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: ituro@ims.u-tokyo.ac.jp 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 (http://www.rcsb.org/pdb/) 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. 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Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain Yoko Kobayashi, Toshiaki. 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,