Patkar3, Jesper Vind3, Allan Svendsen3and Robert Verger1 1 Laboratoire de Lipolyse Enzymatique CNRS-IFR1, Marseille, France; 2 Structural Biology Laboratory, Chemistry Department, Univer
Trang 1Binding of Thermomyces ( Humicola ) lanuginosa lipase to the mixed
Fluorescence resonance energy transfer and crystallographic study
Ste´phane Yapoudjian1, Margarita G Ivanova1, A Marek Brzozowski2, Shamkant A Patkar3, Jesper Vind3, Allan Svendsen3and Robert Verger1
1 Laboratoire de Lipolyse Enzymatique CNRS-IFR1, Marseille, France; 2 Structural Biology Laboratory, Chemistry Department, University of York, UK;3Enzyme Research, Novozymes A/S, Bagsvaerd, Denmark
The binding of Thermomyces lanuginosa lipase and its
mutants [TLL(S146A), TLL(W89L), TLL(W117F,
W221H, W260H)] to the mixed micelles of cis-parinaric acid/
sodium taurodeoxycholate at pH 5.0 led to the quenching of
the intrinsic tryptophan fluorescence emission (300–380 nm)
and to a simultaneous increase in the cis-parinaric acid
fluorescence emission (380–500 nm) These findings were
used to characterize the Thermomyces lanuginosa
lipase/cis-parinaric acid interactions occurring in the presence of
sodium taurodeoxycholate.The fluorescence resonance
energy transfer and Stern–Volmer quenching constant
values obtained were correlated with the accessibility of the
tryptophan residues to the cis-parinaric acid and with the lid
opening ability of Thermomyces lanuginosa lipase (and its
mutants) TLL(S146A) was found to have the highest
fluorescence resonance energy transfer In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open con-formation of TLL(S146A) Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments.As
a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosalipase for mimicking the interaction between the wild-type enzyme and lipids
Keywords: lipase; X-ray crystallography; cis-parinaric acid; fluorescence resonance energy transfer
Lipases (EC 3.1.1.3) can be defined as enzymes that catalyze
the hydrolysis of long-chain acyl-glycerols [1] In addition to
playing an important role in fat catabolism, they have
numerous applications in the food, cosmetics, detergent and
pharmaceutical industries [2–5]
In recent years, the three-dimensional structures of lipases
and lipase–inhibitor complexes have been determined using
X-ray crystallographic methods [6–11] All lipases show a
common a–b hydrolase fold [12] and a catalytic triad
composed of a nucleophilic serine, which is activated via
hydrogen bonds as part of a charge relay system, along with the histidine and the aspartate or glutamate residues [6,7] The crystal structures of some lipases have shown that the active site is covered by a helical surface loop or ÔlidÕ that renders the active site inaccessible to substrate This is referred to as the closed conformation of the lipase On the other hand, the three-dimensional structures of lipases complexed with inhibitors shows a rearrangement of the lid, allowing free access to the active site in the so-called open conformation, in which a large hydrophobic surface around the catalytic triad is exposed
Thermomyces lanuginosa lipase (TLL) has four trypto-phan residues located in positions 89, 117, 221 and 260 The side chains of W117, W221 and W260 are buried into the protein core, whereas the W89 residue is located in the central part of the helical lid [13] In the crystal structures of the open forms of TLL, W89 is in close van der Waals contact with the acyl moiety of an inhibitor mimicking the transition state [14,15]
The fluorescence technique was used previously to study the binding of TLL to small or large unilamellar vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) and to vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine [16] The authors concluded that TLL may bind with a similar affinity
to all types of phospholipid vesicles and may adopt a catalytically active conformation and be involved in inter-facial activation processes only when small unilamellar vesicles of POPG are used Furthermore, molecular
Correspondence to R Verger, Laboratoire de Lipolyse Enzymatique,
CNRS-IFR1, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20,
France Fax: + 33 91 71 58 57, Tel.: + 33 91 16 40 93,
E-mail: verger@ibsm.cnrs-mrs.fr
Abbreviations: cis-PnA, cis-parinaric acid; CMC, critical micellar
concentration; FRET, fluorescence resonance energy transfer; K SV ,
Stern–Volmer quenching constant; NaTDC, sodium
taurodeoxycho-late; OA, oleic acid; POPG,
1-palmitoyl-2-oleoylglycero-sn-3-phos-phoglycerol; RFI, relative fluorescence intensity; TLL, Thermomyces
lanuginosa lipase; TLL(S146A), inactive mutant with S146 mutated to
A; TLL(W89L), mutant with W89 mutated to L; TLL(W117F,
W221H, W260H), mutant with only the W89; W117 mutated to F,
W221 mutated to H and W260 mutated to H.
Note: the atomic co-ordinates have been deposited in the Brookhaven
Protein Data Bank with the accession code 1gt6.
