Tài liệu Báo cáo Y học: Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain docx

Molecular modeling of the dimeric structure of human lipoprotein
lipase and functional studies of the carboxyl-terminal domain
Yoko Kobayashi, Toshiaki Nakajima and Ituro Inoue
Division of Genetic Diagnosis, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Lipoprotein lipase (LPL) plays a key role in lipid metabo-
lism. Molecular modeling of dimeric LPL was carried out
using
INSIGHT II
based upon the crystal structures of human,
porcine, and horse pancreatic lipase. The dimeric model
reveals a saddle-shaped structure and the key heparin-
binding residues in the amino-terminal domain located on
the top of this saddle. The models of two dimeric
conformations – a closed, inactive form and an open, active
form – differ with respect to how surface-loop positions
affect substrate access to the catalytic site. In the closed form,
the surface loop covers the catalytic site, which becomes
inaccessible to solvent. Large conformational changes in the
open form, especially in the loop and carboxyl-terminal
domain, allow substrate access to the active site. To dissect
the structure–function relationships of the LPL carboxyl-
terminal domain, several residues predicted by the model
structure to be essential for the functions of heparin binding
and substrate recognition were mutagenized. Arg405 plays
an important role in heparin binding in the active dimer.
Lys413/Lys414 or Lys414 regulates heparin affinity in both
monomeric and dimeric forms. To evaluate the prediction
that LPL forms a homodimer in a Ôhead-to-tailÕ orientation,
two inactive LPL mutants – a catalytic site mutant (S132T)
and a substrate-recognition mutant (W390A/W393A/
W394A) – were cotransfected into COS7 cells. Lipase

activity could be recovered only when heterodimerization
occurred in a head-to-tail orientation. After cotransfection,
50% of the wild-type lipase activity was recovered, indica-
ting that lipase activity is determined by the interaction
between the catalytic site on one subunit and the substrate-
recognition site on the other.
Keywords: lipoprotein lipase; dimeric model structure;
heparin binding; substrate recognition; catalytic activity.
Lipoprotein lipase (LPL) belongs to a mammalian lipase
family that includes pancreatic lipase (PL), hepatic lipase
(HL), gastric lipase, and endothelial lipase [1,2]. The
primary function of LPL is triglyceride hydrolysis in
triglyceride-rich lipoproteins, such as chylomicron and very
low density lipoprotein (VLDL) particles, which are
converted to remnants. LPL is secreted from a variety of
tissues, such as adipocyte, macrophage, and muscle cells,
and is bound to the capillary bed of endothelium via cellular
surface heparan sulfate proteoglycans (HSPG), a function
reflected in LPL’s strong affinity for heparin. LPL defici-
encies in humans are manifested as severe hypertriglyceri-
demia [3–5] and arteriosclerosis [6]. Genetically engineered
mice lacking LPL also exhibit hypertriglyceridemia. In
addition to lipolytic activity, LPL functions as a ligand for
lipoprotein receptors, such as low density lipoprotein (LDL)
receptor, LDL receptor related protein (LRP), GP330/
LRP-2, and VLDL receptor [7–11].
A model structure of LPL had previously been construc-
ted, based on the crystal structure of human PL as a
template [12]. The model structure exhibited two domains –
a large N-terminal domain (1–312 amino acid residues) and

a small C-terminal domain (313–448 residues). The
sequences of PL and LPL are identical at 31% of their
residues in the N-terminal domain (40% similarity) and are
28% identical in the C-terminal domain (38% similarity).
The catalytic efficiency and heparin-binding functions of
the N-terminal domain have been extensively studied
[13,14]. A chimeric enzyme with the N-terminal domain of
LPL and the C-terminal domain of HL (LPL/HL) exhibited
the characteristic catalytic activity of LPL, as well as other
LPL-specific functions, such as activation by ApoC-II
and inhibition by NaCl [15]. Horse PL [16], human PL [17],
and complexes of human PL and procolipase [18,19] have
been crystallized. These studies demonstrated that the active
site in the N-terminal domain has two conformations – an
active, open conformation and an inactive, closed confor-
mation [18]. A surface loop functions as a lid and governs
the interaction of the lipid substrate with the enzyme’s
catalytic site [20]. On the protein surface at a site opposite to
the lid, occurs a cluster of basic amino acids (Arg279,
Lys280, Arg282) that constitutes a high-affinity, heparin-
binding site [14].
The function of the C-terminal domain has also been
addressed with a chimeric enzyme (LPL/HL), which
exhibits an affinity for heparin similar to that of native
LPL [21], suggesting that the major heparin-binding site
occurs in LPL’s N-terminal domain. Recently, however,
several lines of evidence have demonstrated that the
Correspondence to I. Inoue, Division of Genetic Diagnosis, Institute
of Medical Science, The University of Tokyo, Shirokanedai 4-6-1,
Minato-ku, Tokyo 108-8639, Japan.

