Protein Science ~2000!, 9:1783–1790. Cambridge University Press. Printed in the USA. Copyright © 2000 The Protein Society
Crystal structure of the catalytic domain of human bile salt activated lipase
SIMON TERZYAN,1 CHI-SUN WANG,2 DEBORAH DOWNS,2 BRET HUNTER,1 and XUEJUN C. ZHANG 1 1 2
Crystallography Program, Oklahoma Medical Research Foundation, 825 NE 13 th Street, Oklahoma City, Oklahoma 73104 Protein Studies Program, Oklahoma Medical Research Foundation, 825 NE 13 th Street, Oklahoma City, Oklahoma 73104
~Received April 25, 2000; Final Revision June 30, 2000; Accepted July 7, 2000!
Abstract Bile-salt activated lipase ~BAL! is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL ~residues 1–538! with two surface mutations ~N186D and A298D!, which were introduced in attempting to facilitate crystallization, has been determined at 2.3 Å resolution. The crystal form belongs to space group P212121 with one monomer per asymmetric unit, and the protein shows an a0b hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding ~residues 115–125 and 270–285!. Keywords: bile salt activated lipase; crystal structure; esterase; substrate specificity
Bile salt activated lipase ~BAL! plays important roles in the dietary uptake of triacylglycerol and cholesteryl esters. It presents major lipolytic activity that is secreted from vertebrate pancreas into the intestinal tract where it exerts its physiological functions ~Wang & Hartsuck, 1993; Wang et al., 1999!. In humans and a few other mammals, BAL produced from the same gene is also present in the milk to ensure efficient triacylglycerol utilization in breast-fed newborns as their pancreas develops ~Olivecrona & Hernell, 1976; Wang et al., 1989!. Besides the triacylglycerol hydrolase activity shared with other pancreatic lipases, BAL is the only known intestinal lipase that hydrolyzes cholesteryl ester, fat-soluble vitamins, and other fatty acid esters. BAL knock-out transgenic mice have a reduced uptake of cholesteryl ester, indicating that BAL is responsible for mediating intestinal absorption of cholesteryl esters ~Howles et al., 1996!. For this reason, BAL is also called pancreatic cholesterol esterase. Lack of BAL activity also causes incomplete digestion of milk fat and lipid accumulation by enterocytes in the ileum of newborn mice ~Howles et al., 1999!. Besides its roles in fat digestion, BAL is also implicated in the cycle of cellular homeostasis of cholesterol, which is particularly affected in tu-
Reprint requests to: X.C. Zhang, Crystallography Program, Oklahoma Medical Research Foundation, 825 NE 13 th Street, Oklahoma City, Oklahoma 73104; e-mail:
[email protected]. Abbreviations: BAL, bile salt activated lipase; hBAL, human BAL; bBAL, bovine BAL; RMSD, root-mean-square deviation; TC, taurocholate; T. ACE, Torpedo californica acetylocholine esterase; ACE, acetylocholine esterase; CRL, Candida rugosa lipase.