(Received 20 August 2001, revised 5 December 2001, accepted 14
January 2002)
Trang 2dynamics simulations [17] indicated that the replacement of
a single amino acid at the active site (S146A) may lead to
conformational alterations in TLL
The aim of the present study was to investigate the TLL/
fatty acid interactions using the fluorescence resonance
energy transfer (FRET) technique
One of the prerequisites to be able to observe the FRET
between a donor (TLL tryptophans) and an acceptor
(fatty acid) is that there must exist a spectral overlap
between the donor emission and the acceptor absorption
spectra, and the donor and acceptor groups must be
the right distance apart and properly oriented [18]
Therefore 9,11,13,15-cis,trans,trans,cis-octadecatetraenoic
acid (cis-PnA), a naturally fluorescent fatty acid with
thoroughly characterized spectroscopic properties [19], was
chosen for use as a probe It has been previously
established that cis-PnA can act as an acceptor for the
tryptophan fluorescence emission [20], and its
spectroscop-ic properties have been used in studies on fatty acid
binding to various proteins [20,21]
Bile salts are the main detergent-like molecules
respon-sible for the solubilization of lipolytic products
(monoglyc-erides and free fatty acids) during the digestion of dietary
fats Sodium taurodeoxycholate (NaTDC) is a conjugated
bile acid, which forms very small micelles in an aqueous
solution [22] The mixed micelles of cis-PnA/NaTDC turned
out to be a convenient model system for studying the
interactions between a water soluble protein (TLL) and a
fatty acid in the form of mixed micelles
First, we studied the lipase free cis-PnA/NaTDC system
in order to characterize the cis-PnA/NaTDC mixed micellar
system The binding behavior of TLL (and its mutants) to
pure cis-PnA and to mixed micelles of cis-PnA/NaTDC was
then studied using the FRET technique In addition, X-ray
crystallogaphy studies were performed on the S146A
mutant in order to elucidate the particular properties of its
complexes with fatty acids
M A T E R I A L S A N D M E T H O D S
Materials
NaTDC was from Sigma and cis-PnA was from
Molecu-lar Probes A stock solution of 3.2 mM of cis-PnA in
ethanol containing 0.001% (w/v) butylhydroxytoluene
(BHT) as an antioxydant was stored in the dark at
)20 °C under an argon atmosphere These precautions
were taken to ensure that no polyene decomposition
would occur [20]
The TLL wild-type, its single mutants: TLL(S146A),
TLL(W89L), and triple mutant TLL(W117F, W221H,
W260H) were used All enzymes were kindly provided by
A Svendsen and S A Patkar from Novo Nordisk,
Denmark and prepared as described previously [23,24]
The buffers used were 10 mMTris/HCl pH 8.0, 150 mM
NaCl, 21 mM CaCl2, 1 mM EDTA and 10 mM acetate
pH 5.0, 100 mMNaCl, 20 mMCaCl2
UV absorption spectroscopy
Differential absorption spectra were recorded on a Uvikon
860 spectrophotometer from Kontron Instruments All
assays were carried out using two quartz cuvettes (optical
path length 1 cm) of 3.5 mL each: one for the assay and one for the control assay The contents of each cuvette were mixed 5–10 times by gentle inversion of the cuvette capped with Teflon stopper, and were then left unstirred during the measurement procedure Measurements were performed at room temperature Two types of experiments were per-formed (a) Titration of cis-PnA was carried out by the increasing amounts of NaTDC at pH 5.0, in the absence of TLL The assay and control cuvettes were both filled with buffer and NaTDC at the various concentrations tested cis-PnA was subsequently added to the assay cuvette from an ethanolic stock solution and differential absorption spectra were recorded between 200 and 450 mn (b) Absorption spectra of cis-PnA in the presence of TLL at pH 5.0 or
pH 8.0 The assay and control cuvettes were both filled with buffer, NaTDC and TLL cis-PnA was added afterwards into the assay cuvette and the differential absorption spectra were recorded
Fluorescence spectroscopy Fluorescence measurements were carried out at 29°C under constant stirring using a SFM 25 spectrofluorimeter from Kontron Instruments and a 3.5-mL quartz cuvette (optical path length 1 cm) During all the fluorescence measurements, the optical density was < 0.1 in the spectral range between 280 nm and 500 nm to avoid inner filter effect Two types of fluorescence experiments were performed
Titration of cis-PnA at various NaTDC concentrations at
pH 5.0 The cuvette was filled with buffer containing NaTDC at a given concentration cis-PnA was then added
to the cuvette and a fluorescence emission spectrum was recorded at an excitation wavelength of 320 nm by scanning at an emission wavelength ranging from 350 nm
to 500 nm
FRET experiments TLL (wild-type or mutant) was titrated
at pH 5.0 or pH 8.0 by adding increasing amounts of cis-PnA in the presence and absence of NaTDC The excitation wavelength was set to 280 nm and the emission wavelength ranged from 300 nm to 500 nm
The accessibility of tryptophan to cis-PnA was estimated
by measuring the quenching of the TLL fluorescence effected by cis-PnA, according to the Stern–Volmer equa-tion [25]:
F0
where F0and F are the fluorescence emission intensities in the absence and in the presence of a quencher, respectively, [Q] is the molar quencher concentration and KSV is the Stern–Volmer quenching constant KSVis appropriate for collisional quenching in which binding is not involved However, the Stern–Volmer equation fits well our experi-mental results, even though binding is clearly involved Consequently, KSVwill be replaced by ÔKSVÕ
Protein crystallization and crystallography TLL(S146A) solution was washed several times in 10K Centricon in 10 m Tris/HCl pH 8.0 buffer and
Trang 3concen-trated up to 20 mgÆmL)1 Crystallization trials were
performed using the hanging drop technique at 291 K
Screening for the crystallization conditions was performed
simultaneously at pH 8.0 (0.1M Tris/HCl buffer) and
pH 5.0 (0.1Macetate buffer) OA was used instead of
cis-PnA for crystallization experiments to avoid oxidation
during the crystallization OA was dissolved in iso-propanol
and mixed in this form with a protein sample at a 5 : 1
molar ratio (OA/lipase) After a 1-h incubation, the
resulting precipitate was removed by centrifugation in a
Sigma Eppendorf centrifuge (5 min, 18 000 g) and the
remaining protein was used in the crystallization
experi-ments NaTDC was added to the crystallization trials
separately at a concentration of 10 mM Crystals were flash
frozen in the liquid nitrogen and characterized in-house on
a Rigaku RU200 rotating anode source (k¼ 1.5418 A˚),
MAR Research 345 imaging plate scanner, Osmic focusing
mirrors and Oxford Cryosystem set at 120 K The X-ray
data were subsequently collected at the ESRF in Grenoble
on the MAR Research CCD detector at 100 K, processed
withDENZO and scaled and merged withSCALEPACK[26]
The structure was determined using the Molecular
Replacement method The lid was removed by molecular
modelling in QUANTA to get a model for molecular
replacement (lipase minus lid) The AMORE software
program [27] was used and the wild-type TLL structure
[14] (minus the lid) was used as a model The structure was
refined using maximum likelihood techniques withREFMAC
[28]; other calculations were carried out using the CCP4
suite of programs (Collaborative Computational Project,
Number 4, 1994)
Electron density map inspection, model building and
analysis were carried out with the X-FIT options of the
QUANTAsoftware program (Molecular Simulations Inc.)