Fax: + 81 3 5449 5764, Tel.: + 81 3 5449 5325,
E-mail:
Abbreviations: LPL, lipoprotein lipase; PL, pancreatic lipase;
HL, hepatic lipase; VLDL, very low density lipoprotein; HSPG,
heparan sulfate proteoglycans; LDL, low density lipoprotein; LRP,
LDL receptor related protein; DMEM, Dulbecco’s modified Eagle’s
medium; ADIFAB, acrylodated intestinal fatty acid binding protein.
(Received 17 May 2002, revised 26 July 2002, accepted 13 August 2002)
Eur. J. Biochem. 269, 4701–4710 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03179.x
C-terminal domain is also important in heparin binding.
The Arg405, Arg407, and Lys409 residues of avian LPL,
which correspond to the Lys403, Arg405, and Lys407
residues, respectively, in the C-terminal domain of human
LPL, have been demonstrated to be responsible for
heparin binding [22]. In another study with transgenic
mice expressing a human LPL with Asn residues substi-
tuted for basic amino acids at positions 403, 405, and 407,
the mutant LPL displayed normal enzyme activity but
with a reduced affinity for heparin [23]. The investigators
of the latter study concluded that HSPG binding at the
cell surface is required for maintaining LPL stability.
LPL’s C-terminal domain also appears to be involved in
the binding of the enzyme to receptors such as the LDL
receptor related protein (LRP) [24,25], with the critical
LRP-binding site having been demonstrated to be between
residues 340 and 438. Positively charged amino acid
residues in this region were replaced with Ala to test
receptor binding, because LPL is expected to interact with
the negative charges in LRP’s cysteine-rich repeats [26].
Thus, Lys407 was shown to be important for LRP

binding, whereas substitutions for Lys413 and/or Lys414
and Arg405 demonstrated only weakly decreased affinities
for LRP [24]. The C-terminal domain also has an
important function in binding lipid substrate. A cluster
of tryptophans – Trp390, Trp393, and Trp394 – which the
model structure revealed to be on the protein surface,
play a role in orienting the enzyme at the lipid–water
interface [27].
To better understand LPL’s dimeric structure and its
related functions, we constructed a model structure of the
dimeric form of LPL by using the crystal structures of
human, porcine, and horse PL as templates in which the
subunits are in a Ôhead-to-tailÕ orientation. Two dimeric LPL
model structures were constructed and were based on the
two PL forms in the protein database – the open form with
bound procolipase and the closed form without it. Amino
acid substitutions in the C-terminal domain were made to
address the functional roles of the C-terminal domain in
heparin binding and in catalytic activity. We also provide
experimental evidence of head-to-tail subunit orientation by
producing a functional heterodimer from two distinct and
inactive mutant subunits.
METHODS
Site-directed mutagenesis and expression of LPL
in cultured cells
A cDNA fragment containing the entire coding region of
human LPL was subcloned into expression vector pMT2 at
the EcoR1 site [14]. Nucleotide substitutions were generated
by the Chameleon double-stranded, site-directed mutagene-
sis kit (Stratagene, La Jolla, CA, USA). All the constructs

were verified by direct sequencing.
COS7 cells were maintained at 37 °Cand5%CO
2
atmosphere in Dulbecco’s modified Eagle’s medium
(DMEM), supplemented with 10% fetal bovine serum,
100 UÆmL
)1
of penicillin, 100 lgÆmL
)1
of streptomycin, and
0.25 lgÆmL
)1
of amphotericin B. Cells (1.8 · 10
6
)were
plated onto a 10-cm dish 1 day before transfection. The
pMT2 vector containing either wild-type or mutant LPL
was transfected with the LipofectAMINE
TM
reagent
(Invitrogen Japan K.K., Tokyo, Japan) according to
the manufacturer’s protocol. Two days after transfection,
the cells were washed twice with DMEM without phenol
red. To release LPL from the cell surface, the cells were
treated with 20 UÆmL
)1
of heparin (Sigma Aldrich Japan
K.K., Tokyo, Japan) at 37 °C for 8 h, and then the
conditioned media were collected.
In vitro

