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The crystal structure of human MRP14 (S100A9), a Ca2+-dependent regulator protein in inflammatory process1
doi:10.1006/jmbi.2001.5340 available online at http://www.idealibrary.com on
J. Mol. Biol. (2002) 316, 265±276
The Crystal Structure of Human MRP14 (S100A9), a Ca2-dependent Regulator Protein in Inflammatory Process Hiroshi Itou1, Min Yao1,2, Ikuko Fujita1, Nobuhisa Watanabe1 Masaki Suzuki3, Jun Nishihira3 and Isao Tanaka1,2* 1
Division of Biological Sciences Graduate School of Science Hokkaido University, Sapporo 060-0810, Japan 2
Division of BioCrystallography Technology RIKEN Harima Institute/ SPring-8, Kouto, Mikazuki Sayo, Hyogo 679-5148, Japan 3
Central Research Institute School of Medicine, Hokkaido University, Sapporo 060-0810, Japan
Human MRP14 (hMRP14) is a Ca2-binding protein from the S100 family of proteins. This protein is co-expressed with human MRP8 (hMRP8), a homologue protein in myeloid cells, and plays an indispensable role in Ca2-dependent functions during in¯ammation. This role includes the activation of Mac-1, the b2 integrin which is involved in neutrophil adhesion to endothelial cells. The crystal structure of the holo form of Ê resolution. hMRP14 is distinguished from hMRP14 was analyzed at 2.1 A other S100 member proteins by its long C-terminal region, and its structure shows that the region is extensively ¯exible. In this crystal structure of hMRP14, Chaps molecules bind to the hinge region that connects two EF-hand motifs, which suggests that this region is a target-binding site of this protein. Based on a structural comparison of hMRP14 with hMRP8 and human S100A12 (hS100A12) that is another homologue protein, the character of MRP8/14 hetero-complex and the functional signi®cance of the ¯exibility of the C-terminal region of hMRP14 are discussed. # 2002 Elsevier Science Ltd.
*Corresponding author
Keywords: calcium-binding protein; crystal structure analysis; human MRP14; human S100A9; S100 family of proteins
Introduction Calcium has been implicated as a regulatory ion in a variety of cellular processes such as muscle contraction, secretion, adhesion, synaptic transmission, cell-cycle progression and cell differentiation. In such processes, the change of intracellular Ca2 concentration acts as a signal mediator, and the signal is transduced as activation or inactivation of the Ca2-binding protein. There is a multigenic family of Ca2-binding proteins that are involved in such signal mediation called the S100 family of proteins.1 The S100 family proteins were found to be expressed in a cell and tissue-speci®c manner, and were associated with different human pathological states such as cancer, neurodegeneration and cardiomyopathies.2 The members of the S100 family of proteins are small Abbreviations used: hMRP14, human MRP14; NIF, neutophil immobilizing factor; NCS, non-crystallographic symmetry; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate. E-mail address of the corresponding author: [email protected] 0022-2836/02/020265±12 $35.00/0
(their molecular weights are in the range of 10-13 kDa), acidic, and have a strong tendency to dimerize. They have two EF-hand motifs as Ca2-binding sites, and are implicated in Ca2-dependent regulation of a variety of intracellular activities such as protein phosphorylation, cell proliferation and differentiation, the dynamics of cytoskeleton constituents, and intracellular Ca2 homeostasis.2 The in®ltrating macrophages express speci®c S100 member proteins MRP14 and MRP8 in the event of acute or chronic in¯ammation.3,4 These proteins were originally isolated from human peripheral blood mononuclear cell cultures as a part of a complex using a monoclonal antibody directed against human macrophage migration inhibitory factor (MIF), and were thus named MIF-related proteins.3,5 MRP14 and MRP8 are expressed with tight regulation in granulocytes, monocytes, neutrophils, and keratinocytes during their differentiation.6,7 Phagocytes expressing these proteins were found in a variety of in¯ammatory conditions including rheumatoid arthritis, allograft rejections, in¯ammatory bowel, and lung diseases.8,9 In¯ammatory disorders such as chronic # 2002 Elsevier Science Ltd.
266 bronchitis, cystic ®brosis, and rheumatoid arthritis are associated with elevated plasma levels of MRP14 and MRP8.10,11 These proteins are coexpressed and associate to form a non-covalent hetero-dimer in a Ca2-dependent manner.12 This fact distinguishes MRP14 and MRP8 from other members of the S100 family that tend to act as homo-dimers. The MRP8/14 hetero-complex exhibits speci®c biological functions such as inhibiting casein kinase,13 exhibiting fatty acid-binding ability,14 ± 18 and changing its localization (translocating to the cytoskeleton and plasma membrane19,20) in phagocytes during the in¯ammatory process. Independent expression and functioning of MRP14 protein have also been observed.3,8,21 ± 25 A recent report has also shown that this protein selectively activates the b2 integrin, Mac-1,26 suggesting that MRP14 plays a direct role in immobilizing leukocytes at the endothelial surface. Human MRP14 (hMRP14) has a molecular mass of about 13 kDa and is composed of 114 amino acid residues. A distinguishing feature of this protein is its extended C terminus, rendering it considerably larger than other S100 family members. This C-terminal region of hMRP14 is homologous in sequence with a region of the plasma glycoprotein known as high molecular weight kininogen (HMWK), and functions similarly.27 It has also been reported that the C-terminal region possesses complete identity with the N terminus of the neutrophil immobilizing factor (NIF).28,29 As mentioned above, MRP14 protein plays an indispensable role in Ca2-dependent functions during in¯ammation, but the functional details of this protein are still unknown. Structural analysis of hMRP14 protein is expected to accelerate the understanding of such functions at the molecular level. Here we report the three-dimensional structure of the Ca2-bound (holo) form of hMRP14 as Ê resolution. A number of crysdetermined at 2.1 A tal structures of the holo form S100 family of proteins have been reported: bovine S100B,30 calbindin D9 K,31 human S100A7,32 S100A10,33 S100A11,34 S100A12,35 and MRP8.36 Investigating the hMRP14 molecule and comparing it with structures of the S100 family of proteins has revealed a possible target interaction site and the functional signi®cance of its C-terminal region.