R E S U L T S
Absorption spectroscopy
The UV absorption spectrum of cis-PnA was determined in
an ethanolic solution (95%) and found to be identical to
that obtained by Sklar et al [19] As cis-PnA is prone to
oxidation, the absorption spectrum of the stock solution
was checked regularly and no changes in the cis-PnA
absorption spectra were observed in the ethanolic solution
upon storage
The UV absorption spectrum of cis-PnA at pH 5.0 and
pH 8.0 as well as the fluorescence emission spectrum of
TLL (excited at 280 nm, at pH 5.0) in the presence of 1 mM
NaTDC are shown in Fig 1 At pH 5.0, the cis-PnA UV
absorption spectrum overlapped the TLL emission
spec-trum in the 290–380 nm wavelength range, whereas no
overlap can be observed at pH 8.0 No significant changes
in the TLL emission spectrum were detected between
pH 5.0 and pH 8.0 (data not shown)
The effects of NaTDC on the UV absorption spectrum of
cis-PnA at pH 5.0 are shown in Fig 2 In the absence of
NaTDC, the cis-PnA solution was slightly turbid As soon
as the NaTDC concentration reached at least 1 mM, the
solution became optically clear changing simultaneously the
absorption spectrum of cis-PnA Three main absorption
peaks appeared at 298 nm, 304 nm and 326 nm and
increased in proportion to the NaTDC concentration This
increase in the attenuence of cis-PnA leveled off at NaTDC concentrations above 4 mM
Fluorescence spectroscopy
No significant NaTDC fluorescence was recorded under our experimental conditions The excitation and emission spec-tra of cis-PnA were recorded at various NaTDC concen-trations at pH 5.0 The maximum of the excitation and the emission spectra were found to occur at 320 nm and
410 nm, respectively In order to estimate the critical micellar concentration (CMC) of NaTDC, the relative fluorescence intensity (RFI) of cis-PnA at 410 nm (excita-tion wavelength at 320 nm) was measured as a func(excita-tion of the NaTDC concentration at pH 5.0 At NaTDC concen-trations lower than 1 m , the fluorescence of cis-PnA was
Fig 1 Fluorescence Emission spectra of TLL (––) and UV absorption spectra of cis-PnA (- - -) TLL and cis-PnA concentrations were 0.8 l M
and 10 l M , respectively The buffer used was 10 m M acetate (pH 5.0)
100 m M NaCl, 20 m M CaCl 2 or 10 m M Tris (pH 8.0) 150 m M NaCl,
21 m M CaCl 2 , 1 m M EDTA NaTDC concentration was 1 m M The excitation wavelength used to obtain the fluorescence emission spectra was 280 nm.
Fig 2 Effects of various NaTDC concentrations on the UV absorption spectra of a solution of cis-PnA The cis-PnA concentration was kept constant at 10 l M Buffer was 10 m M acetate (pH 5.0) 100 m M NaCl,
20 m M CaCl 2 (- - -) 0 m M NaTDC, (– - –) 1 m M NaTDC, (– –)
2 m M NaTDC, (––) 4 m M NaTDC The schematic diagram on the right illustrates the experimental protocol used.
Trang 4negligible The RFI increased in parallel with the rise in the
NaTDC concentration above 1 mM This increase leveled
off at NaTDC concentrations higher than 4 mM(data not
shown)
The results of the FRET recordings obtained between
TLL and cis-PnA, at wavelengths ranging from 300 to
500 nm in the presence of NaTDC at pH 5.0, are presented
in Fig 3 As the molar ratio (R) between cis-PnA and TLL
increased, the RFI decreased at wavelengths ranging from
300 to 380 nm and increased simultaneously at wavelengths
ranging from 380 to 500 nm
From the data presented in Fig 3, the decrease in RFI
(%), measured at the maximal emission wavelength (kmax),
as well as the increase of RFI (%), measured at 410 nm, as a
function of cis-PnA concentration are presented in Fig 4 A
good quantitative correlation between increase and decrease
of RFI as a function of increasing concentration of cis-PnA
can be seen Furthermore, a plateau value is reached when
one molecule of TLL is added to one molecule of cis-PnA
(R¼ 1) Similar curves as those presented in Fig 4 were
also obtained for TLL(S146A), TLL(W117F, W221H,
W260H) and TLL(W89L) (data not shown)
Similar FRET experiments were also performed with
TLL, TLL(S146A), TLL(W117F, W221H, W260H),
TLL(W89L) and cis-PnA in the presence and absence of
NaTDC (Fig 5) In the presence of NaTDC, the FRET was
observed between TLL, TLL(S146A), TLL(W117F,
W221H, W260H), TLL(W89L) and cis-PnA In the absence
of NaTDC, the FRET was negligible Surprisingly, in the
absence of NaTDC, a clear-cut quenching process was
observed only with TLL(S146A) and TLL(W89L) Similar
experiments were performed at pH 8.0, in the presence of
Fig 3 FRET between TLL and cis-PnA The numbers refer to the
values of the molar ratio R of cis-PnA to TLL In all the assays, the
excitation wavelength was 280 nm The dotted line corresponds to
the fluorescence emission spectra of cis-PnA (1 l M ) recorded in the
absence of TLL under the same experimental conditions The dashed
line corresponds to the arithmetic sum of the TLL and cis-PnA spectra
recorded separately The correlation between quenched tryptophan
RFI (325 nm) and sensitized RFI of cis-PnA (410 nm) is presented in
Fig 4 TLL concentration was 0.8 l M and cis-PnA concentration
varied from 0 to 1 l M For the sake of clarity, only spectrum
corres-ponding to three cis-PnA concentrations (0, 0.4 and 0.8 l M ) are shown
(plain lines) The NaTDC concentration was 1 m M Buffer was 10 m M
acetate (pH 5.0) 100 m M NaCl, 20 m M CaCl 2
Fig 4 RFI decrease (d) at k max as well as RFI increase (s) at k 410 nm
as a function of cis-PnA concentration Data from Fig 3.