translation of the LPL C-terminal domain
A cDNA segment corresponding to the C-terminal domain
of LPL was placed under the control of the T7 promoter in
the pET4 vector (Novagen Inc., Madison, WI, USA).
In vitro transcription/translation reactions were performed
with TNT T7 quick-coupled transcription/translation sys-
tem in rabbit reticulocyte lysate (Promega Co., Madison,
WI, USA) labeled with [
35
S]-methionine (Amersham Bio-
sciences K.K., Tokyo, Japan).
Determination of LPL heparin affinity
The heparin affinities of whole-molecule LPL or the
C-terminal domain alone were determined by running a
heparin FPLC system as previously described [14]. The
proteins were separated by heparin-Superose chromatogra-
phyandelutedbya0–1.5
M
linear NaCl gradient. NaCl
concentration was directly monitored by flame spectropho-
tometry [14].
LPL concentration determination
LPL concentration was determined with a MARKIT-F
LPL kit (Dai-Nippon Pharmaceutical Co., Osaka, Japan),
which is based on the sandwich-ELISA developed for
human LPL. The kit contains two kinds of LPL monoclo-
nal antibody against human LPL purified from postheparin
plasma. One of the monoclonal antibodies, has an epitope
similar to that of the 5D2 monoclonal antibody and
was conjugated with b-

D
-galactosidase, and the other was
coupled with insoluble bacterial cell walls. LPL concentra-
tion was measured as the catalytic activity with 4-methyl-
umbelliferyl b-
D
-galactoside as a substrate. Fluorescence
emission signals at 450 nm after excitation at 365 nm were
monitored with a spectrofluorometer FP750 (JASCO Co.,
Tokyo, Japan).
Assay of lipase and esterase activities
Acrylodated intestinal fatty acid binding protein (ADI
FAB; FFA science LLC, San Diego, CA, USA) was used
to determine lipase activity [28]. The lipase substrate was a
mixture of triolein and phosphatidylcholine at a weight
ratio of four to one [29]. The lipid mixtures were dissolved
in chloroform and dried under nitrogen gas. The triolein/
phosphatidylcholine mixture was emulsified by sonication
in 20 m
M
Hepes, 150 m
M
NaCl, 5 m
M
KCl, 1 m
M
Na
2
HPO
4

, pH 7.4. The enzyme was incubated in the
same buffer with 10 nmolÆmL
)1
triolein at 37 °Cfor
30 min. Adding NaCl to a final concentration of 1
M
stopped the reaction. ADIFAB responds to fatty-acid
binding by shifting its fluorescence emission from 432 nm
to 505 nm [28]. The product of the lipase reaction, free
fatty acid, was measured after the addition of ADIFAB to
4702 Y. Kobayashi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
final 0.2 l
M
, and the ratio of the fluorescence intensity at
505 nm to that at 432 nm was determined at an excitation
wavelength of 386 nm. Oleic acid was used as a standard
for free fatty acid.
Esterase activity was measured using p-nitrophenyl
butyrate as a substrate. Samples were incubated at 37 °C
for 20–40 min in 150 m
M
NaCl, 0.5% Triton X-100,
100 m
M
sodium phosphate buffer (pH 7.0), and 500 l
M
p-nitrophenyl butyrate. Absorbance of the product,
p-nitrophenol, was measured at 400 nm with a DU640
spectrophotometer (Beckman Coulter Inc., Los Angeles,
CA, USA).

Western blotting
Proteins in the conditioned media were separated by
electrophoresis on a 12.5% polyacrylamide gel, followed by
electrotransfer onto a poly(vinylidene difluoride) membrane
(Nihon Millipore Ltd., Tokyo, Japan). The membranes were
blocked to prevent the binding of nonspecific proteins with
Block Ace (Dai-Nippon pharmaceutical). Anti-LPL mono-
clonal antibody 5D2 (Calbiochem-Novabiochem Co., San
Diego, CA, USA), anti-(bovine LPL) polyclonal lg (Ab7640)
provided by P H. Iverius (University of Utah, Salt Lake
City, UT, USA), or anti-(His-tag) polyclonal Ig (Medical
and Biological Laboratories Co., Nagoya, Japan) was used
to detect LPL or His-tagged proteins. Bound antibody was
reacted with HRP conjugated anti-rabbit or anti-mouse IgG
and developed with enhanced chemiluminescence reagents
(Amersham Biosciences K.K., Tokyo, Japan) on an LAS-
1000 plus image analyzer (Fuji Film, Tokyo, Japan).
Construction of the LPL model structure
The LPL model structure was constructed using the
molecular modeling system
INSIGHT II
version 2000 (Accelrys
Inc., Burlington, MA, USA) on a Silicon Graphics
workstation and was based on the structures of the human
and porcine pancreatic lipases for the open form and on
the horse pancreatic lipase for the closed form [16,18,19,30].
For homology modeling, the 11 C-terminal residues of
LPL were removed from the model because no homolo-
gous region occurred in PL. The crystal structures of
human and porcine PL (Protein Data Bank accession