Results and Discussion Structure description Overall structure A view of the overall structure of the holo form hMRP14 protein is shown in Figure 1(a). The hMRP14 monomer consists of four a-helices (H1H4), and these helices form a pair of helix-loophelix motifs (EF-hand motifs). The N-terminal EFhand (EF-1) consists of helix H1 (Gln7-Ser23), a Ca2-binding loop (Val24-Asn33; loop 1) and helix H2 (Gln34-Asp44). Following H2, there is a hinge
The Crystal Structure of Human MRP14
Figure 1. (a): A ribbon representation of the hMRP14 dimer, viewed parallel to the 2-fold symmetry axis. The two monomers are drawn in different colors (blue and red) and calcium atoms are shown in yellow. Dotted lines show the direction of the C-terminal loop region. All of the ribbon models representing three-dimensional structures in this paper were drawn using the programs MOLSCRIPT65 and Raster3D.66 (b) A presentation of the arrangement of four independent pairs of hMRP14 dimers (DIM1, DIM2, DIM3, DIM4) and Chaps molecules. hMRP14 and Chaps molecules are drawn in a ribbon model and ball-and-stick model, respectively. Two Chaps molecules interact with each other and bridge two hMRP14 dimers.
region that joins the two EF-hand motifs. The hinge region consists of 11 residues (Leu45-Asn55). The C-terminal EF-hand (EF-2) begins with helix H3 (Glu56-Leu66) followed by the Ca2-binding loop (Asp67-Ser75; loop 2) and ®nally helix H4 (Phe76-Arg85). As shown in Figure 2, hMRP14 has the longest C-terminal region in its sequence among the S100 family of proteins. Here, we could not build a model of the C-terminal 28 residues from residue Thr87 because the electron density of this region was poor. Inspection of the amino acid sequence of hMRP14 suggests that its C-terminal region is not likely to form a rigid secondary structure such as an a-helix because the region mainly consists of
267
The Crystal Structure of Human MRP14
Figure 2. Amino acid sequence alignment of the human S100 family of proteins (S100 beta chain and calbindin D9 K are bovine). Proteins whose three-dimensional structures have already been analyzed are marked by an asterisk. Secondary structure elements of hMRP14 are given. Residues that coordinate calcium ions are marked as follows: m, main-chain carbonyl group; s, mono-dentate side-chain of Asp or Asn; b, bi-dentate side-chain of Glu. The residues highlighted in blue are well-conserved residues the side-chains of which coordinate calcium ions (S100A10 is a protein of a permanently calcium-loaded state though the protein cannot bind calcium ions67). The residues highlighted in yellow represent highly conserved hydrophobic residues forming an intra-monomer hydrophobic cluster. Residues that interact with target molecules are marked with $. The residues highlighted in pink are other conserved hydrophobic residues that form an inter-monomer hydrophobic cluster (calbindin D9 K exists as a monomer). All sequences were obtained from the SWISS-PROT protein sequence database.68
hydrophilic residues and has many Gly and Pro residues (Figure 2). The mass spectrometry analysis showed that the molecular mass of hMRP14 protein in the native crystal is slightly smaller than that calculated (experimental, 13,115 Da; calculated, 13,242 Da). This difference is equivalent to the mass of a methionine residue. This ®nding con®rms the presence of the C-terminal region, while indicating the lack of Met at position 1 due to the post-translational processing. These results suggest that the C-terminal 28 residues (Thr87-Pro114) of hMRP14 are ¯exible and disordered in the crystal. The electron density of the N terminus of this protein (Thr2-Lys4) was also poor.
