Crystal Structure of Lactoperoxidase at 2.4 Å Resolution

doi:10.1016/j.jmb.2007.12.012

J. Mol. Biol. (2008) 376, 1060–1075

Available online at www.sciencedirect.com

Crystal Structure of Lactoperoxidase at 2.4 Å Resolution Amit Kumar Singh, Nagendra Singh, Sujata Sharma, S. Baskar Singh, Punit Kaur, A. Bhushan, A. Srinivasan and Tej P. Singh⁎ Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029, India Received 21 September 2007; received in revised form 5 December 2007; accepted 6 December 2007 Available online 14 December 2007

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to Rcryst and Rfree factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by antiphosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined α-helices of varying lengths including a helix, H2a, unique to LPO, and two short antiparallel β-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 Å from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 Å. Two histidine residues and six buried water molecules are connected through a hydrogenbonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry. © 2007 Elsevier Ltd. All rights reserved.

Edited by M. Guss

Keywords: antimicrobial activity; heme; iodide binding site; peroxidase; crystal structure

Introduction *Corresponding author. E-mail address: [email protected]. Abbreviations used: ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-sulfonic acid); EPO, eosinophil peroxidase; GLPO, caprine lactoperoxidase; HEPO, human eosinophil peroxidase; HMPO, human myeloperoxidase; HTPO, human thyroid peroxidase; LPO, lactoperoxidase; MAN, mannose; MPO, myeloperoxidase; NAG, N-acetyl glucosamine; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TPO, thyroid peroxidase.

Lactoperoxidase (LPO; EC 1.11.1.7) is a hemecontaining glycoprotein with a single chain that has a molecular mass of ≈ 68 kDa. It catalyzes the inactivation of a wide range of microorganisms.1–3 The other members of the mammalian peroxidase family include eosinophil peroxidase (EPO), thyroid peroxidase (TPO), and myeloperoxidase (MPO).4,5 LPO, EPO, and MPO contribute to the nonimmune host defense system by oxidizing halide and pseudohalide ions to produce potent antimicrobial

0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.

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Crystal Structure of Caprine Lactoperoxidase

agents. LPO carries out this function in exocrine secretions including milk, tears, and saliva,2 while EPO and MPO play similar roles in the phagosomes of eosinophils5 and neutrophils,6 respectively, during engulfment of microorganisms. The fourth member of the mammalian peroxidase family, TPO is an intracellular membrane-bound protein, which is involved in the catalysis of the iodination and coupling of thyroglobulin moieties in the biosynthesis of thyroid hormones thyroxine and triiodothyronine. The mammalian peroxidases also participate in the oxidative metabolism of xenobiotics responsible for hypersensitivity reactions and other toxic sequelae.6–8 As reported in the literature, LPO,9,10 EPO,11,12 and TPO13 are monomeric proteins while MPO14–17 is a covalently linked dimer of two identical halves each consisting of two polypeptide chains of 108 and 466 amino acid residues as a result of a posttranslational deletion of 6 amino acid residues. In contrast, LPO is a single chain of 595 amino acid residues with an extension of 12 and 2 residues with respect to MPO at the N- and C-termini, respectively. There are four potential glycosylation sites in LPO and EPO, while MPO and TPO have five glycosylation sites in each. There are 15 cysteine residues in LPO, 14 each in MPO and EPO, while TPO contains 17 cysteine residues. All the four members of mammalian peroxidases contain a covalently linked heme group, which is a derivative of protoporphyrin IX. In LPO, EPO, and TPO, the heme is involved in two ester linkages through Glu258 and Asp108 (the numbering scheme used is that of LPO),13,18–22 whereas MPO contains an additional linkage involving the sulfonium ion of Met243. The residues corresponding to Met243 of MPO are Gln, Thr, and Val in LPO, EPO, and TPO, respectively. The amino acid sequence identities of LPO with MPO, EPO, and TPO are in the range of 50–56%. So far, structural information is available only on MPO.15–17 Here, we present the first crystal structure of LPO at 2.4 Å resolution.

Results

tains 15 cysteine residues to form seven disulfide bridges between Cys6 and Cys167, between Cys15 and Cys28, between Cys129 and Cys139, between Cys133 and Cys157, between Cys237 and Cys248, between Cys456 and Cys573, and between Cys554 and Cys579. Cys441 is unpaired. Although the structure of canine MPO is also known,15 for comparison purposes, the 1.8-Å-resolution HMPO structure will be used. It is important to note that GLPO is a monomeric protein whereas HMPO is a covalently linked homodimer. It is also pertinent to note here that the sequence of matured HMPO (Fig. 1b) does not contain the first 12 residues of GLPO, which also include an important cysteine at position 6. Apparently, Cys6 is responsible for the monomeric form of GLPO because it forms an intramolecular disulfide bridge with Cys167. Since Cys6 is absent in HMPO, the residue corresponding to Cys167 (Cys153) in HMPO forms an intermolecular disulfide bridge with the same residue of the second molecule, resulting in the formation of a covalently linked homodimer. HEPO is reported to be a monomeric protein11,12 (Fig. 1b), although the absence of Cys6 from its N-terminal region seems to suggest that it could be a dimeric structure similar to that of HMPO.16,17 Unfortunately, the crystal structure of HEPO is not yet known. There is an excision of a hexapeptide in HMPO as the first chain terminates at residue number 106 and the second chain begins at 113. Thus, its dimeric half contains two polypeptide chains of 108 (after deletions) and 466 amino acid residues. All the four peroxidases contain one covalently linked heme group per monomer. However, the number of covalent linkages in HMPO is three, while in other peroxidases, there are only two covalent bonds. The additional covalent bond in HMPO is provided by Met243 through the interaction between the sulfonium ion and the terminal βcarbon of the vinyl group on pyrrole ring A. The residue corresponding to Met243 is not methionine in other peroxidases (Fig. 1b). There is yet another unexpected difference between the sequences of GLPO and HMPO where Ser198 is found to be phosphorylated in GLPO, while the corresponding residue in HMPO is proline and, hence, cannot be phosphorylated.