numbers 1LPA and 1ETH) obtained from the protein
structure database ( include
bound procolipase, and these structures represent the
active form (open form) of the enzyme. Because no
sequence homology between procolipase and ApoC-II
was observed, procolipase was dissociated from the struc-
ture to model the open form of LPL. The crystal structure
of horse PL (Protein Data Bank accession number 1HPL)
exhibited a dimeric structure in its inactive form in which a
surface loop concealed the active site from the solvent.
After the homology modeling, addition of hydrogen,
modification of bonds, potentials of forcefield, and fixation
of heavy atom, backbone, and Ca, were performed for
molecular mechanics calculations. and then the energy
minimization of the model was iterated 300 cycles using the
conjugate gradient methods with the program
DISCOVER
_3
in
INSIGHT II
. To construct the dimeric form of LPL, the
model structure of monomeric LPL was duplicated and
superimposed on the crystal structure of the horse PL
dimer. After the energy minimization by setting at iteration
of 300 cycles or energy level of the final convergence at
0.002 kcalÆmol
)1
ÆA
˚
)1

was achieved using conjugate gradi-
ent method after fixation of heavy atom, backbone, and
Ca, the dimeric structure of LPL was finalized. The
averaged distance between Ser132 and Trp393 was calcu-
lated using the viewer module in
INSIGHT II
. In addition, a
mutation model of LPL was constructed after substituting
Ala for each basic amino acid, after performing the energy
minimization at final convergence at 0.002 kcalÆmol
)1
ÆA
˚
)1
or
by iteration of 300 cycles. The substitution model structures
were then used for the molecular dynamics simulations.
The velocity verlet method implemented in the
DISCOVER
_3
module of
INSIGHT II
was used at a constant temperature of
298.0 K for 5000 steps of 1.0 fs time-step.
RESULTS
Model structure of human LPL
All model structures of dimeric human LPL were construc-
ted using the
INSIGHT II
program and were based on the

crystal structures of the human, porcine, and horse PLs
(Fig. 1). A frontal view illustrates the overall saddle shape of
the dimeric structure and the key heparin-binding residues
in the N-terminal domain held coordinately by the two
subunits on the top of the hollow (Fig. 1A). The model
structure of the C-terminal domain illustrates two features.
One is a lining-up of basic residues (Arg405, Lys407,
Lys413, Lys414, Lys422, Lys428, and Lys430) oriented in
the same direction with the heparin-binding domain at the
N-terminal end [14]. The other is a cluster of tryptophans
(Trp390, Trp393, and Trp394) exposed to the surface
(Fig. 1A). The cluster of basic residues in the C-terminal
domain may constitute heparin binding-site residues that
coordinate with heparin-binding residues in the N-terminal
domain. The cluster of hydrophobic residues may function
in substrate recognition, as has been reported previously
[27].
LPL forms a homodimer and the dimer is conceivably in
a head-to-tail orientation. As has been demonstrated with
PL, the LPL model contains a surface loop covering the
catalytic pocket that modulates substrate access to the active
site. Two dimeric conformations – a closed, inactive form
and an open, active form – were modeled so that the
surface-loop positions differ (Fig. 1B). The figure of
the closed form illustrates that the surface loop covers the
catalytic site, which makes it inaccessible to solvent. This
observation suggests that substantial conformational chan-
ges must take place before substrate can bind to the active
site. The active LPL structure (open form) was modeled
from the structures of human and porcine PL cocrystallized