Two EF-hands The hMRP14 protein has two EF-hand motifs per monomer: N-terminal EF-1 and C-terminal EF2. The EF-2 of the S100 family of proteins is usually composed of 12 amino acid residues and is referred to as the canonical EF-hand. In this EF-hand, the Ca2-ligands are mostly carboxyl groups of acidic residues that are well-conserved among the S100 family of proteins (Figure 2). In the case of hMRP14, seven O atoms (the side-chain O atoms of Asp67, Asn69, Asp71 and Glu78, the main-chain carbonyl O atom, and a water molecule) bind to Ca2 (Figure 3(a)). The water molecule that ligates
268 to Ca2 forms a hydrogen bond with the sidechain of Asp71. On the other hand, the EF-1 is usually composed of 14 amino acid residues. The Ca2-ligands of this EF-hand are mostly mainchain carbonyl O atoms, and the residues are relatively divergent as shown in Figure 2. In the present case, seven O atoms (the main-chain carbonyl groups of Ser23, Leu26, His28 and Thr31, the carboxyl group of Glu36, and a water molecule) bind to Ca2 (Figure 3(b)). The water molecule as a seventh ligand also forms a hydrogen bond with the side-chain of Thr31. MRP14 dimer and its crystal packing Human MRP14 forms a dimeric structure. Two monomers are related with 2-fold symmetry, in the same way as in other members of the S100 family of proteins.30,32 ± 38 Dimerization is achieved by anti-parallel interactions between helices H1 and H4 of each monomer (Figure 1(a)). The present crystal contains four hMRP14 dimers (DIM1, DIM2, DIM3 and DIM4) in an asymmetric unit (Figure 1(b)). As previously reported, the quality of the hMRP14 crystal was highly improved by the addition of the detergent compound Chaps,39 suggesting that there were some interactions between Chaps and the hMRP14 molecule. The
Figure 3. Stereo representation of two EF-hand motifs of hMRP14 protein. Calcium ions (yellow) in both EFhands are ligated by seven ligands. (a) Canonical EFhand (EF-2) ligates the calcium ion mainly though the carboxyl group of the side-chain. (b) Non-canonical EFhand (EF-1) ligates the ion by main-chain carbonyl O atoms.
The Crystal Structure of Human MRP14
electron density of four Chaps molecules was clearly observed in the asymmetric unit. Each Chaps molecule binds to a hydrophobic patch at the surface of the hinge region of hMRP14. In the asymmetric unit, two hMRP14 dimers are arranged by electrostatic interaction along the crystallographic b-axis. Along the a-axis, two hMRP14 molecules are stabilized by hydrophobic interaction between two Chaps molecules, and as a consequence of this, as shown in Figure 1(b), four dimers of hMRP14 and Chaps molecules are arranged in 222 symmetry in the asymmetric unit. The electron density corresponding to one monomer of DIM3 was poor. This poor density results in rather high temperature factors for this monomer (Table 3). Intra-monomer hydrophobic interactions; predicted target interaction site of hMRP14 The hMRP14 monomer has four a-helices, and spatial arrangements of these helices are maintained by a hydrophobic cluster that is formed in the interior of the monomer (Figure 4: residues colored in yellow). As shown in Figure 2, these hydrophobic residues (Phe19, Tyr22, Leu32, Phe37, Leu40, Val41, Phe48, Leu49, Val58, Ile59, Ile62, Met63, Leu66, Leu74, Phe79, and Leu82 in hMRP14) are highly conserved among the S100 family of proteins. Inspection of the three-dimensional structures of hMRP14 and other members of the S100 family such as calbindin D9 K,31 S100B,30,37 S100A10,33 S100A11,34 and S100A1235 shows that this intra-molecular hydrophobic cluster is partly exposed to solvent at the hinge region and the inter-helix area between helices H3 and H4, and the cluster forms a hydrophobic patch at the molecular surface. Recently, the three-dimensional structures of S100A10 and S100A11 proteins bound to their target molecule annexin fragment were reported (Figure 5(b) and (c)).33,34 In both cases, the target molecules are bound to this hydrophobic patch. These facts indicate the importance of this hydrophobic patch as a target-binding site for the S100 family of proteins. In the present analysis of hMRP14, Chaps molecules are bound to this hydrophobic patch formed among the hinge region and helices H3 and H4 (Figure 5(a)). The steroid group and hydrophobic carbon-tail of the Chaps molecule adhere by hydrophobic interactions to the exposed hydrophobic residues Leu45, Phe48, and Leu49 in the hinge region, Val58 and Ile 62 in helix H3, and Leu86 at the end of helix H4 of hMRP14 (Figure 5(a)). In the case of S100A11, the solventexposed hydrophobic residues Leu45 and Phe48 in the hinge region, Val57 and Met 61 in helix H3, and Leu85 in helix H4 interact with hydrophobic residues of annexin I (Figure 5(b)). In the case of S100A10, Phe38 and Phe41 in the hinge region and Leu78 in helix H4 are involved in target-binding by hydrophobic interactions (Figure 5(c)). In the complex structure of S100A10, Ala50 and Ile54,
The Crystal Structure of Human MRP14
269
Figure 4. Stereo representation of hydrophobic clusters in hMRP14 dimer, from the direction of the hinge region (viewed from the bottom in the direction of Figure 1(a)). The residue color is presented as in Figure 2. Dotted lines are the direction of the C-terminal regions.