moral cells leading to cholesteryl ester storage within cytosolic lipid droplets ~Le Petit-Thevenin et al., 1998; Pasqualini et al., 1998!. BAL remains inactive during its travel from the pancreas to the small intestine. It has been recently shown that an interaction between BAL and chaperone Grp94 plays essential roles in the processes of BAL folding and secretion ~Bruneau et al., 1998!. This complex remains associated in order for BAL to survive the duodenal environment until arriving at the intestinal lumen where it then becomes activated in the presence of bile salts. As a cofactor, a bile salt may have several effects on BAL. Hydrolytic activity of BAL toward emulsified long-chain triacylglycerol or cholesteryl ester is achieved exclusively with primary bile salts ~i.e., bile salts containing a 7a-hydroxyl group! ~Hernell, 1975!, indicating specific interactions are required for the enzyme activation. Arginine residues are involved in bile salt binding through electrostatic interactions ~Blackberg & Hernell, 1993!. Although BAL possesses basal activities toward short fatty acid chain substrates ~e.g., p-nitrophenyl acetate!, enhanced enzymatic activity can be obtained with various bile salts ~Wang & Hartsuck, 1993!. The bile salt dependence of BAL allows the enzyme to accommodate both hydrophilic and hydrophobic substrates, thus placing its function between that of triacylglycerol lipases and esterases. Bile salts may also protect BAL from intestinal proteolysis, as indicated by the fact that both primary and secondary bile salts protect BAL from chymotrypsin digestion ~Vahouny et al., 1965; Hernell, 1975!. The amino acid sequences of BALs are highly homologous across species, particularly in their catalytic domains. Human bile
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1784 salt-activated lipase ~hBAL! contains 722 amino acid residues ~Hui & Kissel, 1990; Baba et al., 1991!. The N-terminal 538 or so residues comprise the catalytic domain, which belongs to the a0b hydrolase fold superfamily ~Ollis et al., 1992!, followed by a mucinlike flexible C-terminal tail ~Downs et al., 1994!. In hBAL, this tail consists of 16 highly analogous proline-rich motifs. The tail varies in its repetitive numbers of the motifs across species and is often highly glycosylated through Ser0Thr residues. Although it is not required for the enzymatic activity the C-tail has multiple functions, including: to protect the enzyme from proteolysis ~e.g., by pancreatic proteases! during transportation ~Loomes, 1995!, to interact with soluble heparin ~Spilburg et al., 1995!, to mediate substrate delivery ~DiPersio et al., 1994!, and to prevent BAL aggregation ~Loomes & Senior, 1997a!. The C-terminal nineresidue segment of BAL is conserved across species, which is proposed to function in an inhibitory mechanism by binding and distorting the active site of BAL ~Chen et al., 1998!. Within the catalytic domain, BAL contains a conserved N-glycosylation site at Asn187. It has been shown that, however, attachment of an oligosaccharide to this residue is not required for the enzymatic activity ~Downs et al., 1994; Chen et al., 1998!. To gain insight into the distinct bile-salt activation mechanism of BAL and its broad substrate specificity, we pursued structural studies on BALs from different species. Previously, we reported 2.8 Å resolution crystal structures of bovine BAL ~bBAL! in both the presence and absence of bile salts ~Wang et al., 1997!, in which the C-terminal tail was disordered. In spite of available information from these structures and others ~Chen et al., 1998!, controversy exists over some detailed mechanisms such as the roles of a BAL specific insertion loop near the active site and dimerization. We present here the crystal structure of the apo-protein of human BAL. This 2.3 Å resolution structure represents the catalytic domain of a recombinant hBAL. The structure indicates that in the absence of bile salts, the BAL-specific insertion loop does not possess a predominant close conformation, and that dimerization is not a general property of BAL. Furthermore, structural consistency between hBAL and bBAL and difference with other lipases suggest functional significance of structural features of BAL. Results Overall structure A crystal structure of the catalytic domain ~residues 1–538! of hBAL is solved at 2.3 Å resolution with the molecular replacement method based on the structure of bBAL. The crystal form of hBAL belongs to the space group P212121 and is different from all previously reported bBAL crystal forms ~Wang et al., 1997; Chen et al., 1998!. One crystallographic asymmetric unit contains one hBAL molecule with a 50% solvent content ~Matthews, 1968!. A model structure of hBAL catalytic domain has been refined to a crystallographic R factor of 21.1% ~Rfree 5 26.6%! using 2.3 Å resolution data. A summary of the data collection and refinement statistics is shown in Table 1. The N-terminal Met residue adapted from the expression vector and the C-terminal residues beyond position 533 were disordered in the electron density map and were not included in the refined model. Figure 1 shows the overall folding of hBAL catalytic domain. Secondary structural elements are named following our previously reported bBAL crystal structures ~also see Fig. 2!. The peptide folding belongs to the a0b hydrolase fold family that is one of the
S. Terzyan et al. Table 1. Data collection and refinement statistics A. Data statistics Space group Unit cell ~a, b, and c in Å! Resolution ~Å! Rmerge ~%! No. of unique reflections Completeness ~%! Mean redundancy ^I0s~I !&
P212121 64.7, 89.0, 104.3 50.0–2.3 5.0 ~44.3! 25,287 91.2 ~87.8! 3.5 19.3 ~2.5!