with procolipase, for which drastic conformational changes,
especially in the loop and the C-terminal domain, are
necessary to allow substrate access to the active site
(Fig. 1B). The loops of both peptides form regular helix-
turn-helix structures and lie close to each other, and a
substantial conformational change in the C-terminal
domain is induced. The key heparin-binding site of basic
amino acids in both peptides is located at a site opposite to
that of the active center, suggesting that heparin binding
regions may not play a direct role in LPL catalytic activity
(Fig. 1B).
Ó FEBS 2002 Dimeric model structure and function of lipoprotein lipase (Eur. J. Biochem. 269) 4703
Fig. 1. Molecular modeling of the dimeric structure of LPL. Two dimeric model structures of LPL were constructed using
INSIGHT II
version 2000 on
a Silicon Graphics workstation. The model structure of the closed form was generated from horse pancreatic lipase as a template (left), whereas the
model structure of the open form was from human and porcine pancreatic lipase (right). The structural views are from the side (A) or the top (B).
The N-terminal heparin-binding site (residues 279–282) is in yellow, the basic amino acids in the C-terminal domain are in green, and the cluster of
hydrophobic residues Trp390, Trp393, and Trp394 is in red. The catalytic site is enlarged in B and illustrates the conserved disulfide bridge between
Cys239 and Cys216 (black) and two distinct structures of the lid (orange).
4704 Y. Kobayashi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Heparin affinity of
in vitro
translated C-terminal
domain of LPL
Because C-terminal truncated proteins purified from a
prokaryotic system were eluted from the heparin affinity
column at extremely low efficiency, the proteins produced in
the in vitro system were measured for heparin affinity
(Table 1). The C-terminal domain of wild-type LPL (amino

acid residues 313–448) or its mutants, K381A, R405A,
K407A, K413A/K414A, K422A, K428A, and K430A, was
applied to a heparin-Superose FPLC system and eluted by a
linear NaCl gradient. The wild-type C-terminal domain was
eluted at 0.48
M
NaCl, whereas the substituted mutants,
R405A and K407A, demonstrated reduced affinities for
heparin by eluting at 0.33
M
and 0.39
M
NaCl, respectively.
The K413A/K414A mutant demonstrated a low affinity for
heparin by eluting at 0.26
M
NaCl. Other mutants (K381A,
K422A, K428A, and K430A) did not exhibit any altered
affinity for heparin by this method.
Impact of C-terminal substitutions on heparin
binding to full-length LPL
The affinities of heparin for full-length LPLs after muta-
genesis of residues in the C-terminal domain were examined
(Fig. 2) by heparin-affinity chromatography of the condi-
tioned media collected from cells transfected with either
wild-type or substitution mutant LPL. Chromatography
yielded two distinct immunoreactive peaks with a linear
NaCl gradient. The high-affinity peak corresponds to active
LPL homodimer, whereas the low-affinity peak corresponds
to inactive monomer together with some intermediate

degradation products. In the K413A/K414A mutant, the
two LPL peaks both eluted at lower salt concentrations
than those of wild-type LPL. The R405A mutant exhibited
an earlier elution of the high-affinity peak, whereas the
elution of the low-affinity peak was unchanged.
To investigate the effect of substitutions in the tryptophan
cluster of the C-terminal domain on heparin affinity, the
W393A/W394A (WW) mutant was applied to the heparin
FPLC column (Fig. 3A). Because the epitope recognized by
the monoclonal antibody in the MARKIT-F LPL kit is near
the peptide sequence containing Trp393 and Trp394
Table 1. Heparin affinity of C-terminal domain of LPL.
In vitro product of
C-terminal domain
Heparin affinity
NaCl (
M
)
Wild type 0.48
K381A 0.46
R405A 0.33
K407A 0.39
K413A/K414A 0.26
K414A No product
K422A 0.44
K428A 0.46
K430A 0.47
W393A/W394A 0.49
Fig. 3. Heparin binding of wild-type LPL and the WW mutant.
Wild-type LPL (left) and WW mutant (right) were applied to heparin-

Superose FPLC and eluted by a linear NaCl gradient. (A) LPL
concentration (j), lipase activity (h). The concentrations of the WW
mutant could not be detected with the MARKIT-F LPL kit, and
chromatography fractions were analyzed by Western blotting with
LPL polyclonal antibody 7640 (B).
Fig. 2. Heparin affinity of wild-type and basic amino acid–substituted
LPL. Proteins, released from LPL or LPL mutant–expressed COS7
cells, were subjected to heparin-Superose FPLC as described in [14].
Bound LPL was eluted by a linear NaCl gradient (broken line), and
concentrations of LPL (j) and lipase activities (h) were measured in
each fraction as described in the Materials and methods. The upper,
middle, and lower panels are the wild-type LPL, the K413A/K414A
mutant, and the R405A mutant, respectively.
Ó FEBS 2002 Dimeric model structure and function of lipoprotein lipase (Eur. J. Biochem. 269) 4705
(information from supplier), where the epitope of monoclo-
nal antibody 5D2 exists [27], no detectable LPL concentra-
tion was recovered for the WW mutant. Only a trace peak of
LPL activity was detected at the same position as the high-
affinity peak of wild-type LPL. The LPL in the eluate was
detected by Western blot with LPL polyclonal antibody
7640. The fraction number of the high-affinity peak was the
same for the wild type and the WW mutant (Fig. 3B).
Catalytic activities with long- and short-chain
fatty acid substrates
To study the functional impact of the LPL C-terminal
domain on catalytic activity, wild-type and mutant proteins
R405A, K413A, K413A/K414A, K414A, and S132T were
obtained from the conditioned media of transfected COS7
cells (Fig. 4A,B). Lipase activity was measured with a long-
chain fatty acid, triolein, as a substrate. The K413A mutant