which are other hydrophobic residues that correspond to Val58 and Ile62 in hMRP14 (Val57 and Met61 in S100A11), do not participate in targetbinding, though these hydrophobic residues are
also exposed and form the hydrophobic area at the molecular surface (Figure 5(c)). Inspection of the three-dimensional structures of S100 family proteins reveals that there is variety in this patch, both
Figure 5. Ribbon and surface representations of the ligand-binding site of (a) hMRP14, (b) S100A11 and (c) S100A10 in the hinge region. The color coordinates are as follows; green, ligand molecule; yellow, solvent-exposed conserved hydrophobic residues. The coordinates for S100A10 (1bt6) and S100A11 (1qls) were obtained from the Protein Data Bank.69 All of the protein surface presentations in this paper were generated by the program GRASP.70
270 in shape and in size. Figure 2 also shows the variety in the hinge region in terms of amino acid sequences. Members of the S100 family of proteins have their own target molecules. The differences observed in this region may explain the divergence of targets and the way in which the protein interacts with them. These results suggest that the hydrophobic patch formed among the hinge region and helices H3 and H4 of hMRP14 protein works as a target-binding site. The Chaps binding structure indicates the target interaction residues of hMRP14 protein, though the true ligand for hMRP14 has not been identi®ed yet. Comparison with other calgranulin proteins and its implications for the complex formation of MRP proteins MRP14 (S100A9) is a protein that is expressed in myeloid cells. There are two other S100 family proteins called MRP8 (S100A8) and S100A12 that are co-expressed in the cell with this protein. These proteins are also called calgranulin A (MRP8), calgranulin B (MRP14), and calgranulin C (S100A12), and are included in the calgranulin sub-family.4,40 The amino acid sequence similarities of human MRP8 (hMRP8) and human S100A12 (hS100A12) with hMRP14 are 40 % and 46 %, respectively. The crystal structure of homo-dimeric hMRP8 has been analyzed by our group,36 and that of hS100A12 has also been reported recently.35 All of the analyzed holo form S100 family proteins exhibit a similar three-dimensional structure,30 ± 34,37,38 and the structures of hMRP14, hMRP836 and hS100A1235 are the most similar to each other (Figure 6; the r.m.s. differences between hMRP14 and hMRP8, and hS100A12 for the 66 Ca atoms excluding the hinge Ê and 0.757 A Ê , respectively). The region are 0.884 A major structural differences are localized in the
The Crystal Structure of Human MRP14
hinge region and the C-terminal helix H4 (Figure 6). As discussed in the previous section, these areas are thought to form the target-binding sites for these proteins, and these structural differences may re¯ect the divergence in both the target molecules and in the area's interactions with their target molecules. Figure 7 shows electrostatic potential and hydrophobicity distributions on the surfaces of hMRP14, hMRP8 and hS100A12. As common features of these proteins, the negatively charged nature of EF-2 Ca2-binding loops caused by the conserved acidic residues Glu and Asp is obvious (Figure 7(a)). On the opposite side of EF-2 (Figure 7(b)), positive and negative charges are scattered over the whole surface with no obvious localization. On the other hand, there were also important differences among these three proteins. For example, the side of EF-2 exhibits signi®cant differences in charge and hydrophobicity distribution around their dimeric center and hinge regions. As shown in Figure 7(a), this side of hMRP14 is strongly charged negative. This localization of negative potential around the dimeric center is due to the exposed side-chains of acidic residues in helices H3 and H4. The corresponding area in hMRP8 has larger hydrophobic surfaces (Figure 7(c)) and exhibits a neutral potential (Figure 7(a)). Human S100A12 shows a tendency similar to that of hMRP14, but its dimeric center does not show a strong negative potential like that of hMRP14 (Figure 7(a)). Figure 7(a) and (c) also shows the obvious differences in surface condition in the hinge region among these three proteins. Human MRP8 has no hydrophobic patch in this region and is the most basic, while the other two calgranulins have large hydrophobic patches that can bind other hydrophobic molecules and exhibit a rather acidic nature. All of these differences
Figure 6. The main-chain superposition of the human calgranulin proteins. The Figure is drawn in the same direction as Figure 1(a). Human MRP14 (calgranulin B) dimer is shown in red and orange, hMRP8 (calgranulin A) monomer is shown in blue, and hS100A12 (calgranulin C) is shown in yellow. The coordinates for hMRP8 (1mr8) and hS100A12 (1e8a) were obtained from the Protein Data Bank.
The Crystal Structure of Human MRP14
271
Figure 7. Electrostatic potential ((a) and (b)) and hydrophobicity (c) representations on the surface of human calgranulin proteins. (a) View from the same direction as Figure 1(a). (b) View from the opposite direction of (a). Red represents a negative charge (acidic), and blue represents a positive charge (basic). (c) View from the same direction as (a), with the exposed hydrophobic surface drawn in yellow. The Chaps molecule that binds to the hydrophobic patch of hMRP14 is indicated by the green stick-model.
among hMRP14, hMRP8 and hS100A12 proteins are assumed to re¯ect the differences in the roles they play in exhibiting biological functions. From the structural point of view, the EF-2 side is likely to play an important role as an interaction interface with target molecules for these proteins. MRP14 and MRP8 form a stable hetero-dimer complex (MRP8/14).12,41,42 It has also been reported that the hetero-dimerization of these proteins occurs more easily than does homodimerization.43 The crystal structures of the homo-dimers of hMRP836 and hS100A1235 revealed that their dimerization is achieved by hydrophobic interactions between paired monomers. Dimerization of hMRP14 is also stabilized by a hydrophobic interaction between paired monomers. The N-terminal helix H1 and the Cterminal helix H4 are respectively arranged in an anti-parallel manner to form a dimer structure (Figure 1(a)). In the case of hMRP14, residues Leu8, Ile12, Ile15, and Ile16 at helix H1, Leu45 and Phe48 in the hinge region, and Phe76, Ile80, Met81, Met83 and Ala84 at helix H4 are involved in this hydrophobic interaction between two monomers, and maintain the dimeric structure (Figure 4: residues colored in pink). As shown in Figure 2, these hydrophobic residues that take part in this inter-monomer hydrophobic interaction are highly conserved among the S100 family of proteins, suggesting that these hydrophobic residues play a key role in the dimerization of these proteins. The three-
dimensional structures of other S100 family members also show that similar hydrophobic interactions commonly take part in their dimerization.30,32 ± 34,37,38 As shown in Figure 6, the structural similarity between hMRP14 and hMRP8 monomers is likely to allow their heterodimerization to occur in the same way as their homo-dimerization. The results of some antibody recognition experiments also support the proposal that the MRP8/14 complex is formed in such a way.44 The crystal structures of homo-dimeric hMRP14 and hMRP836 provide the possible features of this biologically important MRP8/14 molecule from the structural point of view. As mentioned above, notable differences between hMRP14 and hMRP8 monomers are obvious around the hinge region and dimeric center, so the hetero-dimeric molecule may exhibit a polar nature due to the obvious differences between the hMRP14 and hMRP8 monomers. In the hetero-dimer, half of the molecule is expected to be charged negative (hMRP14 monomer) and the other half positive (hMRP8 monomer). Not only the electrostatic potential, but also the hydrophobicity distribution on the surface is also expected to be different in the two halves of the dimer. This hetero-dimer molecule may have two different target-binding sites in its hingeregions, and those of hybrid characters may play the key role in exhibiting hetero-dimer-speci®c functions.