B. Refinement statistics Rworking ~%! Rfree ~%! No. of nonhydrogen atoms Protein Solvent RMSD from ideal values Bond length ~Å! Bond angle ~deg! Average B-factor ~Å2 ! Protein Solvent
21.1 for 22,303 reflections 26.6 for 1,171 reflections 4,149 167 0.011 1.60 57.4 49.9
a Rmerge 5 (hkl (i |Ihkl, i 2 ^Ihkl &60 (hkl ^Ihkl &, where Ihkl,i is the intensity of the i th measurement and ^Ihkl & is the weighted mean of all measurements of Ihkl . Rworking ( free) 5 ( 66Fo | 2 |Fc |60( 6Fo |. Numbers in parentheses are the corresponding numbers for the highest resolution shell ~2.38–2.30 Å!.
largest known families of related enzymes and includes various lipases ~Ollis et al., 1992!. A major domain consists of a 11-strand major b-sheet ~ b2, 3, 5, 7, 6, & 8–13 ! gradually twisted and flanked by helices, loops, and short b-strands from both sides. The core of this major a0b domain has relatively low B-factors and provide the scaffold for the active site apparatus. A helix bundle formed by the segment of residues 330–396 ~aI , aJ , and aK ! and the last helix within the catalytic domain ~aN ! is loosely packed against the major a0b domain. This helix bundle domain can be considered as an insertion between strands b10 and b11 relative to the canonical structure of the a0b hydrolase fold ~Ollis et al., 1992!. The core of this helix bundle contains seven aromatic residues, thus stabilizing it by hydrophobic interactions. The entire domain, however, has higher than average B-factors. A 29-residue a-helix, aK , kinks in the middle at Pro396, leaving the N-terminal half in the helix bundle and the C-terminal part in the major a0b domain. Sequence analysis shows that the amino acid residue identity between the catalytic domains of hBAL and bBAL is 80%, with no insertion ~or deletion! in this region ~see Fig. 2!. Consistent with this sequence homology, the overall three-dimensional ~3D! structure of the catalytic domain of hBAL closely resembles that of previously reported bBAL. The RMSD between hBAL and apo-bBAL ~Protein Data Bank ~PDB! # 1akn! ~Wang et al., 1997! is 0.84 Å for 503 Ca atoms if a 3.0 Å distance cutoff is used in the structural superposition. For hBAL and the two bBAL molecules cocrystallized with the bile salt taurocholate ~TC! ~PDB # 1awp! ~Wang et al., 1997!, the RMSD is 0.79 and 0.82 Å, respectively, for 510 Ca atoms. The RMSD is 1.11 Å for 492 Ca atoms between hBAL and a bBAL structure that has a disrupted active site ~PDB # 2bce! ~Chen et al., 1998!.
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Structure of human bile salt activated lipase
Fig. 1. Stereoview of a ribbon representation of the crystal structure of the catalytic domain of hBAL. The hBAL molecule is viewed toward the active site. b-Strands are shown as curved arrows in blue, and a-helices are shown as magenta ribbons. Ca atom positions of the catalytic triad residues are marked with spheres. Around the active site, four loops, Lp70 ~i.e., 70-loop!, Lp120, Lp270, and Lp420, are colored in brown. This figure was produced with the program MolMol ~Koradi et al., 1996!.