Sequence analysis Quality of the final model The complete amino acid sequence determination of caprine (goat) LPO (GLPO) (EF363153) shows that it is a single-chain polypeptide of 595 amino acid residues including 15 cysteines. The sequence of 20 amino acid residues from the N-terminus of GLPO determined using automated protein sequencer established the correct sequence of the starting N-terminal residue of the matured protein. The amino acid sequence of GLPO has been compared with sequences of LPOs from other species (Fig. 1a) and with those of human MPO (HMPO), human EPO (HEPO), and human TPO (HTPO) (Fig. 1b). The GLPO sequence has four potential glycosylation sites with Asn-X-Ser/Thr sequence motifs, which are at Asn95, Asn205, Asn241, and Asn332. It con-

The final model of GLPO consists of an entire polypeptide chain with residues from 1 to 595, one covalently linked heme group, a calcium ion, four glycan chains containing 8 N-acetyl glucosamine (NAG) and 3 mannose (MAN) residues, and 421 water oxygen atoms. The overall mean B-factor is 27.3 Å2. The entire structure is well defined. A Ramachandran plot of the main-chain torsion angles (ϕ,ψ)23 shows that 86.5% of the residues are in the core regions as defined in the program PROCHECK.24 The 11 sugar residues included in the model are remarkably well defined in the electron density map. Four hundred twenty-one water positions fulfill the criteria of good electron density in the 2Fo − Fc map at

1062 2.5σ cutoff and interactions with protein atoms or with other water molecules. Overall molecular structure The structural organization for the polypeptide chain of GLPO is shown as a ribbon diagram in Fig. 2. The monomeric structure is largely α-helical with only two small antiparallel β-strands (residues 357–359 and residues 373–375). The slightly elongated molecule of GLPO is packed with at least 20 α-helices of varying lengths, while the structure of HMPO in its dimeric half contains only 19 α-helices.16,17 An important α-helix, H2a (residues 124–133) is absent in HMPO, whereas it is one of the very important features of GLPO. Four out of six residues of the deleted hexapeptide in HMPO are part of helix H2a. The central core of the molecule consists of five long α-helices, H2, H5, H6, H8, and H12, with a covalently attached heme group. The N-terminus of GLPO does not form any repetitive secondary structure until residue 75 of helix H1. H1 is a short helix and is connected to H2 through a long chain. Helix H2 is a part of the core, and one of the residues of this helix, Asp108, is covalently linked to the heme group. Helix H2 is connected to H2a (124– 133) through an extended chain. In HMPO, the residues corresponding to 119 and 120 were not observed in the electron density map while the next six residues are absent from the sequence. As a result, helix H2a is absent in HMPO. Helix H2a is almost perpendicularly aligned to the plane of the heme moiety and is located in the proximity of the substrate channel. This helix is followed by two short helices, H3 and H4. Helices H5 and H6 are connected with each other by a V-shaped loop that is flanked by two extended chains. A core helix, H8, forms a triangle with helices H5 and H6. The heme group is sandwiched between helices H2 and H8. This is connected to the region consisting of helices H13, H14, H15, and H16 and parts of helices H17, H18, and H19. This region represents the crown of the back face of the core region. The three helices H2, H5, and H6 also form a triangle, below which lies the heme group. The other two core helices, H8 and H12, which run parallel with each other, form the lower wall on which the heme moiety rests. Role of disulfide bonds in the structure of GLPO The sequence of GLPO contains 15 cysteine residues that form seven intrachain disulfide bonds, Cys6–Cys167, Cys15–Cys28, Cys129–Cys139, Cys133–Cys157, Cys237–Cys248, Cys456–Cys513, and Cys554–Cys579. In the structure of HMPO, there are six intramolecular disulfide bonds and one intermolecular disulfide bond. It may be noted that the disulfide bond formed between Cys6 and Cys167 in GLPO tethers the N-terminal segment and a rigid loop, Phe165–Arg177. In HMPO, the N-terminal segment including Cys6 is absent and the corresponding loop Pro151–Arg161 containing Cys153 is shorter by two residues. As indicated by