exhibited the same level of lipase activity as wild type. The
lipase activities of the other mutants, R405A, K413A/
K414A, and K414A, were lower. Esterase activity measured
with the short-chain fatty acid substrate, p-nitrophenyl
butyrate, indicated that the esterase activities of the R405A
and K413A mutants were reduced to about half those of the
wild type, whereas the K413A/K414A and K414A mutants,
like the S132T mutant, lost esterase activity.
Next, the effects of the surface tryptophan substitutions at
the C-terminal domain on the lipase and esterase activities
were examined (Fig. 4C,D). Because the LPL concentration
of the mutants, WW and W390A/W393A/W394A (WWW),
could not be determined with the MARKIT-F LPL kit, their
lipase and esterase activities are not expressed as specific
activities. To confirm the LPL levels in the assay, Western
blot with anti-(His-tag) Ig was carried out in order to detect
the His-tagged LPL. Lipase activity was not detected in WW
or WWW mutants, whereas both mutants catalyzed the
short-chain fatty acid at the same rate as wild-type LPL.
Addition of the His-tag to the LPL C-terminus did not affect
enzyme activities (shown in Fig. 6B).
Conformational changes in the LPL mutant models
The model structures of R405A, K413A, and K414A
mutants were performed using the molecular dynamic
Fig. 4. Lipase and esterase activities of amino
acid–substituted mutants. The catalytic acti-
vities were measured as described in the
Materials and methods. LPL, released from
transfected COS7 cells by adding heparin to
the media, was assayed for catalytic activity.

The lipase (A) and esterase (B) activities of the
substituted mutants, R405A, K413A, K413A/
K414A, K414A, and S132T, are presented as
specific activities. Catalytic activities for the
WW mutant (Ala substituted for Trp393 and
Trp394) and the WWW mutant (Ala substi-
tuted for Trp390, Trp393 and Trp394) (C,D).
Protein expression of the wild type, the WW
mutant, and the WWW mutant tagged with
polyhistidine were evaluated by Western
blotting with anti-(His-tag) polyclonal anti-
body (insert in D).
4706 Y. Kobayashi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
simulation after substitution of the residues, and the LPL
mutant models were superimposed on wild-type LPL
(Fig. 5). The K413A mutant retains normal lipase and
esterase activities, and the structure of the K413A mutant
is similar to that of wild-type LPL. The model of the
R405A mutant, which has low lipase activity and a normal
esterase activity, displays a substantial conformational
change in the substrate-binding site in the C-terminal
domain (red arrow). Modeling of the K414A mutant,
which exhibits decreased lipase and esterase activities,
reveals substantial conformational changes in the N-ter-
minal heparin-binding site (blue arrow) and the substrate
recognition site (red arrow).
Recovery of lipase activity after cotransfection
of S132T and WWW mutants
Two possible subunit orientations have been postulated for
LPL, head-to-head and head-to-tail. The crystal structure of