Fig 5 FRET between TLL (and its mutants) and cis-PnA in the presence and absence of NaTDC The protein concentration was 0.8 l M
and the cis-PnA concentration was varied stepwise from 0 to 1 l M
(0, 0.2, 0.4, 0.8, 1 l M ) Excitation wavelength: 280 nm Buffer pH 5.0
as in Fig 2 The data in the graph at the uppermost left hand corner are identical to those shown in Fig 3.
Trang 5NaTDC (data not shown) Quenching was observed only
between TLL(S146A), TLL(W89L) and cis-PnA
The maximum fluorescence emission wavelengths (kmax)
of TLL, TLL(S146A), TLL(W117F, W221H, W260H), and
TLL(W89L) (excitation at 280 nm, pH 5.0) with or without
NaTDC, in the presence or absence of cis-PnA are
summarized in Table 1 In the absence of cis-PnA, the
addition of NaTDC led to a blue shift of the kmaxof all the
lipases tested, except for TLL(W89L) Furthermore, in
contrast to what occurred with TLL(W89L), the addition of
cis-PnA in the presence of 1 mM NaTDC also led to a
significant blue shift in the case of TLL, TLL(S146A) and
TLL(W117F, W221H, W260H) It is worth noting that
TLL(W89L) displayed no significant blue shift under any of
the experimental conditions tested
Stern–Volmer plots for the fluorescence quenching of
TLL (mutants) by cis-PnA were calculated from the data
presented in Fig 5, in the presence of 1 mMNaTDC (data
not shown) The ÔKSVÕ constants calculated for TLL,
TLL(S146A), TLL(W117F, W221H, W260H) and
TLL(W89L) were 3.2.106, 4.6.106, 3.4.106and 1.5.106M )1,
respectively
X-ray crystallography
Good X-ray quality crystals of TLL(S146A) were obtained
in the presence of OA in 0.1M Tris/HCl pH 8.0 buffer,
10 mMNaTDC, 25% w/v poly(ethylene glycol) 5K MME,
25 mMMgCl2 Crystallization at pH 5.0 and control setups
were unsuccessful under similar conditions with wild-type
TLL Crystals of the TLL(S146A) mutant were found to
belong to the P21212 space group and to have two
molecules in the asymmetric unit, a packing density of
2.64 A˚3ÆDa)1and a solvent content of 53% The final X-ray
data are 97.8% complete up to 2.20 A˚ resolution (96.2% in
the 2.28–2.20 resolution shell) with an overall Rmerge of
0.075 (0.44), I/r(I) of 12.2, and a mean multiplicity of 3.2
observations per reflection The final model has a
crystal-lographic factor of 21.4 and a Rfree of 23.9 against all
reflections in the resolution range of 20–2.20 A˚ The overall
root mean square deviations (rmsd) from geometrical
ideality are 0.009 A˚ in bond lengths, 1.3° in bond angles,
and 1.2 A˚2for the DB between bonded atoms This model
is composed of all the atoms of all the residues between E1
and L269 in the case of molecule A (chain A) and molecule
B (chain B), with a rmsd of 0.17 A˚ between the
corres-ponding Ca atoms of molecule A and B However, due to
the high mobility and the resulting lack of clarity of the
electron density maps, occupancies of only a few residues
were set at zero during the refinement procedure and consequently in the final model as well This was the case in particular with loop 24–44 in molecule B and few residues
of this loop in molecule A 262 water molecules were identified and refined Both molecules have open (Ôfully activeÕ) conformations, as discussed previously [15] After satisfactory convergence of the refinement of the protein and water molecules, the remaining positive electron density
in the surroundings of the active site cavities was analyzed This made it possible to model and refine the full-length OA molecule in the active site of molecule B Due to the residual electron density present in the active site of molecule A, the modeling of the ligand was restricted to its carboxylic group and the alkyl chain between C2 and C9 The remaining atoms of the OA in molecule A, C10–C18, were included in the final protein model for the sake of overall clarity but their occupancies were set to zero, as the electron density of these atoms was negligible The OA compound was trapped
in the active site of molecule B in an unexpected manner (Fig 6A); it was rotated by 180° with respect to the main sn-1TLL alkyl chain binding site [14,15], which is referred
to here as antisn1 Structural studies of fatty acids bound to human serum albumin also revealed this unexpected behaviour [29] The OA carboxylic group of lipid molecule
is thrust deeply into this alternative binding cleft of molecule B, where it is anchored via hydrogen bonds between its carboxylic group and the carbonyl oxygen of N92 (2.7 A˚) and NE2 of H110 (Fig 6A) The C9–C10 cisdouble bond lies near the b carbon of A146 ( 3.0 A˚), causing this alkyl chain to bend and become wrapped around the W89 residue of the lid, and the C18 carbon is finally wedged between CD1 of I255 and CH2 of W89 The partially defined electron density map of the OA in molecule A binding site cavity was used to model the carboxylic group lying on the top of A146, which is stabilized by a hydrogen bond with the carbonyl oxygen of this residue (2.8 A˚) and the NE2 atom of H256 (2.8 A˚) The fact that C2-C9 carbon atoms of the lipid occupy the sn-1 position in the active site indicates that the lipid binds according to the Ôconventional modeÕ in this molecule The location of the remaining OA atoms is not very clear, due to the very weak electron density but the position of the first atom of the cis C9–C10 bond was used to model the remaining part of thsis moiety in a similar manner to the model of one of the alkyl chains in the TLL complex with di-dodecyl phosphatidylcholine [15] (not presented in Fig 6B for the sake of clarity), and with dodecyl phospho-nate inhibitor [14], which occupies the main sn-1 binding site
as depicted in Fig 6B
Table 1 Effects of NaTDC (1 m M ) and/or cis-PnA (1 l M ) on k max (nm) of the RFI of TLL and its mutants Data from Fig 5 Buffer was 10 m M
acetate (pH 5.0) 100 m M NaCl, 20 m M CaCl 2 The protein concentration was 0.8 l M and the excitation wavelength was 280 nm.