Crystal Structure of Caprine Lactoperoxidase

B-factors, it is a relatively flexible loop also because it interacts poorly with other parts of the protein. Presumably, it helps it to protrude out of the main body of the molecule, facilitating the formation of an intermolecular Cys153–Cys153 disulfide bridge, resulting in the formation of a covalently linked homodimer. Heme environment The heme group is a derivative of protoporphyrin IX26 in which the methyl groups on pyrrole rings A and C are modified to allow formation of ester linkages with the carboxyl groups of Glu258 and Asp108, respectively (Fig. 3a). Unlike in HMPO where Met243 also forms a covalent bond giving rise to a sulfonium ion linkage with the β-carbon of the vinyl group on pyrrole ring A, GLPO lacks this interaction because the corresponding residue is glutamine (Fig. 3b). As seen from Fig. 3b, the environment in the proximity of Met243 in HMPO favors it to move closer to the heme group to form a covalent linkage as the adjacent residues Pro244 and Glu245 and the distant residue Met343 do not allow enough space for flexibility. On the other hand, in GLPO, Gln259 turns toward the distal heme cavity while the adjacent residues Ile260 and Leu261 move toward the hydrophobic pocket containing Val358 and other hydrophobic residues. The noncovalent interactions between the protein and the heme moiety that are similar in both GLPO and HMPO are given in Fig. 3c. There are also some noncovalent interactions between protein residues and the heme group in GLPO (Fig. 3d), which are not present in HMPO. This might compensate partly for the lack of sulfonium ion linkage of HMPO. Gln259 in GLPO is a part of the type III β-turn formed by the segment Gln259-Ile260-Leu261-Leu262. The observed β-turn conformation is stabilized by a hydrogen bond between Gln259 O and Leu262 NH. The corner residues Ile260 and Leu261 protrude into the adjacent hydrophobic pocket formed with residues Leu98, Val358, Leu376, Leu379, Phe384, Leu395, and Leu399. Apart from stabilizing the turn conformation, it also enriches the core structure of LPO with hydrophobic interactions. Gln259 is also involved in a hydrogen-bonded network with Gln102 and Gln105 in the distal heme region. Gln105 forms a hydrogen bond with the nitrogen atom of pyrrole ring B of the heme moiety. Such a hydrogen-bonded network and the interaction with the heme group are absent in HMPO. It may be noted that the sequence of the corresponding segment in HMPO is Met243-Pro244-Glu245-Leu246, which is remarkably different from that in GLPO. Although it also adopts a β-turn conformation, the side chains of corner residues Pro244 and Glu245 are in different orientations. Met243 is covalently linked to the heme group. Pro244 is not involved in any specific interaction, while Glu245 forms a hydrogen bond with its own NH group. The corresponding Leu262 in GLPO is a part of one of the sides of the distal heme cavity. The heme moiety is deeply buried

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Crystal Structure of Caprine Lactoperoxidase

inside the protein molecule, while the heme cavity is surrounded by a number of helices from three sides. It is open only from one side. The two strands, S1

and S2 (Fig. 2), are situated on the upper portion of the opening to the heme cavity. Apart from the two strands, H4 and H9 are the two short helices that are

Fig. 1 (legend on next page)

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Crystal Structure of Caprine Lactoperoxidase

Fig. 1. (a) Sequences of mammalian LPOs from goat (GLPO), cow (CLPO), buffalo (BLPO), sheep (SLPO), camel (ULPO), and human (HLPO). The residues have been numbered from 1 to 595. The residues in LPO from other sources have been aligned with the sequence of GLPO. The sequence identities vary from 84% to 95%. Cys residues are indicated in yellow. The covalently linked residues to the heme moiety are shown in blue. The residues involved in the hydrogenbonded interactions with the heme group are shown in red. Phosphorylated Ser was indicated in pink, and glycosylated Asn residues are shown in green. The differences in the sequences are shown in gray. (b) Sequences of four mammalian peroxidases (GLPO, HMPO, HEPO, and HTPO) are shown. The numbering schemes original to individual proteins have been indicated. The molecular mass of HTPO is of the order of 100 kDa, and its sequence is extended on both sides of Nand C-termini of the other three peroxidases. Cys residues are indicated in yellow. The covalently linked residues to the heme moiety are shown in cyan. The residues involved in the hydrogen-bonded interactions with the heme group are shown in red. Phosphorylated Ser is shown in pink, and glycosylated Asn residues are shown in green. The differences in sequences are highlighted in gray.

present at the upper position of the opening of the cavity. H11 is another short helix at the lower portion of the opening of the heme cavity. The heme protoporphyrin IX is slightly distorted from planarity. Pyrrole rings A, C, and D are essentially planar. Pyrrole ring B is considerably distorted from planarity. It forms a boat-shaped structure, with atoms N1, C4, and C2 on one side of the plane while C1 and C3 are on the other side. The corresponding ring in HMPO is planar. The iron position is shifted

only slightly (0.1 Å) toward the proximal site. It forms a coordinate covalent bond (2.14 Å) with Nå2 of His351 on the proximal site, whereas the nearest atom on the opposite side is a water oxygen atom at a distance of 2.65 Å. The β-carbon of the vinyl group on pyrrole ring A is located in the hydrophobic pocket formed by Val354, Leu376, Phe380, Leu417, and Leu433, while that of pyrrole ring B also makes several van der Waals contacts with Gly104, Tyr312, Phe347, and Phe349. Most of the residues that

