
considered for the calculations; cstandard error of mean; dPAL = palmitic
acid.
For the binary complexes, the results of our computational
studies revealed that the affinity of 1, as well as that of palmitic
acid to sub-domain IIIB is higher than to IIIA. More importantly,
the binding of a second 1 molecule to the free sub-domain IIIA of
HSA-IIIB/1 complex to afford complex B is 24.5 kcal more
favorable than opposite case. In addition, our MD simulation
studies also showed that the resulting complex is more stable
than the HSA-IIIB/1 one (Figures 7D and 7E). In fact, for the
complex A, after 60 ns of dynamic simulation the ligand is no
longer interacting with sub-domain IIIB. Therefore, it seems that
the conformational changes that the protein undergoes as a
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Acknowledgements
Financial support from the Spanish Ministry of Economy and
Competiveness (CTQ2013-47872-C2-1-P, CTQ2016-78875-P,
SAF2013-42899-R, SAF2016-75638-R), Generalitat Valenciana
(PROMETEOII/2013/005), the Xunta de Galicia (Centro singular
de investigación de Galicia accreditation 2016-2019,
ED431G/09) and the European Union (European Regional
Development Fund -ERDF) is gratefully acknowledged. E. L.
thanks the Xunta de Galicia for a postdoctoral fellowship. We
are grateful to the Centro de Supercomputación de Galicia
(CESGA) for use of the Finis Terrae II supercomputer. The
proteomic analysis was performed in the proteomics facility of
SCSIE University of Valencia that belongs to ProteoRed PRB2-
ISCIII and is supported by grant PT13/0001, of the PE I+D+i
2013-2016, funded by ISCIII and FEDER.
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Keywords: Ezetimibe • Human Serum Albumin • Molecular
Dynamics Simulations • Photoallergy • Photobinding
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Raúl Pérez-Ruiz, Emilio Lence,
Inmaculada Andreu, Daniel Limones-
Herrero, Concepción González-Bello,
Miguel A. Miranda* and M. Consuelo
Jiménez*
Page No. – Page No.
A Novel Pathway of Protein
Haptenation by β-Lactams
A new haptenation mechanism of β-lactams drugs, which constitute the key step in the sequence of events leading to photoallergy, has
been identified. Selecting human serum albumin as target protein and ezetimibe as probe, the covalent modification of Lys414 and Lys525
residues (located in sub-domains IIIA and IIIB) were identified. The process involves the photochemical ring splitting of the β-lactam ring of
ezetimibe giving a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues.
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