Protein (0.8 l M )
[NaTDC] ¼ 0 m M [NaTDC] ¼ 1 m M
[PnA] ¼ 0 l M [PnA] ¼ 1 l M Dk max [PnA] ¼ 0 l M [PnA] ¼ 1 l M Dk max
TLL(W117F, W221H, W260H) 335 335 0 324 304 )20
Trang 6D I S C U S S I O N
Mixed micelles ofcis-PnA/NaTDC
Adding NaTDC to an aqueous solution of cis-PnA resulted
in a drastic change in the UV absorption spectra of this fatty
acid (Fig 2) This spectroscopic property of cis-PnA is
probably associated with the transformation of cis-PnA
aggregates into mixed micelles of cis-PnA/NaTDC The
presence of mixed micelles of cis-PnA/NaTDC was also
suggested by the increase in the fluorescence emission
intensity of cis-PnA recorded with increasing amounts of
NaTDC (data not shown) The results obtained using
cis-PnA as a fluorescent reporter, indicate that the increase in
the cis-PnA fluorescence intensity was due to its
incorpor-ation into the NaTDC micelles, resulting in a drastic change
in the microenvironment to which cis-PnA was exposed This
phenomenon has already been used as the basis of a sensitive,
continuous and specific lipase assay involving the use of the naturally fluorescent oil from Parinari glaberrimum [30] The CMC ( 1 mM) of NaTDC measured using cis-PnA
as reporter is in good agreement with the previously published values [22] However, it is worth noting that cis-PnA cannot be used as a general fluorescent probe to evaluate the CMC of synthetic detergents such as Tween 20, Chaps, and Nansa (alkyl benzene sulfonate) In contrast to what occurred with NaTDC, no sharp increase was observed in the RFI of cis-PnA at the respective CMCs of the above mentioned detergents Therefore, NaTDC appeared to be the most suitable detergent for the present studies, as FRET measurements can be performed after the incorporation of cis-PnA into mixed micelles
We have observed by direct excitation at 280 nm an increase of the RFI of cis-PnA with increasing concentra-tions of NaTDC (data not shown) Consequently, we have selected a NaTDC concentration of 1 m to perform the
Fig 6 Oleic acid binding modes revealed by TLL-OA complex crystal structure (A) Anti sn-1 position of the OA in the hydrophobic catalytic cleft
of the open conformation of TLL(S146A) The REFMAC 2Fo-Fc electron density map of the active site surroundings in molecule B (contoured at
1 r level), with the ligand – oleic acid (OA) – omitted from this calculation; dashed lines indicate hydrogen bonds between OA and the protein (B) Comparisons between several ligand binding modes in the catalytic cleft of TLL The stereoview of OA binding modes in molecule A and B (OA_A and OA_B, respectively, thick lines); C9 and C10 correspond to the position of these atoms in the OA_A ligand, where the electron density was visible from its carboxylic group up to the C9-C10 carbon atoms: the alkyl chain of OA_A is included in the model beyond this double bond for the sake of clarity The corresponding part of the covalent complex of the C12 posphonate inhibitor–TLL [14], in which the C12 alkyl moiety occupies sn-1 site of the active center, is also shown (protein shown by a thin line, ligand marked C12), for comparisons.
Trang 7FRET experiments in order to optimize the signal to noise
ratio Under these conditions (NaTDC concentration of
1 mM), some cis-PnA molecules are not incorporated into
the mixed micelles of cis-PnA/NaTDC, as already indicated
from the data presented in Fig 2 It is known that the
micelle formation process of bile salts is a complex process
Pre-micellar aggregates of various sizes have been described
previously [22,31] This may explain why at 1 mMNaTDC,
we observed a clear and characteristic UV absorption
spectrum of cis-PnA, different from the one recorded in the
absence of NaTDC (see Fig 2) Despite this limitation
(a high RFI background resulting from a direct cis-PnA
excitation at 280 nm), we observed a significant FRET at a
NaTDC concentration of 4 mM(data not shown)
Further-more, the addition of aliquots of pure ethanol (up to a
concentration of 10%, v/v) to a mixed solution of cis-PnA/
NaTDC (1 lM/1 mM) does not significantly change the RFI
of the cis-PnA (data not shown)
Binding of TLL (and mutants) tocis-PnA and NaTDC
In the presence of NaTDC, withoutcis-PnA The presence
of NaTDC at a concentration of 1 mM resulted in a blue
shift of the kmaxof the RFI of TLL (4 nm), TLL(S146A)
(5 nm) and TLL(W117F, W221H, W260H) (11 nm) (see
Table 1), which was probably due to the decreasing polarity
of the local environment of W89 in these three molecules
The lack of any wavelength shift observed for TLL(W89L)
and the highest blue shift observed for TLL(W117F,
W221H, W260H) indicate that the lid’s W89 was involved
in the interaction with NaTDC micelles
These findings are in an agreement with the data obtained
from other studies [16,32], showing that W89 is the only
accessible tryptophan of the TLL and that the lid region is
directly involved in the binding of TLL to micelles of the
pentaoxyethylene octyl ether (C8E5) detergent [32]
In the absence of NaTDC, withcis-PnA The addition of
cis-PnA to TLL (or mutants) did not lead to any significant
changes in the kmaxof the RFI of the proteins (see Table 1)
Moreover, no FRET (Fig 5) was observed for TLL or for
its mutants upon addition of cis-PnA, probably due to the
physico-chemical state of cis-PnA in the absence of NaTDC
micelles However, the presence of cis-PnA led to a
quenching of the fluorescence emission only with
TLL(S146A) and TLL(W89L) The S146A and W89L
mutations are located either in the catalytic triad or in the
lid, respectively, and favor the open structure of the lipase in
solution, enhancing its interaction with cis-PnA Hence, the
mutant TLL(S146A) is not an appropriate substitute for the
wild-type TLL for mimicking the interaction between lipase
and lipids
In the presence of NaTDC andcis-PnA The absence of
significant FRET at pH 8.0 with an excitation wavelength
ranging from 250 to 290 nm (data not shown), may be
attributed to the lack of spectral overlap between the UV