Crystal Structure of Caprine Lactoperoxidase

1065

Fig. 2. Schematic diagram of the caprine LPO molecule. α-Helices are represented as cylinders, and β-sheets are indicated by arrows. The iron atom is shown as a brown-colored sphere, iodine ions are shown as purple-colored spheres, and the calcium ion is shown as a gray-colored sphere. The heme moiety is indicated in CPK representation (green), and the four carbohydrate chains attached to Asn95, Asn205, Asn241, and Asn332 are shown in ball-and-stick representation. The helices have been numbered. The figure was drawn using PyMOL.25

interact with the heme group belong to core helices H2, H5, H6, H8, and H12. The carboxyl group of the pyrrole ring D propionate interacts with the guanidinium groups of both Arg348 and Arg440 and forms a hydrogen bond with a water molecule, W355. In contrast, the pyrrole ring C propionate interacts with Asp112 Oδ2, Ala114 N, and W78. (Fig. 3c). Calcium coordination Calcium ion is coordinated to seven oxygen atoms, resulting in the formation of a slightly distorted

pentagonal bipyramidal coordination polyhedron. The coordination distances vary from 2.41 to 2.68 Å, which are slightly longer than the distances generally observed for calcium coordinations in protein structures.15,16 Ser190 Oγ and the peptide carbonyl oxygen atom of Phe186 provide the axial ligands, whereas the other five ligands, Asp110 carboxyl oxygen atom Oδ1 and carbonyl oxygen atom O, Thr184 Oγ and carbonyl oxygen atom O, and Asp188 Oδ1, are arranged in a planar environment. It is noteworthy that five of these ligands belong to one large loop (181–196) while the remaining two linkages are from Asp110, which is next to the distal

1066 His109 in the sequence, indicating the significance of calcium ion coordination in the structure and function of LPO. Proximal interacting residues The proximal side of the heme moiety shows that the amino acid side chains from two of the core helices, H8 and H12 (Fig. 2), are involved in interactions. His351 Nε2 of helix H8 is coordinated to the iron atom (2.14 Å), while Nδ1 forms a hydrogen bond with Asn437 Oδ1 (2.78 Å) from helix H12. Asn437 O forms a hydrogen bond with peptide NH of Cys441. The side chain of Cys441 is free in the structure of LPO and is buried in the hydrophobic pocket built by Phe345, Ala434, Leu438, and Trp493. In contrast, the corresponding site is significantly less hydrophobic in HMPO as it has not only Asn345 and Ser434 in place of Phe345 and Ala434 but also serine at position 441. This shows that Cys441 is very tightly packed in the hydrophobic pocket and is protected well from getting oxidized. However, the significance of free Cys441 is not yet clearly understood. The carboxylic group of the ring D propionate not only interacts with the guanidinium groups of both Arg348 and Arg440 but also forms a hydrogen bond with a water molecule. The ring C propionate interacts with Asp112 Oδ2, Ala114 N, and W38. Gln423 interacts with the carboxyl oxygen of the propionate through water molecule W355. This interaction is absent in HMPO as the corresponding residue is Glu423 and its side chain is oriented in the opposite direction. It is interesting to note that Cys441 with a free side chain is located in the region of proximal cavity. Distal heme cavity The distal heme cavity is located on the opposite side of the proximal cavity and is known to be the site of catalysis in heme-containing peroxidases. It extends from the heme pyrrole rings to the surface of the protein in a well-formed channel. Its sides are marked with the side chains of Phe113, Leu199, Asn230, Pro234, Pro236, Phe381, and Pro424. It is remarkable to observe that helix H2a (124–133) is involved in stabilizing the shape of the distal heme cavity through interactions with loops that are part of the cavity walls. Helix H2a is a very stable structure with two disulfide bridges through Cys129 and Cys133. The helix corresponding to H2a is not present in HMPO.16 Instead, it forms a loop with a very different orientation. There is another loop, 420–430, which is of considerable interest because it has a remarkably different conformation from that of the corresponding segment in HMPO. This loop supports a part of one of the sides of the distal cavity, resulting in the difference in the shape of distal cavities of GLPO and HMPO. The residue at 430 is Gly in both structures, which can impart flexibility to this loop. The residue next to it is His429 in GLPO, which forms a hydrogen bond

Crystal Structure of Caprine Lactoperoxidase

(His429Nε2⋯His377 O = 2.81 Å) with a well-ordered loop structure containing His377. The residue corresponding to His429 is absent in HMPO. Yet another important interaction is observed in GLPO between His426 and Glu130 Oε1 (His426 Nδ1⋯ Glu130 Oδ1 = 2.51 Å). Glu130 is part of a very stable helix, H2a. On the other hand, the residue corresponding to His426 is Met411 in HMPO. Loop 420– 430 is also characterized by a tight β-turn for the residues Gln423-Pro424-Thr425-His426. In this arrangement, Gln423 turns toward the heme group and interacts with the propionate carboxylate of pyrrole ring A through a water molecule. It also imparts structural uniqueness to the conformation of loop 420–430 as well as to the heme moiety. It is clearly shows that the interactions involving the heme group (Fig. 3c) and the residues that contribute to the size and shape of the distal cavity are not identical in GLPO and HMPO. The channel on the side of the distal heme cavity is filled with water molecules as well as with the side chains of Gln105, His109, and Arg255. In the present structure, the distal heme cavity contains one iodide ion I1, which is hydrogen bonded to W1, which, in turn, is hydrogen bonded to the heme iron atom and distal His109 Nε2. His109 Nδ1 forms a very strong hydrogen bond (2.45 Å) with W2. W2 forms two other hydrogen bonds, one with His266 Nε2 and the other with Asp253 Oδ2. His266 Nδ1 forms another hydrogen bond with W3, which is involved in two other hydrogen bonds with W4 and His266 O. W4 is hydrogen bonded to W5, which is further hydrogen bonded to W6 and Gln250 O. W6 forms two hydrogen bonds with surface residues Ala214 O and Leu203 O. The linking of Asn437–His351 (on the proximal site)–heme iron–W1–His109–W2–His266– W3–W4–W5–W6–Ala214/Leu203 (Fig. 4) is an important structural feature of peroxidase enzymes, which facilitates proton relay from distal histidine away to the surface of the protein. Characterization of halide binding The solution of native protein was incubated in the protein buffer solution containing 2 mM NH4I for 24 h. The iodide-saturated GLPO was crystallized. The structure revealed 10 positions of iodide ions (I1–I10) (Fig. 2). Out of 10 iodide ions, I1 is found in the distal heme cavity. It is liganded to His109 and three water molecules including W1. A similar halide position has also been observed in HMPO.16 A second iodide has been located in the proximal site, which is hydrogen bonded to the peptide NH groups of Trp46 and Val342. It may be noted that this site is located in the proximity of the amino-terminus of the core helix H8. The 3rd and 4th halide ions are found in shallow clefts on the surface of the protein but are liganded well to the protein NH/NH2 groups and water molecules. All these halide positions were also reported in the HMPO structure.16 Additional halide ions (I5–I10) have been observed in the LPO structure. I5 is observed in a cleft on the surface. It forms two hydrogen bonds