Active site apparatus
Molecular recognition
The active site of hBAL, including both the catalytic triad and oxyanion hole, is essentially identical to that of our previously reported bBAL structure and appears to have a functional conformation, thus different from the disrupted active site of the structure reported by Chen et al. ~1998!. The catalytic triad, Ser194–His435– Asp320, resides at the bottom of a deep surface depression. The nucleophile Ser194 is in the tip of a g-shaped turn ~nucleophilic elbow! between the strand b8 and helix aD . It is the only residue in the entire structure located in an energetically unfavorable region in the Ramachandran plot and is consistent with the equivalent residue observed in the structures of other a0b hydrolases ~Ollis et al., 1992!. His435 is located in the so-called “His-loop” at the N-terminus of the helix aL , and Asp320 is located in the “acidturn” at the N-terminus of the helix aH ; thus, the charge-relay function of both residues are reinforced by helix-dipoles to some extent. While Ser194 and His435 are partially exposed to the solvent in the absence of a substrate, Asp320 is completely shielded from solvent by the peptide backbone of the His-loop. The sidechain carboxyl group of Asp320 form hydrogen bonds with backbone amide group of Asn317 and side-chain hydroxyl group of Ser220, the later of which further hydrogen bonds with the buried Glu193 and partially buried Asp438 through two buried water molecules. In addition, two surface acid residues, Asp434 and Glu437, are located near the active site. Such a concentrated acidic residue distribution renders the active site region the most negatively charged in the whole catalytic domain of BAL. The oxyanion hole in hBAL is formed by backbone amide groups of conserved residues Gly107, Ala108, and Ala195. In the hBAL crystal structure, the oxyanion hole is occupied by a well-ordered solvent molecule that forms a hydrogen bond network with the side-chain hydroxyl group of the nucleophile Ser194 and the amide groups of Gly107 and Ala108. In an ester hydrolysis reaction, this water molecule is likely to be replaced by the oxyanion of the tetrahedral intermediate ~Grochulski et al., 1994a!.
In the N-terminal region of hBAL, a group of positively charged residues, including Lys31, Lys56, Lys58, Lys61, Lys62, and Arg63, composes a tentative heparin binding site about 30 Å from the active site. This is the only conserved positively charged surface patch within the N-terminal heparin-binding fragment of BAL ~Baba et al., 1991!. The bile salt TC is present in the crystallization solution at a concentration of about 1.0 mM, which is higher than the 400 mM concentration required for half-maximal activation in the case of water soluble ester hydrolysis, but significantly lower than the 5.0 mM concentration required for half-maximal activation in the case of long fatty acid chain substrate hydrolysis ~Chen et al., 1998!. No ordered TC molecule is identified in the electron density map of hBAL, particularly at the corresponding positions where TC molecules were observed in the previously reported bBAL crystal form ~Wang et al., 1997!. In the bBAL-TC complex structure, one of the TC binding sites was identified in a cleft between the major a0b domain and helix bundle domain and distal from the active site. In the hBAL crystal form, this cleft is compressed and contains only one ordered water molecule. Surface loops Around its active site, hBAL possesses several surface loops. Most of these loops have higher than average B-factors and represent the largest differences between the crystal structures of hBAL and bBAL. In Figure 1, to the north of the catalytic triad, the 420-loop ~residues 423– 433! moves away from the active site in hBAL relatively to bBAL, with a Ca RMSD of 5.3 Å after an overall superposition. Since this loop is involved in a crystal contact in the hBAL crystal form, the structural difference may reflect a crystal environment difference. To the east, the 270-loop ~residues 270–285!, including residues Val272, Leu274, Leu277, Met281, and Val285 together with Ile323, Phe324, and Ile327 from the helix aH , forms a very hydropho-
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Fig. 2. Three-dimensional structure based sequence alignment of the catalytic domain of human BAL against homologous proteins. Included are bovine BAL ~bBAL, PDB # 1akn!, C. rugosa lipase ~CRL, PDB # 1lps!, and T. californica acetylcholine esterase ~T. ACE, PDB # 2ace!. The secondary structural elements observed in hBAL are named following those described in bBAL crystal structures ~Wang et al., 1997! and are shown on the top. Helices ~as cylinders! are named alphabetically; b-strands ~as arrows! are named numerically. Amino acid sequences in a-helices and b-strands, according to the crystal structures cited, are also highlighted with dark and light shading, respectively. The catalytic triad residues are marked with open triangles. Residue numbers obtained from the crystal structures are shown at the beginning of each line and at the C-termini in addition to the hBAL residue numbers above the sequences. This figure was produced with the program ALSCRIPT ~Barton, 1993!.