PL suggests that a head-to-tail dimeric structure is the most
likely, but experimental evidence in support of this hypo-
thesis are meager. In the head-to-tail orientation, the
catalytic site in the N-terminal domain of one subunit and
the substrate recognition site in the C-terminal domain of
the other face each other, thereby bringing together the
substrate-binding and catalytic functions in proximity for
effective enzyme activity.
Two LPL mutants lacking the lipase activity – one a
catalytic site mutant (S132T) and the other a substrate
recognition site mutant (WWW) – were cotransfected into
COS7 cells (Fig. 6). If the head-to-tail model is correct, then
50% of the normal LPL activity should be recovered
stoichiometrically after the cotransfection experiment
(Fig. 6A). If head-to-head dimerization occurs, no lipase
activity is expected. Lipase and esterase activities (Fig. 6B)
are expressed per mL because the concentration of the LPL
WWW mutant could not be quantified. The lipase activity
of the His-tagged, wild-type LPL was comparable to that of
wild-type LPL. WWW and His-tagged WWW mutants
both exhibited only a trace amount of lipase activity, similar
to that of the S132T mutant, but both retained the esterase
activity. After the cotransfection of S132T and His-tagged
WWW mutants, almost half of the lipase activity was
recovered, indicating a head-to-tail subunit orientation
(Fig. 6B). A similar result was obtained for the esterase
activity. A Western blot confirmed that the two mutant
proteins were expressed in equivalent amounts (Fig. 6C).
DISCUSSION
LPL exerts its biological role at a lipid/water interface.

Consequently, its catalytic function requires a unique
structure that exposes the lipid-binding site. In the absence
of a crystal structure for LPL, any investigation into LPL
Fig. 5. Model structure of basic amino acid–substituted mutants. The mutant models after Ala substitutions for the Arg405, Lys413, and Lys414 of
wild-type LPL were constructed after the molecular dynamic simulation in the Discover_3 interface module of
INSIGHT II
.Indicatedare:wild-type
LPL (pink); mutant models (blue) superimposed on wild-type LPL backbone (pink trace); residues in the hydrophobic cluster (red); N-terminal
heparin-binding sites (yellow); the C-terminal heparin binding site (green); and substituted residues (orange).
Ó FEBS 2002 Dimeric model structure and function of lipoprotein lipase (Eur. J. Biochem. 269) 4707
structure–function relationships requires reliance upon a
model structure and functional expectations derived from
that model. The crystal structure of human pancreatic
lipase has provided a framework for such directed
approaches to the study of LPL structure and function.
LPL shares a high degree of primary-sequence similarity
with pancreatic lipase, and the conservation of most of
the disulfide bonds between LPL and PL suggests a
similar tertiary structure.
For the two dimeric models of LPL (the closed and open
forms, Fig. 1), the active dimeric form of LPL in particular
was based on the cocrystallized structures of the complexes
of human and porcine pancreatic lipases with procolipase.
The PL cofactor, procolipase, binds to the C-terminal
domain of PL and interacts with the surface loop, presum-
ably stabilizing the open form [18,19]. LPL requires the
cofactor apoC-II for triolein hydrolysis, but not for
tributyrin [31]. Despite the fact that the level of sequence
conservation between apoC-II and procolipase is low, it is
possible that a similar mechanism leading to a conforma-

tional change also produces the LPL open form. Recently,
apoC-II binding to the LPL C-terminal domain has been
Fig. 6. Recovery of lipase and esterase activi-
ties after coexpression of S132T and WWW
mutants. Schematic dimeric structures of LPL
in head-to-tail configuration after cotransfec-
tion of inactive LPL mutants and recovery of
catalytic activity (A). Lipase activity is not
detected in COS7 cells transfected with only
the S132T or the WWW mutant. If the cata-
lytic site and the lipid recognition site of dif-
ferent subunits regulate the lipase activity,
then lipase activity is recovered when head-to-
tail dimerization occurs after cotransfection of
S132T and WWW mutants. The lipase and
esterase activities after transfection of vectors
were assayed (B). Gray bar indicates the
recovery in LPL activity after cotransfection
of S132T and WWW mutants. The expression
levels of wild-type and mutant LPLs were
detected by Western blotting with anti-(His-
tag) polyclonal antibody or 5D2 monoclonal
antibody (C).
4708 Y. Kobayashi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
demonstrated with a cross-linking experiment using the
HL-LPL chimeric protein [21]. However, a previous study
with the LPL/HL chimera enzyme had suggested that the
LPL N-terminal domain was responsible for apoC-II
binding [15]. These conflicting results suggest that apoC-II
interacts simultaneously with complementary regions