Crystal Structure of Caprine Lactoperoxidase

with Asn80 Nδ2 and W304. This position is close to the N-terminus of helix H1. This halide binding site is not feasible in the structure of HMPO as the cleft is not formed because the conformation of a large peptide loop (143–157) protrudes into the region and reduces the space for halide binding. In addition to it, the side chain of the Glu67 residue in HMPO is in the proximity of this position. The corresponding residue in GLPO is Lys81. I6 has been observed in the highly positive environment in the protein. It is buried in a hole-like structure and is surrounded by residues Asn95, Arg96, and Arg504 and two NAG residues of the glycan chain attached to Asn95. The corresponding site in HMPO is occupied by the side chain of Glu81, and the support from one of the sides is completely absent because glycosylation is

1067 not possible at this site in HMPO (Fig. 1b). Therefore, this halide binding site is unique for the LPO. The 7th iodide ion is observed on the surface and interacts with the Nε and NH2 atoms of Arg202. In HMPO, Arg202 is replaced by Asn186 and the halide ion is not observed. The 8th iodide ion is observed in a shallow pocket on the surface and is liganded to Arg310 N and Trp530 Nε. The corresponding site in HMPO is empty, although there is apparently no reason for not having a halide ion at this site. The 9th iodide ion is present on the surface and is linked to Lys462 NH and Thr463 Oγ. This site is not formed for halide binding in HMPO as Gln450 side chain fills the space occupied by I9. The residue corresponding to Gln450 is Gly466 in LPO. The 10th halide ion I10 is liganded to Phe567 NH, Ala566 NH,

Fig. 3. (a) Stereoview of the electron density (2Fo − Fc) for the heme moiety contoured at the 1.2σ level. The residues Asp108 and Glu258, covalently linked to the heme, are also shown. (b) The environment surrounding extra covalent linkage involving the Met243 residue in MPO (blue) and the corresponding residue (Gln259) in GLPO (yellow). (c) The commonly observed noncovalent interactions between the heme group and protein are indicated by broken lines. (d) The additional noncovalent interactions observed only in GLPO are indicated.

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Crystal Structure of Caprine Lactoperoxidase

Fig. 3 (legend on previous page)

W166, and W383. The iodide ion is surrounded by helices H6 and H19 and a large loop (loop 562–571). The corresponding site is poorly formed in HMPO as the conformation of loop 546–556 is different and is not favorable to halide binding. Furthermore, Asn550 occupies this halide binding site in HMPO. The large-scale binding of halide ions in GLPO indicates more favorable stereochemical and charge distribution properties of GLPO as compared to those found in HMPO. Phosphorylation of Ser198 The phosphorylation of Ser198 was first indicated by the structure determination of GLPO (Fig. 5). Then, it was confirmed by Western blotting using anti-phosphoserine antibodies.27 This is the first time that a mammalian peroxidase has been found phosphorylated. The residue corresponding to phosphorylated Ser198 is proline in HMPO, which is unsuitable for phosphorylation. It is a remarkable observation that the position of phosphorylated Ser198 in the structure is at the favorable site so as to allow a better adsorption of calcium ion. As shown in Fig. 6a, the calcium ion is attracted to the calcium binding site via phosphorylated Ser198. Therefore, the phosphorylation of Ser198 in LPO appears to be a desirable modification of the protein for an

efficient intake of calcium ion. It may be mentioned here that a calcium binding protein, Orchestin, is phosphorylated at the serine residue and that calcium binding occurs in this protein only via the phosphoserine residue.28 Similar observations were also made while evaluating calcium uptake in dihydropyridine-sensitive calcium channels from rabbit skeletal muscle.29 It is also important to note that the calcium binding site is in the proximity of the distal heme cavity. Asp110 is coordinated to the calcium ion, which is the only residue next to the distal heme cavity His109, which is involved in catalytic action in LPO. Furthermore, Gln105, which is a part of the calcium coordination loop, interacts with heme pyrrole ring A. Therefore, calcium ion coordination produces a direct effect in maintaining the stereochemical environment of the heme moiety with respect to the substrate binding site in the protein. In contrast, the corresponding site in HMPO is considerably wider and shallower (Fig. 6b) than the one observed in GLPO.