bic pocket. The B-factors of this loop are rather high, suggesting adaptability of this loop upon interacting with molecules approaching from the solvent. These properties make the 270-loop fit nicely to the criteria for a surface region capable of binding
to a wide range of substrates ~DeLano et al., 2000!. In fact, the corresponding region was found to interact with substrate analogous in the crystal structures of several homologous lipases ~Grochulski et al., 1994a; Ghosh et al., 1995!. To the south of the
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Structure of human bile salt activated lipase hBAL active site, the 70-loop ~residues 65–75! is truncated as compared to other triacylglycerol lipases ~Grochulski et al., 1994b!. Such a truncation renders BAL lacking in ability to change the conformation of a surface amphipathic lid over the active site in the so-called water-oil interfacial activation mechanism ~Grochulski et al., 1994b!. This truncated loop, together with its anchoring disulfide bond ~Cys64–Cys80!, is conserved in BAL across species. To the southwest of the active site, the 120-loop ~residues 115–125! corresponds to a conserved six-residue insertion in the BAL family relative to other a0b hydrolases ~e.g., Torpedo californica acetylcholine esterase ~T. ACE!!. In our previous apo-bBAL crystal structure, this loop was observed in a closed conformation which might hinder substrate binding. It was in an open conformation in the bBAL-TC complex crystal structure, stabilized by an active site proximal TC molecule ~Wang et al., 1997!. In the hBAL crystal form the electron density of this region is not well defined, and the loop was modeled in an open conformation with high B-factors. Solvent molecules and crystal packing About 170 solvent molecules were included in the refined model. All were modeled as water molecules. About half of them ~58! are buried inside the protein molecule and completely inaccessible to bulk solvent in the static model. About 35 solvent molecules are superimposable ~within 1.0 Å! with solvent molecules from the 1.6 Å resolution bBAL crystal structure ~PDB # 2bce! reported by Chen et al. ~1998! following superposition of the corresponding protein molecules. Some of these “conserved” solvent molecules are likely to play important structural or functional roles. For example, the above-mentioned two solvent molecules, which bridge an extensive hydrogen bond network near the catalytic triad, are consistently observed underneath the backbone of His-loop in the crystal structures of hBAL, bBAL, and T. ACE ~PDB # 2ace!. Restricted by the single copy of hBAL molecule per asymmetric unit and the crystal symmetry, there is no twofold symmetry related hBAL dimer in this crystal form. The hydrophobic surface immediately surrounding the active site is not involved in crystal contacts. This is in contrast to our previous bBAL crystal forms ~Wang et al., 1997! but consistent with the contention that hBAL functions as a monomer ~Loomes & Senior, 1997b!. The mutation site at the position 186 is located at the N-terminus of the strand b8 and on the surface opposite from the active site. The other mutation site at position 298 is in a surface loop and also away from the active site. None of these two sites are directly involved in an intermolecular salt bridge in the crystal lattice. However, Asp186 participates in a positively charged patch formed by Asp97, Asp184, and Asp186, which appears to interact with Lys334 from a neighboring protein molecule. Next to this point mutation is the potential glycosylation site, Asn187 ~Baba et al., 1991!, on the protein surface. Since the recombinant hBAL protein was purified from Escherichia coli, Asn187 was not glycosylated. An oligosaccharide attached to this site would be unlikely, however, to interfere with substrate binding because they occur on opposite sides of the protein molecule. Discussion Crystallization The catalytic domain of wild-type hBAL ~residues 1–538! was cloned, purified ~Downs et al., 1994!, and crystallized ~X. Zhang,