272 In contrast to these hMRP14 and hMRP8 proteins, hS100A12 does not form a complex with hMRP14 or hMRP8,42,45 though the hS100A12 monomer shows a higher similarity with hMRP14 than hMRP8 monomer both in terms of amino acid sequence (Figure 2) and three-dimensional structure (Figure 6). In this sense, it may be worth noting that hMRP14 and hMRP8 monomers choose each other exclusively as partners in dimer formation, while three calgranulin proteins are coexpressed and exhibit similar localization in the cell.42,45 These facts may suggest the existence of some special mechanisms for the selection of a partner in MRP14 and MRP8 proteins. The details of hetero-dimerization of these proteins are still unclear at present. Further study from both the biological and structural perspectives is required to elucidate the functions of this biologically important hetero-dimer of two MRPs at the molecular level. Long C-terminal tail; importance of its flexibility MRP14 protein is distinguished from other S100 family members by its long C-terminal region. In the present crystal, the C-terminal region has a poor electron density, suggesting that this region has an extensively ¯exible character. The poor electron density still allows the main-chain trajectory to be traced up to a point, and this tracing shows that the C-terminal region of hMRP14 makes a turn at the end of helix H4 and turns toward the solvent region of the inter-molecular space (Figures 1(a) and 4). As shown in Figure 1(b), the crystal of hMRP14 has a relatively large solvent area around the side of helix H4 of all of the dimers. Two Chaps molecules bridge two hMRP14 dimers, thus creating enough volume of free space to allow random conformation of this C terminus of each of the monomers without interfering with any neighboring molecules. The importance of this C-terminal tail has been reported as an effector region to target molecules other than MRP8,46,47 including acting as a Ca2binding ability inducing switch,20 a regulator of translocation of MRP8/14 complex,19 and a nuetrophil immobilization inducer.48 To perform such biological functions, the C-terminal ¯exible tail may adapt its conformation for various targets. In this sense, it is assumed that the ¯exibility of the C-terminal extended region of hMRP14 is functionally indispensable. The C-terminal region including helix H4 of S100 family members is assumed to be involved in the interaction with target molecules.33,34,49 ± 51 Three-dimensional structures of several S100 family members show structural differences in the C-terminal extension region between biologically equivalent monomers of hS100A6,38 hS100A7,32 hMRP8,36 and hS100A12.35 These structural differences may also represent the ¯exible nature of this region. Human MRP14 selectively activates the b2 integrin Mac-1 that is involved in the adhesion of neu-
The Crystal Structure of Human MRP14
trophil to the endothelial surface.26 This activation of Mac-1 is achieved only by the homo-dimer of hMRP14; the hetero-dimer of hMRP8 and hMRP14 does not activate Mac-1. In this activation, hMRP8 plays a regulatory role; it suppresses hMRP14's activation of Mac-1 through the formation of the hetero-dimer.26 As shown in Figure 2, the amino acid sequence of the hMRP14 C-terminal region (Ala89-Gly107) exhibits complete identity with the N-terminal region of NIF,29 an inhibitor which acts directly and irreversibly on neutrophils to prevent their random migration and chemotaxis in a noncytotoxical way.28,52 The homo-dimeric hMRP14 molecule has two NIF-identical regions, while the hMRP8/14 molecule has only one. Previous biological studies have shown that the region Arg85Met94 of hMRP14 is involved in hetero-dimer interaction between hMRP14 and hMRP8 monomers.44,46,47 This means that the former half of the unique NIF-identical sequence of the hMRP14 molecule will be involved in the interaction with hMRP8, and the region would be expected to lose its ability to interact with target molecules. Thus, hetero-dimerization is likely to inhibit free movement of the NIF-identical region of hMRP14. This may partly explain the regulation by hMRP8 of the activation of Mac-1 by hMRP14. To understand functional activities of the unique C-terminal tail region of hMRP14 in greater detail, further investigation including structural analysis of MRP8/14 hetero-dimer will be necessary.