absorption spectrum of cis-PnA and the fluorescence
emission spectrum of TLL (Fig 1)
The FRET between TLL and mixed micelles of cis-PnA/
NaTDC at pH 5.0 can be illustrated by comparing the
separately taken spectra of TLL and cis-PnA (dashed curve
from Fig 3 corresponding to their arithmetic sum) with
spectra obtained after mixing TLL and mixed micelles of cis-PnA/NaTDC Moreover, as the molar ratio (R) between cis-PnA and TLL increased, the RFI recorded at wave-lengths ranging from 300 to 380 nm decreased, while increasing simultaneously at wavelength ranging from 380
to 500 nm (Fig 4) This behavior indicates that a FRET occurred between TLL and cis-PnA An isobestic point was observed at 380 nm The distance between the donor and the acceptor can be estimated to be around 25 A˚ [20] Similar experiments were performed with a TLL mutant in which all four tryptophan residues were mutated to nonfluorescent amino acids (data not shown) In this case, the RFI decreased by 10-fold and no significant FRET was observed This indicates that any contributions of the tyrosine residues to the FRET were negligible under our experimental conditions
When mixed micelles of cis-PnA/NaTDC were added, a blue shift of the kmax of the RFI of TLL (7 nm), TLL(S146A) (14 nm) or TLL(W117F, W221H, W260H) (20 nm) was observed, which indicates that the environment
of their tryptophan residues became less polar (see Table 1) The greatest blue shift which occurred with TLL(W117F, W221H, W260H) and the smallest one with TLL(W89L) (2 nm) suggest that the lid is involved in the binding of the mixed micelles of cis-PnA/NaTDC On the other hand, these shifts might result from the quenching of the tryptophan fluorescence in the presence of the mixed micelles, which simultaneously reveals the fluorescence of the tyrosine residues However, this explanation can be ruled out, as no significant shift was observed with the TLL(W89L) mutant
in the presence of the mixed micelles Similar experiments were performed with BSA in a control experiment and no blue shift was observed, although FRET showed a characteristic decrease in the RFI at 330 nm and a simultaneous increase at 410 nm (data not shown) If the quenching of the tryptophan emission had revealed the tyrosine emission, then we would also have observed a spectral shift in the experiments performed with BSA and TLL(W89L), which was not the case We can therefore attribute the spectral blue shifts observed with mutants TLL(S146A) and TLL(W117F, W221H, W260H) to a change in the local surroundings of their W89 residues towards a less polar environment
The accessibility of the tryptophan residues to the cis-PnA quencher was used to estimate the changes taking place
in the lid region of TLL and its mutants As the values of
ÔKsvÕ calculated with TLL and TLL(W117F, W221H, W260H) were the same, we can assume that W89 is the only tryptophan side chain accessible to cis-PnA This conclusion was also confirmed by the lowest ÔKsvÕ obtained for TLL(W89L)
The blue shift observed in the case of TLL(W117F, W221H, W260H) confirms that the lid is involved in the interaction between TLL and the mixed micelles of cis-PnA/ NaTDC The highest accessibility of W89, assessed by ÔKSVÕ values, observed with TLL(S146A) indicates that this mutant has a higher binding affinity towards mixed micelles of cis-PnA/NaTDC than the wild-type TLL The S146A mutation presumably destabilizes the closed conformation of the lipase, exposing a cluster of hydrophobic amino acids, including L206, F95, F113, F211, Y21, A146, L147 and A146, and consequently enhances the interaction between the lipase and the lipid aggregates, resulting in an efficient
Trang 8FRET This is in agreement with the molecular dynamics
simulations data obtained by Peters et al [17], which
indicated that the mobility of the lid was enhanced in the
TLL(S146A) mutant Furthermore, the results of
independ-ent direct binding measuremindepend-ents carried out on TLL and
TLL(S146A) with monomolecular films of substrate
ana-logues have shown that TLL(S146A) has the highest affinity
for these substrates (S Yapoudjian, M Ivanova, I Douchet,
A Ze´nath, M Sentis, W Marine, A Svendsen and R Verger,
unpublished data) This confirms that TLL(S146A) is not an
appropriate substitute for the wild-type TLL for mimicking
the interaction between lipase and lipids
Crystal structure of TLL(S146A) complexed with OA
The crystal structure of TLL(S146A) in a complex with OA
is virtually identical to other complexed structures of this
enzyme [14,15], and can be classified as Ôfully activeÕ
conformation of TLL according to Brzozowski et al [15]
The lids in both molecules (A and B) are well defined in the
electron density maps and are in the same fully opened
conformations They are not involved in the interactions
between molecule A and B, and are almost free from
intermolecular contacts For example, the W89 in molecule
A is 8 A˚ from its nearest crystal lattice neighbour (F211)
and 16 A˚ from W89 of symmetry related molecule The
nearest ( 4 A˚) and only intermolecular contact of the lid in
molecule A is between its E87 and symmetry linked D111
There is only one (nondirect) hydrogen bond via water
molecules between E87 and N95–D96 of the symmetry
related molecule The lid in molecule B is also exposed to a
large crystal cavity and is free from strong intermolecular
interactions However, the proximity ( 4 A˚) of L90 from
L90 and D94 of symmetry related molecule might have
some stabilizing effect on the lid in this molecule This
would explain much better definition of the OA electron
density in the molecule B in comparison with higher
disorder of the ligand in the molecule A However, despite
of the lack of strong lattice contacts that may affect the
conformation of the lid, its high mobility (described in other
TLL crystal structure with unrestricted lid positioning [9]) is
not observed in the reported structure
The OA has been found in the exposed, catalytic cleft of
the enzyme in two completely different orientations:
Ôclas-sicalÕ sn-1 position and, unexpectedly, in a antisn1 binding
mode These two different OA binding modes may shed
some light on the unusual spectroscopic properties of the
Ser146fiAla mutant described and discussed above Firstly,
the Ser146fiAla mutation abolishes OG Ser146–NE2
His258 hydrogen bond (2.7 A˚), which stabilizes 144–148
loop with the active serine at its apex [6,14,15] As the
Ala146 residue is released from this interaction it collapses
slightly deeper ( 0.62 A˚ S146Ca–A146Ca distance) into
the protein core creating more space and flexibility for the
putative ligand in the active site region This relaxation,
together with the lack of steric hindrance created usually by
the hydroxyl group of Ser146 in the wild-type enzyme,
results in a larger binding cavity, capable of accommodation
of lipid analogues normally not acceptable by the wild-type
active site Secondly, the local perturbation caused by
Ser146fiAla mutation may affect the stability of its
neighbouring residue: L147 This can lead to a disruption
of the weak ( 3.8 A˚), but likely stabilizing, van der Waals
contact of L147 with W89 of the lid that is present in all closed wild-type TLL structures, resulting in a higher mobility of the lid in the S146A mutant Moreover, the removal of the potential structural strain of Ser146–His258 hydrogen bond may be propagated through freed His258 into the C-terminal (262–269) region of Tl lipase, which is thought to be one of the crucial structural element of this enzyme involved in the first steps of interfacial activation [15,16] Any small changes in this C-terminus/Arg84 switch area may therefore contribute substantially to the destabil-ization of the closed (low activity or activated form [15]) forms of Tl lipase, facilitating faster opening of the lid and its transformation into the fully active form It is also likely, that the lack of a hydroxyl group of Ser146 diminishes the role of this residue in the stabilization of the substrate– ligand molecule, leading to the ÔconfusionÕ of the enzyme in the process of the ligand recognition This may result in the two alternative OA binding modes observed here The better defined electron density for OA in molecule B may than in molecule A suggests that the antisn1 binding mode of this lipid is favored by the mutant This is probably due to the stabilizing effect of the hydrogen bonds of D92 and H110 with carboxyl group of the OA molecule, specific for this particular ligand conformation Whether this form of OA binding is enhanced further by the more stable conforma-tion of the lid in molecule B is difficult to assess as generally very weak crystal contacts of the lid in molecule B are only marginally stronger than in molecule A, and the tempera-ture factors of these regions are similar in both molecules The control crystallizations of the wild-type of TLL under the conditions used for the S146A mutant, have been unsuccessful This deficiency of crystals of the wild-type TLL–OA complex may result from the lack of interactions between TLL and OA in the crystallization conditions that
is in an agreement with the FRET data observed here Hence the physiological relevance of the two binding modes
of OA observed in S146A mutant should be interpreted with caution, as they may result from the small changes in the ligand binding cavity caused by the S146A mutation Micellar or molecular binding?
It is worth noticing that the FRET technique used in the present study cannot, in principle, distinguish a micellar from
a molecular binding mode In other words, one could expect
to observe comparable fluorescence signals whether a lipase molecule binds to the mixed micelle of cis-PnA/NaTDC or toa single molecule of cis-PnA incorporated into the mixed micelles These two alternative models are reminiscent to a long standing discussion about the surface dilution pheno-menon concerning the interaction of phospholipase A2 with mixed micelles of Triton X-100/phospholipid [33]
As seen in Fig 4, the FRET signal reaches a plateau value when a molecule of TLL is added per molecule of cis-PnA This stoechiometry is more in favour of a molecular recognition rather than a micellar binding mode More-over, we determined the three-dimensional structure of the open TLL(S146A), co-crystallised with mixed OA/NaTDC micelles We identified one OA molecule per TLL mono-mer lying in the catalytic site, in contact with W89, according to two binding modes (see Fig 6) These structural considerations suggest that the FRET was probably due to a molecular binding of TLL to cis-PnA
Trang 9Thus FRET can be taken as an index of the open
conformation of TLL
A C K N O W L E D G E M E N T S
This research was carried out with financial support of the BIOTECH
program of the European Union under contract no BIO4-CT97-2365.
We would like to thank the staff and beamline managers at the
European Synchrotron Radiation Facility (ESRF) (Grenoble) and SRS
Daresbury for their assistance with the data collection The
infrastruc-ture of the Structural Biology Laboratory in York is supported by the
Biology and Biotechnology Science Research Council (BBSRC) We
would like to thank Dr Antonie J VISSER (Wageningen Agricultural
University, MicroSpectroscopy Centre, Laboratory of Biochemistry,
the Netherlands) and Prof J Sturgis (LISM, CNRS, Marseille, France)
for fruitful discussions The assistance of Dr Jessica Blanc is
acknowledged for revising the English manuscript.
R E F E R E N C E S
1 Verger, R (1997) Interfacial activation of lipases: facts and
arti-facts Trends Biotech 15, 32–38.
2 Schmid, R.D & Verger, R (1998) Lipases: interfacial enzymes
with attractive applications Angew Chem International
1608–1633.
3 Faber, K (1992) Bio-Transformations in Organic Chemistry.
Springer-Verlag, Berlin.
4 Alberghina, L (2000) Protein Engineering in Industrial
Biotech-nology, Harwood Academic Publishers, Australia, Canada, France.
5 Bornscheuer, U.T & Kazlauskas, R.J (1999) Hydrolases in
Organic Synthesis: Regio- and Stereo-selective Biotransformations.