unpubl. result!. However, so far, the available crystals were not suitable for crystallographic studies because of poor diffraction. Two surface mutations were introduced in attempting to facilitate a better crystallization. In our previously reported apo-bBAL crystal structure, Asp186 forms a salt bridge with Arg239 from a neighboring protein molecule. Similarly, Asp298 contacts with Arg458 in the crystal lattice. By mutating both Asn186 and Ala298 into Asp in hBAL, we hoped similar favorable intermolecular interactions could promote crystallization of the latter. These surface mutations appear helpful in crystallization of hBAL to some extent. During screening the crystallization conditions, we found that one of the proposed salt bridges occurred in a P1 crystal form, which contained four hBAL molecules per asymmetric unit and diffracted to 2.5 Å resolution ~X. Zhang, unpubl. results!. Meanwhile, we obtained a P212121 crystal form that has one hBAL molecule per asymmetric unit and diffracts to 2.3 Å resolution. Although the side chains of the mutated residues failed to form direct crystal contacts in this orthorhombic crystal form, one of the mutations was indeed involved in electrostatic interactions between symmetry related protein molecules. Furthermore, crystallization the wild-type recombinant hBAL ~residues 1–538! under similar conditions gave negative results, which may suggest some subtle structural difference between the wild-type and mutant. Although protein engineering has been widely used to improve crystallization ~Price & Nagai, 1995!, unexpected results can arise because of versatility of protein–protein and protein–solvent interactions. The crystal structure reported here is the P212121 crystal form because of its favorable resolution and number of crystallographically independent protein molecules. Substrate specificity BAL has a broad substrate specificity. In general, a substrate of BAL comprises two parts: the alcoholic part and the fatty acid part, linked by an ester bond. Both parts can be very hydrophobic, thus requiring hydrophobic binding sites on BAL. Because of the similarity of their overall peptide folding and identical active site apparatuses, the hydrolysis reaction catalyzed by BAL is very likely to be similar to that catalyzed by other a0b hydrolases ~Ollis et al., 1992!. The reaction consists of two steps of nucleophilic attack to the ester bond of the substrate. The first attack is by the catalytic residues Ser194, and the second one is by a water molecule. Chemically, the first reaction step releases the alcoholic product, and the second step releases the fatty acid. Although currently no BALsubstrate analog complex crystal structure is available, significant insights can be obtained by comparing BAL crystal structures with homologous lipase structures complexed with substrate analogs ~see Fig. 3!. For example, the crystal structures of Candida rugosa lipase ~CRL, PDB # 1lps, and 1cle! ~Grochulski et al., 1994a; Ghosh et al., 1995! illustrate that the alcoholic part of the substrate binds to a surface depression right above the catalytic serine residue, and that the fatty acid chain of the substrate inserts into a tunnel running between the helix bundle domain and the major a0b domain. Correspondingly, in BAL a binding pocket for the alcoholic part is formed when the 120-loop assumes an open conformation ~Wang et al., 1997!, and the hydrophobic pocket formed by the 270-loop and helix aH may serve as the acyl chain binding site at least for short fatty acid chain substrates. What is the structural role of bile salts in BAL activation? Bile salts are required for the hydrolysis of substrates of long acyl chains or bulky alcoholic motifs ~such as cholesterol! ~Wang et al., 1988; Wang & Hartsuck, 1993!. Bile salts bind to BAL through
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Fig. 3. Structural comparison between hBAL and CRL around the substrate binding site. CRL coordinates ~PDB # 1lps! are superimposed to that of hBAL and only the substrate binding site region is shown. The Ca trace of hBAL is colored in white, and CRL is in blue. The catalytic triad residues are shown in red stick models. Selected loops and helices of hBAL are labeled. It is shown that the cover lid responsible for the interfacial activation in CRL is truncated to a short loop, Lp70 ~i.e., 70-loop!. The acyl chain binding tunnel in CRL underneath the helix aH and Lp270 equivalents is blocked by Trp227 in hBAL.