Experimental Procedures Sample preparation and crystallization The full-length (1-114 amino acid residues) recombinant hMRP14 protein was prepared by the overexpression system using Escherichia coli. Sample preparation and crystallization of native protein have been reported.39 Se-Met-substituted hMRP14 was also prepared for the multiple wavelength anomalous diffraction method (MAD) following the previously described method with slight modi®cations. The best crystallization condition for the Se-Met-substituted hMRP14 was the same as that of native hMRP14. The dimensions of the Se-Met crystal were 0.05 mm 1.0 mm 0.4 mm, and the crystals were isomorphous with native ones. Se-Met-substituted crystal belongs to space group P21 with cell Ê , b 178.59 A Ê , c 61.24 A Ê, dimensions of a 57.62 A and b 113.45 , and an asymmetric unit contains four dimers of hMRP14. The crystal volume per protein mass Ê 3 Daÿ1, and the solvent content was (VM)53 was 2.7 A 54.8 %. To con®rm the molecular mass of the protein that was contained in the crystal, MALDI-TOF mass spectrometry analysis (Voyager DE Pro, PE biosystems) was performed on dissolved native and Se-Met-substituted crystals of hMRP14. Data collection All data collections were carried out using SPring-8 (Hyogo, Japan) under 100 K by ¯ash cooling after soaking in the cryo-protect solution with 30 % (v/v) glycerol. Native data were collected using a wavelength of
273
The Crystal Structure of Human MRP14 Table 1. Summary of data collection
Ê) Wavelength (A Ê) Resolution (A Space group Number of observed Reflections Unique reflections Completeness(%) Averaged redundancy Averaged I/s (I) Rmeas (%)b Rl (%)c
Se-MAD (peak)
native
(edge)
(remote)
0.7000 40 2.1 (2.21 2.10) P21
0.9794
0.9792 402.3 (2.422.30) P21
0.9000
251,869 (37,031) 65,554 (9,583) 99.7 (99.7) 3.8 (3.9) 6.5 (2.5) 6.6(33.9) -
239,355(20,212) 49,806(7,035) 99.4(99.4) 5.2(2.9) 11.3(2.9) 5.6(30.6) 6.9(14.5)
261,277(20,381) 49,818(7,043) 99.4(99.4) 5.2(2.9) 11.1(2.7) 6.3(32.0) 6.3(14.8)
284,544(40,391) 50,075(7,288) 99.9(99.9) 5.7(5.5) 10.5(2.4) 6.2(36.9) 0(0)
a
Values in parentheses are for the outermost resolution shell. Rmeas h[m/(m ÿ 1)]1/2jjhIih ÿ Ih,jj/hjIh,j, where hIih is the mean intensity of symmetry-equivalent re¯ections and m is redundancy. c Rl hjjFljj ÿ jFlojj/hjFloj, where Flj is the structure factor of the data collected at lj and Flo is the structure factor of the data collected at remote wavelength. b
Ê on beamline BL44B2. MAD data were collected 0.7000 A using three different wavelengths (0.9792, 0.9794 and Ê ) from a single Se-Met-substituted crystal on 0.9000 A beamline BL41XU. The data were processed using the program MOSFLM,54 and SCALA55 in the CCP4 program suite.56 Data-collection statistics are given in Table 1. Phase calculation The full-length hMRP14 protein has six Met residues per monomer in its amino acid sequence. The results of mass-spectrometry analysis suggested that the N-terminal Met of the recombinant hMRP14 did not exist due to the post-translational processing, so the Se-Met-substituted proteins had ®ve Se-Met residues per monomer. The asymmetric unit contains four dimers of hMRP14, each of which has ten Se-Met residues. Eight of 40 Se sites were found by the program SHELXS in the SHELX97 package.57 Initial phases with the eight sites were calculated, and 20 additional sites were located by the difference-Fourier methods using the SHARP program.58 The initial electron density map was obtained after phase improvement by non-crystallographic symmetry (NCS) averaging using the program SOLOMON.59 The phasing statistics are summarized in Table 2. Model building and refinement Based on the initial electron density map, 85 residues out of 114 from one hMRP14 monomer were built using the graphic program O,60 and the other monomer that comprises the dimer was generated by NCS operation. Three other hMRP14 dimers were also generated by NCS operation from the ®rst dimer model. A total of 28 carboxyl-terminal residues of each monomer were disordered and could not be identi®ed in the electron density map. After the ®rst round of re®nement, including rigidbody re®nement, positional re®nement and temperaturefactor re®nement with the program CNS61 using MAD Ê resolution, those models were re®ned data at 2.3 A Ê resolution. To improve the using native data up to 2.1 A electron density map of the C-terminal region, a density modi®cation technique with NCS averaging was applied using the program DM62 in the CCP4 program suite.
Simulated-annealing re®nement using slow-cooling protocols, positional re®nement, and temperature-factor Ê ) with re®nement were performed using CNS (10-2.1 A NCS restraints. A manual model ®tting was carried out between re®nement rounds using the program O. At ®rst, the NCS restraints were applied to main-chain atoms of all monomers. After several rounds of re®nement, NCS restraints were used for only main-chain atoms of secondary structures among the NCS equivalent monomers, and Chaps molecules were identi®ed from the 2Fo ÿ Fc and Fo ÿ Fc maps. The initial model, topology, and parameter ®les of Chaps for structure re®nement were obtained from the HIC-up,63 and its carbon-tail was elongated to ®t the electron density map according to the progress of re®nement. Water molecules were found using the CNS program. Further positional re®nement and temperature-factor re®nement were performed without NCS restraints. Analysis of protein geometry was performed using the program PROCHECK.64 The ®nal re®nement statistics are given in Table 3.