Wiley-VCH, Weinheim, New York.
6 Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E.,
Dodson, G., Tolley, S., Turkenburg, J.P., Christiansen, L.,
Huge-Jensen, B., Norskov, L., Thim, L & Menge, U (1990) A
serine protease triad forms the catalytic centre of a triacylglycerol
lipase Nature 343, 767–770.
7 Winkler, F.K., d’Arcy, A & Hunziker, W (1990) Structure of
human pancreatic lipase Nature 343, 771–774.
8 van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N.,
Verger, R & Cambillau, C (1993) Interfacial activation of the
lipase–procolipase complex by mixed micelles revealed by X-ray
crystallography Nature 362, 814–820.
9 Derewenda, U., Swenson, L., Wei, Y.Y., Green, R., Kobos, P.M.,
Joerger, R., Haas, M.J & Derewenda, Z.S (1994)
Conforma-tional lability of lipases observed in the absence of an oil–water
interface Crystallographic studies of enzymes from the fungi
Humicola lanuginosa and Rhizopus delemar J Lipid Res 35,
524–534.
10 Brzozowski, A.M., Derewenda, U., Derewenda, Z.S., Dodson,
G.G., Lawson, D.M., Turkenburg, J.P., Bjorkling, F.,
Huge-Jensen, B., Patkar, S.A & Thim, L (1991) A model for interfacial
activation in lipases from the structure of a fungal lipase–inhibitor
complex Nature 351, 491–494.
11 Grochulski, P., Li, Y., Schrag, J.D & Cygler, M (1994) Two
conformational states of Candida rugosa lipase Protein Sci 3,
82–91.
12 Ollis, D.L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F.,
Franken, S.M., Harel, M., Remington, S.J., Silman, I., Schrag, J.,
Sussman, J.L., Verschueren, K.H.G & Goldman, A (1992) The
a/b hydrolase fold Protein Eng 5, 197–211.
13 Derewenda, U., Swenson, L., Green, R., Wei, Y., Yamaguchi, S.,
Joerger, R., Haas, M.J & Derewenda, Z.S (1994) Current
pro-gress in crystallographic studies of new lipases from filamentous
fungi Protein Eng 7, 551–557.
14 Lawson, D.M., Brzozowski, A.M., Rety, S., Verma, C &
Dodson, G.G (1994) Probing the nature of substrate binding in
Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling Protein Eng 7, 543–550.
15 Brzozowski, A.M., Savage, H., Verma, C.S., Turkenburg, J.P., Lawson, D.M., Svendsen, A & Patkar, S.A (2000) Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase Biochemistry 39, 15071–15082.
16 Cajal, Y., Svendsen, A., Girona, V., Patkar, S.A & Alsina, M.A (2000) Interfacial control of lid opening in Thermomyces lanugi-nosa lipase Biochemistry 39, 413–423.
17 Peters, G.H., Svendsen, A., Langberg, H., Vind, J., Patkar, S.A., Toxavaerd, S & Kinnunen, P.K.J (1998) Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase Biochemistry 37, 12375–12383.
18 Cantor, C.R & Schimmel, P.R (1980) Fluorescence Spectroscopy
in Biophysical Chemistry, pp 433–465 W.H Freeman, San Francisco.
19 Sklar, L.A., Hudson, B.S., Petersen, M & Diamond, J (1977) Conjugated polyene fatty acids as fluorescent probes: spectro-scopic characterization Biochemistry 16, 813–818.
20 Sklar, L.A., Hudsen, B.S & Simoni, R.D (1977) Conjugated polyene fatty acids as fluorescent probes: binding to bovine serum albumin Biochemistry 16, 5100–5108.
21 Dansen, T.B., Westerman, J., Wouters, F.S., Wanders, R.J.A., van Hoek, A., Gadella, T.W.J & Wirtz, K.W.A (1999) High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2) Biochem J 339, 193–199.
22 Small, D.M (1971) The physical chemistry of cholanic acids In: The Bile Acids (Nair, P.P & Kritchevski, D., eds), pp 249–356 Plenum Press, N.Y.
23 Svendsen, A., Clausen, I.G., Patkar, S.A., Borch, T & Thellersen,
M (1997) Protein engineering of microbial lipases of industrial interest Methods Enzymol (Dennis, E & Rubin, B., eds),
pp 317–340.
24 Holmquist, M., Martinelle, M., Clausen, I.G., Patkar, S., Svendsen, A & Hult, K (1994) Trp89 in the lid of Humicola lanuginosa lipase is important for efficient hydrolysis of tributyrin Lipids 29, 599–603.
25 Lehrer, S.S (1971) Solute perturbation of protein fluorescence The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion Biochemistry 10, 3254–3263.
26 Otwinowski, Z & Minor, W (1997) Processing of X-ray diffrac-tion data collecdiffrac-tion in oscilladiffrac-tion mode Methods Enzymol 276, 307–326.
27 Navaza, J (1994) AMORE: an automated package for molecular replacement Acta Crystallogr A50, 157–163.
28 Murshudov, G.N., Vagin, A.A & Dodson, E.J (1997) Refine-ment of macromolecular structures by the maximum-likelihood method Acta Crystallogr D53, 240–255.
29 Bhattacharya, A.A., Gru¨ne, T & Curry, S (2000) Crystal-lographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin J Mol Biol.
303, 721–732.
30 Beisson, F., Ferte´, N., Nari, J., Noat, G., Arondel, V & Verger, R (1999) Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings J Lipid Res 40, 2313–2321.
31 Small, D.M (1968) Size and structure of bile salt micelles Advan Chem Series 84, 31–52.
32 Jutila, A., Zhu, K., Patkar, S.A., Vind, J., Svendsen, A & Kinnunen, P.K.J (2000) Detergent-induced conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy Biophys J 78, 1634–1642.
33 Deems, R.A., Eaton, B.R & Dennis, E.A (1975) Kinetic analysis
of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes J Biol Chem 250, 9013–9020.