specific interactions, presumably reshaping the substrate binding site~s!. They also accelerate the process of releasing product through nonspecific interactions between BAL and micellar bile salts; a tightly bound fatty acid product may otherwise inhibit the continuation of the reaction. For the alcoholic binding site, the flexibility of the 120-loop shown in the hBAL crystal structure suggests that this loop is in a dynamic equilibration between the closed and the opened forms. The binding of a bile salt molecule to the active site proximal site may stabilize the loop in the opened conformation, as indicated in our previous bBAL-TC complex crystal structure. Structural comparison between BALs and CRL-linoleate complex and molecular modeling suggests that the open conformation of the 120-loop is necessary for a cholesteryl ester substrate to bind to BAL. For the acyl chain binding site, the surface hydrophobic pocket formed by the 270-loop and a-helix aH can easily accommodate a fatty acid chain of up to C6 in length. Kinetic studies have shown that BAL has basal activities towards substrates that are not longer than C8 in length in the fatty acid chain ~Wang et al., 1988!. For substrates of fatty acid chains longer than C8 , the tail of the fatty acid chain may have to either insert into a hydrophobic tunnel like that in CRL or protrude into the solvent. In hBAL ~and bBAL!, the acyl chain binding tunnel is not preformed in the absence of the substrate. A buried loop, 220-loop ~i.e., residues 222–229!, which forms a part of the tunnel wall in CRL, assumes a less extended conformation and blocks the tunnel path with the side chain of Trp227 in hBAL. To form an acyl chain binding tunnel in BAL similar to that of CRL, the surface 270-loop may have to assume a different conformation. The conformational flexibility of the 270-loop is likely to play a key role in accommodating different substrates. Structural breath between the major a0b
domain and the helix bundle domain may facilitate the conformational change of the 270-loop and the formation of the binding tunnel. It is possible that a specific bile salt binding may be involved in reshaping this acyl chain binding site. The TC binding site distal to the active site observed in our bBAL crystal is close to the 220-loop and between the helix bundle domain and the a0b domain ~Wang et al., 1997!. Its location argues that this bile salt binding site may facilitate the formation of or stabilize the acyl chain binding tunnel. Such a tunnel may not be necessary for soluble substrates or substrates of a short fatty acid chain ~e.g., shorter than C8 !. This would explain the basal activity of BAL towards such substrates in the absence of bile salts ~Wang & Lee, 1985!. Releasing the reaction product is also crucial for the continuation of the reaction. For hBAL, it has been shown that micellar bile salts serve as a fatty acid acceptor in releasing the fatty acid product. The exact exit for the released product is not clear at this time. However, it does not have to be the entrance of the acyl chain binding tunnel, which is close to the binding site for the alcoholic part if similar binding modes are assumed between BAL and CRL. Therefore, the physical order of product releasing may not follow the chemical order of the hydrolysis reaction ~Wang & Hartsuck, 1993! as long as remaining of the alcoholic product near the active site does not inhibit the second nucleophilic attack. In addition, two Asp residues are buried in this hypothetical binding tunnel for long acyl chains in hBAL. Asp328 is conserved in all BALs across species, and Glu388 appears in four out of seven BAL species known to date ~Sbarra et al., 1998!. Such an energetically unfavorable setting may implicate some functional roles of these residues; for example, in releasing the product from the mostly hydrophobic tunnel. Thus, the hBAL crystal structure, to-
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Structure of human bile salt activated lipase gether with bBAL structures, are providing insight into the substrate specificity of BAL and the requirement of bile salts for its hydrolase activity.
Materials and methods Cloning, mutagenesis, and protein purification Human BAL was cloned and truncated at position 538 as previously described ~Downs et al., 1994!. In an attempt to obtain X-ray quality hBAL crystals by adopting intermolecular salt bridges from an apo-bBAL crystal form ~Wang et al., 1997!, we introduced two point mutations on the surface of hBAL at the positions of 186 ~Asn r Asp! and 298 ~Ala r Asp!. The gene of the double substitution mutant was constructed using an overlap extension polymerase chain reaction technique ~Ge & Rudolph, 1997!. The recombinant human BAL, containing residues 1–538 of the mature protein and an extra N-terminal Met residue adopted from the cloning vector pET11a ~Novagen, Madison, Wisconsin!, was expressed