Discussion Mammalian peroxidases including MPO, LPO, EPO, and TPO have been classified into a separate group of the peroxidase superfamily. The three-

1069

Crystal Structure of Caprine Lactoperoxidase

Fig. 3 (legend on page 1067)

dimensional structure of MPO is already known, while the first structure of LPO is reported here. Structures of EPO and TPO are not yet known. The mode of heme binding in LPO involves two ester linkages through Glu258 and Asp108. These two residues are conserved in all the four members of the mammalian peroxidase family. In this regard, LPO, EPO, and TPO are identical while MPO is different as it has an additional covalent linkage through the sulfonium ion of Met243. The heme group is also involved in a number of noncovalent interactions with the protein. The particularly notable interactions unique to LPO involve Gln105 on the distal heme cavity and Gln423 on the proximal site. Gln105 forms a hydrogen bond with the nitrogen atom of pyrrole ring B of the heme moiety, while Gln423 interacts with the pyrrole ring A propionate through a solvent water molecule. As a consequence of these differences, the heme ring in MPO assumes a significantly different distortion from the planarity in the heme group as compared to that in LPO. Similarly, the out-of-plane location of iron in the heme group of MPO (0.2 Å) is more pronounced than that observed in GLPO (0.1 Å). It may also be noted that the plane of pyrrole ring B is considerably

distorted in GLPO. The corresponding ring in HMPO is essentially planar. All these differences should form the basis for the observed variations in the spectral characteristics between MPO and LPO.20 As reported in the literature,20,22 both EPO and TPO exhibit similar optical properties as recorded in LPO. There is also a report that suggests that the interactions between the heme and the protein and the associated heme conformation contribute to the observed spectral behavior of the two proteins.20,22 The substrate binds to the protein at the distal heme cavity. The first step in the catalytic process involves the formation of a hydrogen bond between H2O2 and the unprotonated Nε2 of the distal histidine His109 before proton transfer occurs. His109 Nδ1 forms a hydrogen bond with water molecule W2, which is further hydrogen bonded to Nε2 of His266. Nδ1 of His266 forms a hydrogen bond with W3, which is hydrogen bonded to the chain of hydrogen-bonded buried water molecules W4–W7. The water molecules W2, W3, W5, and W6 are hydrogen bonded to Asp253 O δ2, His266 O, Gln250 O, Ala214 O, and Leu203 O (Fig. 4). A similar hydrogen-bonded chain of five water molecules was reported in MPO.16

1070

Crystal Structure of Caprine Lactoperoxidase

Fig. 4. Hydrogen-bonded chain involving His109, His266, and six buried water molecules. His351 in the proximal site is also shown.

Fig. 5. Electron density for the phosphorylated Ser198. It was modeled in the omit map. The final (2Fo − Fc) electron density map is contoured at the 1.2σ level.

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Crystal Structure of Caprine Lactoperoxidase

Access to the distal cavity is through a narrow cylindrical channel with an average diameter of approximately 6.0 Å between the nearest Cα atoms (Table 1; Fig. 7a). The channel in MPO has a funnellike shape (Fig. 7b). The inner surface of the channel is made up of hydrophobic residues. The composition of residues indicates a higher degree of hydrophobicity in LPO as compared to that of MPO (Table 1). Due to the presence of an extra stretch of residues from 121 to 126 in LPO, the channel length appears to be longer than that of MPO. The opening of the tunnel in LPO is guided by the residue Lys427 (Cα, β, γ, δ, and ε carbons) on one side and by residues Pro234 and Phe239 on the other side. The corresponding residues in MPO are Gly412, Asp218, and Leu223, respectively. One of the cylinder walls is supported by a well-defined loop (loop 420–430). This loop is firmly held by an ionic interaction involving Glu130 (helix H2a) and His426 loop residue. Since helix H2a is absent in MPO, this important interaction is also absent. Therefore, the loop in HMPO is not only differently oriented but also poorly organized. Furthermore, this loop in MPO is also shorter by 1 residue due to a deletion (Fig. 1b). The N-terminal stretch of 12 residues, which is absent in MPO, also supports the channel formation from behind. As a result, the overall shapes of the channels connecting to the heme group are significantly different in the two

peroxidases. The preferences for different substrates may be determined by the overall shapes of the hydrophobic channel, the planarity characteristic of the heme group, displacement of iron atom from the plane of the heme, interactions between the protein and the heme moiety, and the water structure in the distal cavity. The observed differences pertaining to the above are significant between the structures of LPO and MPO so as to differentiate between the preferences for substrates. The catalytic mechanism involving peroxidases is complex, and the preference for the substrates is based on a number of properties.5,30,31 More crystallographic information for various peroxidase–substrate complexes will be helpful for further understanding of the intricate mechanism.

Materials and Methods Purification of the protein Fresh caprine mammary gland secretions were collected from the Indian Veterinary Research Institute, Izatnagar, India. The samples were skimmed and separated from fat. These were diluted twice with 50 mM Tris–HCl (pH 7.8). Cation exchanger CM–Sephadex C-50 (7 g l− 1) equilibrated in 50 mM Tris–HCl (pH 7.8) was added and stirred slowly for about 1 h with a mechanical stirrer. The gel was

Fig. 6. (a) The site of phosphorylation of Ser198 showing the entry of the calcium ion to the calcium binding site. (b) The corresponding region in HMPO has been drawn using coordinates from the Protein Data Bank (1CXP).