Table 2. Summary of phase calculation Se-Met hMRP14 crystal Ê) Resolution (A Space group MAD phasing RCullis_isoa Phasing power_isob Phasing power_anoc FOMd NCS averaging CCe R-factor
Remote 2.549 0.666
202.3 P21 Edge 0.300 6.020 2.417
Peak 0.392 5.625 3.335
0.793 35.2 %
a RCullis_iso is the mean residual lack of closure error divided by dispersive difference. Values are for centric re¯ections. b Phasing power_iso is the root mean square of FH/E, where FH is the dispersive difference of FH and E is the lack of closure error. c Phasing power_ano is as for Phasing power_iso except that FH is the anomalous difference of FH. d FOM is the mean ®gure of merit. e CC is standard linear correlation coef®cient between observed and calculated structure factor amplitudes.
274
The Crystal Structure of Human MRP14
Table 3. Final re®nement statistics Ê) Resolution range (A Number of reflections Completeness(%)
10.02.1 65,101 99.8
Total number of non-hydrogen atoms Protein Others Solvent R-factor (%)a R-free-factor (%)b r.m.s. deviation from standard values Ê) Bonds(A Bond angles (deg.) Dihedral angles (deg.) Ê 2) Average B-factor (A Ramachandran Residues in Residues in Residues in Residues in a b c
plotc most favored regions (%) additional allowed regions (%) generously allowed regions (%) disallowed regions (%)
5603 140 140 24.49 27.36 DIM1 0.008 1.278 19.36 39.4
DIM2 0.007 1.174 18.94 43.9
DIM3 0.006 1.047 18.08 59.2
DIM4 0.010 1.384 19.52 38.6
Total 0.008 1.228 18.99 47.7
89.4 10.6 0 0
89.7 10.3 0 0
84.1 15.9 0 0
91.8 8.2 0 0
88.8 11.2 0 0
R-factor jFobs ÿ Fcalj/ Fobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. R-free-factor value was calculated for R-factor, using only an unre®ned subset of re¯ections data (10 %). Ramachandran plot was calculated by PROCHECK.64
Atomic coordinates The atomic coordinates of hMRP14 have been deposited in the Protein Data Bank (ID code 1irj).
Acknowledgments We thank M. Kawamoto and S. Adachi of SPring-8 (Hyogo, Japan) for their kind help during data collection. This work was partly supported by the Research for the Future Program (JSPS-RFTF 97 L0051) from the Japan Society for the Promotion of Science. H.I. is a research fellow of the Japan Society for the Promotion of Science.
References 1. Kligman, D. & Hilt, D. C. (1988). The S100 protein family. Trends Biochem. Sci. 13, 437-443. 2. Donato, R. (1999). Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type. Biochim. Biophys. Acta, 1450, 191-231. 3. Odink, K., Cerletti, N., BruÈggen, J., Clerc, R. G., Tarcsay, L., Zwadlo, G. et al. (1987). Two calciumbinding proteins in in®ltrate macrophages of rheumatoid arthritis. Nature, 330, 80-82. 4. Kerkhoff, C., Klempt, M. & Sorg, C. (1998). Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9). Biochim. Biophys. Acta, 1448, 200-211. 5. Burmeister, G., Tarcsay, L. & Sorg, C. (1986). Generation and characterization of a monoclonal antibody (1C5) to human migration inhibitory factor (MIF). Immunobiology, 171, 461-474. 6. Lagasse, E. & Clerc, R. G. (1988). Cloing and expression of two human genes encoding calciumbinding proteins that are regulated during myeloid differentiation. Mol. Cell. Biol. 8, 2402-2410.
7. Zwadlo, G., BruÈggen, J., Gerhards, G., Schlegel, R. & Sorg, C. (1988). Two calcium-binding proteins associated with speci®c stages of myeloid cell differentiation are expressed by subsets of macrophages in in¯ammatory tissues. Clin. Exp. Immunol. 72, 510515. 8. Goebeler, M., Roth, J., Burwinkel, F., Vollmer, E., Bocker, W. & Sorg, C. (1994). Expression and complex formation of S100-like proteins MRP8 and MRP14 by macrophages during renal allograft rejection. Transplantation, 58, 355-361. 9. Rugtweit, J., Brandtzaeg, P., Halstensen, T. S., Fausa, O. & Scott, H. (1994). Increased macrophage subset in in¯ammatory bowel disease: apparent recruitment from peripheral blood monocytes. Gut, 35, 669-674. 10. Roth, J., Teigelkamp, S., Wilke, M., Grun, L., Tummler, B. & Sorg, C. (1992). Complex pattern of the myelo-monocytic differentiation antigens MRP8 and MRP14 during chronic airway in¯ammation. Immunobiology, 186, 304-314. 11. Brun, J. G., Jonsson, R. & Haga, H. J. (1994). Measurement of plasma calprotectin as an indicator of arthritis and disease activity in patients with in¯ammatory rheumatic diseases. J. Rheumatol. 21, 733-738. 12. Teigelkamp, S., Bhardwaj, R. S., Roth, J., MeinardusHager, G., Karas, M. & Sorg, C. (1991). Calciumdependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14. J. Biol. Chem. 266, 13462-13467. 13. Murao, S., Collart, F. R. & Huberman, E. (1989). A protein containing the cystic ®brosis antigen is an inhibitor of protein kinases. J. Biol. Chem. 264, 83568360. 14. Siegenthaler, G., Roulin, K., Chatellard-Gruaz, D., Hotz, R., Saurat, J. H., Hellman, U. & Hagens, G. (1997). A heterocomplex, formed by the calciumbinding proteins MRP8 (S100A8) and MRP14
The Crystal Structure of Human MRP14
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26. 27.