as inclusion bodies in an E. coli BL21 strain. The cell culture ~4 L! was grown at 37 8C and induced at ;0.6 OD600 nm with 1.0 mg isopropyl-b-d-1-thiogalactopyranoside per liter of cell culture. After induction the cells were given 3 h of aerobic growth and then harvested, suspended in 200 mL TN buffer ~i.e., 50 mM Tris-HCl, 100 mM NaCl, pH 8.0!, lysed with lysozyme and frozen at 220 8C overnight. The suspension was thawed and treated with deoxyribonuclease A for 1 h, then centrifuged. After centrifugation, the inclusion bodies were washed by adding in 1 L of TN buffer with 1% ~v0v! Triton X-100, and stirred at 4 8C until resuspended. The washing procedure was repeated several times with 200 mL volume of TN buffer with Triton X-100 until the supernatant became clear, the last wash containing no Triton X-100. The washed inclusion bodies were solubilized in 50 mL 8.0 M urea, 100 mM Tris-HCl, 1.0 mM EDTA, 5% ~v0v! glycerol, 10 mM 2-mercaptoethanol, pH 12.5, and stirred at 4 8C for a few hours. Solubilized inclusion bodies were centrifuged at 27,000 3 g ~1 h! and refolded by dialyzing against 3 L of 1.0 M urea, 10 mM Tris-HCl, 5% ~v0v! glycerol, 1.0 mM sodium-taurocholate ~TC!, 0.1 mM 2-mercaptoethanol, pH 8.0, overnight. After refolding, an equal volume ~50 mL! of saturated ammonium sulfate was added to the protein solution, stirred at room temperature for 5 min, and centrifuged at 27,000 3 g ~1 h!. The precipitated protein was resuspended in 15 mL of 50 mM Tris-HCl, 1.0 mM TC, pH 8.0, and re-centrifuged. It was further purified at room temperature with a heparin-sepharose column ~Pharmacia Biotech, Uppsala, Sweden! using buffers containing 50 mM Tris-HCl, pH 8.0, 1.0 mM TC, and a NaCl gradient from 0.0 to 1.0 M. Fractions with enzymatic activity were collected, dialyzed against TN buffer containing 1.0 mM TC, and concentrated to ;20 mg0mL. The yield of this protein purification procedure is ;5 mg0L cell culture. The enzymatic activity of the double substitution mutant of the catalytic domain of hBAL was similar to that of the wild-type hBAL ~data not shown! using a p-nitrophenyl acetate assay ~Wang & Hartsuck, 1993!.
Crystallization and data collection The initial crystallization condition was obtained using the crystal screen kits from Hampton Research ~Laguna Niguel, California!. Crystals were grown in vapor diffusion plates at 20 8C from sitting
drops of hBAL solution mixed 1:1 in volume with the reservoir solution of 12% ~w0v! polyethylene glycol of 6,000 Da molecular weight ~PEG 6K!, 100 mM NaCl, 100 mM N-~2-acetamido! iminodiacetic acid ~ADA!, pH 6.5. The crystal used for data collection was ;0.5 3 0.2 3 0.2 mm 3 in size. It was equilibrated with a cryo-protection solution of 36% ~w0v! PEG 6K and 100 mM ADA, pH 6.5, and flash-cooled in a stream of nitrogen gas to a temperature of approximately 2180 8C for data collection. X-ray diffraction data were collected with a 0.978 Å wavelength synchrotron source and a CCD area detector at the X12B beam line of the National Synchrotron Light Source at Brookhaven National Laboratory ~Long Island, New York!. A data set of 2.3 Å resolution was collected from the crystal with a 91% completeness. Raw data were processed using the program suite HKL ~Otwinowski & Minor, 1997!. Statistics of the data are summarized in Table 1. Molecular replacement solution and structural refinement Initial phases of the hBAL crystal form were solved with molecular replacement methods using the program AMoRe ~Navaza, 1994! and a bBAL structure ~PDB # 1akn! ~Wang et al., 1997! as the search model. The initial crystallographic R-factor from the molecular replacement solution was 41.9% ~0.57 correlation, 15.0– 3.5 Å!. The refinement was carried out using the program CNS ~Brünger et al., 1998!. A 4% set of reflections was randomly selected prior to refinement for monitoring Rfree ~Brünger, 1992!. The graphic program FRODO ~Roussel & Cambillau, 1989! was used for interactive modeling. Simulated annealing followed by cycles of conjugate gradient minimization dropped both Rworking and Rfree below 30%. About 170 solvent molecules were then gradually included in the refined model. A bulk solvent correction and anisotropic overall B-factors were applied at later stages of the refinement. In the final refined model, 98% of the residues are within either the most favored or additional allowed regions of the Ramachandran plot, as defined by the program PROCHECK ~Laskowski et al., 1993!. The programs EDPDB ~Zhang & Matthews, 1995! and O were used in structural comparison. The atomic coordinates ~ID code 1f6w! have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University ~New Brunswick, New Jersey!. Acknowledgments We thank the X12B beam line staff at Brookhaven National Laboratory for assisting in data collection. This study was supported by American Heart Association Grant 9605507S.
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