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Crystal Structure of Caprine Lactoperoxidase

Fig. 6 (legend on previous page)

allowed to settle, and the solution was decanted. The protein-bound gel was washed with an excess of 50 mM Tris–HCl (pH 8.0) in order to remove the unbound proteins. The washed gel was loaded on a CM–Sephadex C-50 (Pharmacia, Sweden) column (10 cm × 2.5 cm) and equilibrated with 50 mM Tris–HCl (pH 8.0). The elution of LPO was done with a linear gradient of 0.0–0.5 M NaCl using the same buffer. The protein fractions with an Rz value of 0.79 and above were pooled and concentrated using an Amicon ultrafiltration cell. The concentrated protein sample was passed through a Sephadex G-100 column (100 cm × 2 cm) using 50 mM Tris–HCl buffer (pH 8.0). The elution was done at a flow rate of 6.0 ml/h. The fractions with an Rz value of 0.9 and above were pooled and dialyzed against deionized water, lyophilized, and stored at 253 K.

Table 1. Cα distances between residues of the substrate channel in GLPO Residue 1 Ser356 (Pro341) His377 (Ser362) Phe380 (Phe365) Phe381 (Phe366) Pro236 (Pro220) Pro234 (Asp218) Phe239 (Leu223)

Residue 2

Distance (Å)

Gln416 (Gln401) Leu421 (Leu406) Phe422 (Phe407) Pro424 (Gln409) Pro424 (Gln409) Pro424 (Gln409) Lys427 (Arg412)

6.0 (5.4) 6.4 (5.5) 6.4 (5.4) 7.7 (8.8) 5.4 (8.7) 11.9 (14.4) 8.1 (11.9)

The corresponding values for HMPO are enclosed in parentheses.

The protein samples with a molecular mass of 68 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were blotted on a polyvinyl difluoride membrane (Sigma-Aldrich, USA) and were subjected to the N-terminal amino acid sequence determination using an automated protein sequencer PPSQ 21 (Shimadzu, Japan). The sequence of the first 20 N-terminal amino acid residues was found to be Ser-Trp-Glu-Val-Gly-Cys-GlyAla-Pro-Val-Pro-Leu-Val-Thr-Cys-Asp-Glu-Gln-Ser-Pro. It matched with the known sequence of LPO from other species.32 LPO activity measurements The assay was carried out following the procedure of Shindler and Bardsley33 with some modifications. We mixed 3.0 ml of 1 mM 2,2′-azino-bis(3-ethylbenzthiazoline-sulfonic acid) (ABTS) in phosphate buffer (0.1 M, pH 6.0) with 0.1 ml of the sample in phosphate buffer (0.1 M, pH 7.0) containing 0.1% gelatin to initialize the spectrophotometer (Perkin Elmer, USA). We mixed 3.0 ml of 1 mM ABTS solution with 0.1 ml of the sample and 0.1 ml of 3.2 mM hydrogen peroxide solution; the absorbance was measured at 412 nm as a function of time for 2–3 min. The rate of change of absorbance was constant for at least 2 min. One unit of activity is defined as that amount of enzyme catalyzing the oxidation of 1 μmol of ABTS min− 1 at 293 K (molar absorption coefficient, 32,400 M− 1 cm− 1). The peroxidase activity of goat milk was found to be 5.3 U ml− 1. The purity of LPO was determined by the absorbance ratio A412/A280 (Rz

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Crystal Structure of Caprine Lactoperoxidase

Fig. 7. The substrate channel and the hydrogen-bonded His109 and His266 and buried water molecules involved in proton relay. Loops 118–132, 230–240, 246–248, 375–383, and 420–423 in GLPO are indicated in red (a). The corresponding loops in HMPO (loops 101–118, 214–224, 230–232, 360–368, and 405–415) are shown in red (b). The N-terminal segment 1–12 in GLPO is indicated in purple. The dotted line in (b) indicates a break in the chain of HMPO. Helices H2a and H4 in GLPO and H4 in HMPO are also indicated.

value). The Rz value for the purified LPO was found to be 0.932. Complete amino acid sequence determination The mammary gland tissue of lactating goat was obtained from the Indian Veterinary Research Institute. The total RNA was extracted by the phenol/chloroform method.34 The reaction with Moloney murine leukemia virus–reverse transcriptase polymerase chain reaction was used for polymerase chain reaction amplification of the gene. The conserved nucleotide sequences from other proteins of the peroxidase family32 and the N-terminal sequence of GLPO as obtained using Edman degradation were used for the design of primers. The nucleotide sequence was carried out on the cloned double-stranded DNA (pGEM-T) using automatic sequencer model ABI377. The complete nucleotide and deduced amino acid sequences have been deposited in the gene bank with accession number EF363153. Phosphorylation detection Purified LPO was run on SDS-PAGE using 10% polyacrylamide gel as described by Laemmli. The protein was transferred from SDS-PAGE to a polyvinyl difluoride membrane (Sigma-Aldrich, USA) by Western blotting, and nonspecific binding sites were blocked for 2–4 h at room temperature using 5% dried milk, Tris-buffered saline (pH 7.4), and 0.1% Tween 20. The membrane was probed overnight at 4 °C with primary anti-phosphoserine antibody (1:5000) for the serine phosphorylation site in GLPO.