located in the N-terminal domain of one subunit and the
C-terminal domain of the other. This hypothesis was
suggested when Razzaghi et al. demonstrated in a molecular
modeling experiment that the C-terminal domain of apoC-
II interacts with the interface of the N- and C-terminal
domains of LPL and part of the lid surface [32].
Because the functional importance of LPL’s C-terminal
domain is increasingly appreciated, the current study’s
approach to the function of the C-terminal domain is to be
derived from model structures and amino acid substitution
experiments. The C-terminal domain of LPL contains
regions that contribute to heparin affinity – critical basic
residues that line up with residues in the N-terminal
heparin-binding site (Fig. 1A). The truncated C-terminal
proteins of R405A, K407A, and K413A/K414A exhibit
reduced heparin affinities, whereas K381A, K422A,
K428A, and K439A proteins possess heparin affinities
comparable to wild type (Table 1). These observations are
further confirmed by monitoring the enzyme activity of the
entire molecule. The R405A mutant as an LPL dimer
exhibits a low heparin affinity and a decreased lipase
activity but retains esterase activity. On the other hand,
both monomeric and dimeric forms of the K413A/K414A
mutant possess reduced affinities for heparin. This LPL
mutant has very low lipase and esterase activities. The
model structure demonstrates that the side chains of
Lys413 and Lys414 face in opposite directions (Fig. 1B);
that is, Lys413 is exposed to the surface, whereas Lys414
lies buried in the molecule. Therefore, a substantial
conformational alteration occurs in the K414A mutation,

leading to the loss of lipase and esterase activities and
heparin affinity (Fig. 4A and B, 5). The lipase activity of
the K413A mutant is similar to that of wild type LPL,
whereas the esterase activity is reduced to 60% of the
wild type and the model structure displays no major
conformational change (Fig. 5). In the substitution model
structure, the R405A mutation results in a substantial
conformational change in the C-terminal domain, whereas
the N-terminal domain containing the active site is
unchanged (Fig. 5). The overall success of the model
structures in predicting function is a confirmation of their
reliability and accuracy.
Hydrophobic residues in a hydrophilic environment tend
to be held in a protein’s interior, so exposed hydrophobic
residues are uncommon. The model structure of LPL
reveals that the hydrophobic cluster of Trp390, Trp393, and
Trp394 is exposed to the surface, presumably at a lipid/
water interface (Fig. 1). Mutations in these hydrophobic
residues abolish the ability of the C-terminal domain to bind
or to induce VLDL, but this domain retains its capacity to
bind LRP [24,33]. Therefore, the hydrophobic cluster
should be crucial for lipid substrate binding. The fact that
the substitution mutants in this cluster (the WW and WWW
mutants) retain esterase activity but not the lipase activity
and that the normal affinity of these mutants for heparin
(Fig. 3, Table 1) implies conservation of the overall struc-
ture of the WW and WWW mutants – indicate that these
hydrophobic residues are important in determining sub-
strate specificity, a conclusion that the work of Lookene
et al. [27] has already established.

Of two possible dimeric structures of LPL, the head-to-
head and the head-to-tail models, the studies of the chimeric
proteins of hepatic lipase and LPL and the tandem repeat
approach of LPL [21,34] support the head-to-tail configur-
ation. According to the model structures, the distance
between the catalytic site (Ser132) and the substrate
recognition site (Trp393) is 59.2 A
˚
in the same subunit
and 29.3 A
˚
between the two sites on different subunits in
the dimer (data not shown), which implies that a dimer with
a head-to-tail configuration is an efficient catalyst. Here, we
applied a unique experimental approach to examine the
subunit orientation in the dimer. This involved the comple-
mentation of two LPL mutants, the WWW mutant lacking
lipid substrate recognition and the S132T mutant lacking
catalytic activity. If the substrate recognition site and the
catalytic site in the same polypeptide were responsible for
catalyzing the lipid substrate, which is the expectation of the
head-to-head model, then the lipase activity would not be
expected to recover in the LPL heterodimer consisting of
WWW and S132T mutant subunits. But lipase activity is
expected in the heterodimer of these mutants if the catalytic
site and the substrate recognition site are in proximity to
each other on separate subunits. Approximately 50% of the
lipase activity of the transfected wild-type cells is recovered
after cotransfection of WWW and S132T mutants. This
confirms that the dimeric structure of LPL is in the head-to-

tail orientation (Fig. 6).
In summary, the dimer models constructed for the
inactive and active forms of LPL reveal interesting
features of LPL structure, including the conformational
change in the active center, the critical sites for heparin
binding, and the orientation of dimerization. Investiga-
tion into the structure–function relationships of LPL
continues to provide important information for under-
standing the molecular mechanism of LPL action in lipid
metabolism.
ACKNOWLEDGEMENTS
We thank Kenta Kobayashi (SGI Japan Ltd. and Human Genome
Center of IMS, University of Tokyo) for computer system support.
This work was supported in part by a Research for the Future Program
Grant of The Japan Society for the Promotion of Science (II).
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