A secondary horseradish-peroxidase-labeled antibody was raised in rabbit (goat anti-rabbit immunoglobulin G; Jackson Immunochemicals) in combination with enhanced chemiluminescence detection system (SuperSignal West Pico Chemiluminescent Substrate) to visualize the primary antibodies. Crystallization The purified samples of protein were dissolved in 0.01 M phosphate buffer (pH 7.0) to a concentration of 25 mg/ml. A reservoir solution containing 0.2 M ammonium iodide and 20% (w/v) polyethylene glycol 3350 was prepared. Protein solution (5 μl) was mixed with 5 μl of reservoir solution to prepare 10 μl of drops for hanging drop vapor diffusion method. The rectangular, brownish crystals measuring up to 0.3 mm × 0.2 mm × 0.2 mm were obtained after 1 week. X-ray intensity data collection The X-ray intensity data were collected at 287 K using a 345-mm-diameter MAR Research dtb Imaging plate scanner mounted on a Rigaku RU-300 rotating anode X-ray generator operating at 50 kV and 100 mA. The Osmic Blue confocal optics was used for focusing CuKα radiation. The data were indexed and scaled using the programs DENZO and SCALEPACK.35 The crystals belong to monoclinic space group P21 with the following cell parameters: a = 54.2 Å, b = 80.8 Å, c = 77.0 Å, and β = 102.9°. The unit cell contains one molecule in the asymmetric unit. The final data show an overall complete-

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Crystal Structure of Caprine Lactoperoxidase

Table 2. Data collection and refinement statistics of GLPO Space group Unit cell dimensions a (Å) b (Å) c (Å) β (°) Number of molecules in the unit cell Resolution range (Å) Total number of measured reflections Number of unique reflections Overall completeness of data (%) Completeness in the highest-resolution shell (2.44–2.40 Å) (%) Overall Rsym (%) Rsym in the highest-resolution shell (%) Overall I/σ(I) I/σ(I) in the highest-resolution shell (%) Rcryst (%) Rfree (%) Protein atoms Heme group (1) atoms Iodide ions Calcium ion NAG residues (n = 8) (N-linked) MAN residues (n = 3) (N-linked) Water oxygen atoms r.m.s.d. in bond lengths (Å) r.m.s.d. in bond angles (°) r.m.s.d. in torsion angles (°) Mean B-factor for main-chain atoms Mean B-factor for side-chain atoms and waters Mean B-factor for all atoms Residues in the most allowed regions (%) Residues in the additionally allowed regions (%) Residues in the generously allowed regions (%)

P21 54.2 80.8 77.0 102.9 2 20.0–2.4 141,640 24,962 97.9 90.1 12.1 32.3 5.9 2.3 19.6 20.3 4757 43 10 1 112 33 421 0.01 1.8 24.1 25.2 28.3 26.9 89.4 9.0 1.6

ness of 98% with an Rsym of 12.1% for 2.4 Å resolution. Data collection summary is shown in Table 2. Structure determination and refinement The structure was determined with molecular replacement method based on maximum likelihood in PHASER36 using the coordinates of one molecule of HMPO16 as the search model. The rotation and translation search functions were calculated, with data between 12.0 and 4.0 Å yielding a clear solution with a distinct peak. The stacking arrangement of molecules in the unit cell for this solution yielded no unfavorable intermolecular contacts. The transformed coordinates using PHASER were subjected to 20 cycles of rigid-body refinement with REFMAC537 from the CCP4i v4.2 software suit.38 This reduced the Rcryst and Rfree factors to 33.4% and 42.6%, respectively (2% of the reflections were used for the calculation of Rfree and were not included in the refinement). The manual model building of the protein using |2Fo − Fc| Fourier and |Fo − Fc| difference Fourier maps was carried out with graphics program O39 and COOT40 on a Silicon Graphics O2 Workstation. Further refinement cycles reduced Rcryst and Rfree factors to 0.251 and 0.284, respectively. The difference electron density |Fo − Fc| map computed at this stage indicated the presence of four carbohydrate chains at Asn95 (2 NAG residues), Asn205 (2 NAG residues), Asn241 (2 NAG residues + 1 MAN residue), and Asn332 (2 NAG residues). Eleven strong peaks corresponding to 10 iodide ions and 1 calcium ion were also observed. They were included in the subsequent refinement cycles. Further, difference Fourier |Fo − Fc| maps indicated an

extra electron density for the side chain of Ser198 at 3σ cutoff into which a phosphorylated Ser side chain was fitted well and refined (Fig. 6). The subsequent Fourier (2Fo − Fc) and difference Fourier (Fo − Fc) maps revealed the positions of 421 water oxygen atoms. The refinement finally converged to Rcryst and Rfree factors with values of 0.196 and 0.203, respectively. The final data collection and refinement statistics are given in Table 2. Protein Data Bank accession number The refined atomic coordinates of GLPO have been deposited in the Protein Data Bank with accession code 2R5L.

Acknowledgements The authors acknowledge financial support from the Department of Science and Technology, New Delhi. T.P.S. thanks the Department of Biotechnology, New Delhi, for the Distinguished Biotechnologist award. A.K.S. thanks the Council of Scientific Industrial Research, New Delhi, for having been awarded a fellowship.

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