(S100A9) binds unsaturated fatty acids with high af®nity. J. Biol. Chem. 272, 9371-9377. Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U. & Sorg, C. (1997). The heterodimer of the Ca2-binding proteins MRP8 and MRP14 binds arachidonic acid. FEBS Letters, 408, 81-84. Kerkhoff, C., Klempt, M., Kaever, V. & Sorg, C. (1999). The two calcium-binding proteins, S100A8 and S100A9, are involved in the methabolism of arachidonic acid in human neutrophils. J. Biol. Chem. 274, 32672-32679. Roulin, K., Hagens, G., Hotz, R., Saurat, J. H., Veerkamp, J. H. & Siegenthaler, G. (1999). The fatty acid-binding heterocomplex FA-p34 formed by S100A8 and S100A9 is the major fatty acid carrier in neutrophils and translocates from the cytosol to the membrane upon stimulation. Exp. Cell Res. 247, 410421. Kerkhoff, C., Sorg, C., Tandon, N. N. & Nacken, W. (2001). Interaction of S100A8/S100A9-arachidnic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells. Biochemistry, 40, 241-248. Roth, J., Burwinkel, F., van den Bos, C., Goebeler, M., Vollmer, E. & Sorg, C. (1993). MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate ®laments in a calcium-dependent manner. Blood, 82, 1875-1883. van den Bos, C., Roth, J., Koch, H. G., Hartmann, M. & Sorg, C. (1996). Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca2-dependent translocation from cytoplasm to membranes and cytoskelton. J. Immunol. 156, 1247-1254. Sunderkotter, C., Beil, W., Roth, J. & Sorg, C. (1991). Cellular events associated with in¯ammatory angiogenesis in the mouse cornea. Am. J. Pathol. 138, 931939. Delabie, J., de Wolf-Peeters, C., van den Oord, J. J. & Desmet, V. J. (1990). Differential expression of the calcium-binding proteins MRP8 and MRP14 in granulomatous conditions: an immunohistochemical study. Clin. Exp. Immunol. 81, 123-126. Bhardwaj, R. S., Zotz, C., Zwadlo, G., Roth, J., Goebeler, M., Mahnke, K. et al. (1992). The calciumbinding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic in¯ammatory lesions. Eur. J. Immunol. 22, 1891-1897. Akiyama, H., Ikeda, K., Katoh, M., McGeer, E. G. & McGeer, P. L. (1994). Expression of MRP14, 27E10, interferon-alpha and leukocyte common antigen by reactive microglia in postmortem human brain tissue. J. Neuroimmunol. 50, 195-201. Aguiar-Passeti, T., Postol, E., Sorg, C. & Mariano, M. (1997). Epithelioid cells from foreign-body granuloma selectively express the calcium-binding protein MRP-14, a novel down-regulatory molecule of macrophage activation. J. Leuk. Biol. 62, 852-858. Newton, R. A. & Hogg, N. (1998). The human S100 protein MRP-14 is a novel activator of the b2 integrin Mac-1 on neutrophils. J. Immunol. 160, 1427-1435. Hessian, P. A., Wilkinson, L. & Hogg, N. (1995). The S100 family protein MRP-14 (S100A9) has homology with the contact domain of high molecular weight kininogen. FEBS Letters, 371, 271-275.
275 28. Watt, K. W., Brightman, I. L. & Goetzl, E. J. (1983). Isolation of two polypeptides comprising the neutrophil-immobilizing factor of human leucocytes. Immunobiology, 48, 79-86. 29. Freemont, P., Hogg, N. & Edgeworth, J. (1989). Sequence identity. Nature, 339, 516. 30. Matsumura, H., Shiba, T., Inoue, T., Harada, S. & Kai, Y. (1998). A novel mode of target recognition suggested by the 2.0 angstrom structure of holo S100B from bovine brain. Structure, 6, 233-241. 31. Szebenyi, D. M. & Moffat, K. (1986). The re®ned structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins. J. Biol. Chem. 261, 87618777. 32. Brodersen, D. E., Etzerodt, M., Madsen, P., Celis, J. E., Thùgersen, H. C., Nyborg, J. & Kjeldgaard, M. (1998). EF-hands at atmic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing. Structure, 6, 477-489. 33. ReÂty, S., Sopkova, J., Renouard, M., Osterloh, D., Gerke, V., Tabaries, S. et al. (1999). The crystal structure of a complex pf p11 with the annexin II Nterminal peptide. Nature Struct. Biol. 6, 89-95. 34. ReÂty, S., Osterloh, D., Arie, J., Tabaries, S., Seeman, J., Russo-Marie, F. et al. (2000). Structural basis of the Ca2-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I. Structure, 8, 175-184. 35. Moroz, O. V., Antson, A. A., Murshudov, G. N., Maitland, N. J., Dodson, G. G., Wilson, K. S. et al. (2001). The three-dimensional structure of human S100A12. Acta Crystallog. sect. D, 57, 20-29. 36. Ishikawa, K., Nakagawa, A., Tanaka, I., Suzuki, M. & Nishihira, J. (2000). The structure of human MRP8, a member of the S100 calcium-binding protein family, by MAD phasing at 1.9 angstrom resolution. Acta Crystallog. sect. D, 56, 559-566. 37. Smith, S. P. & Shaw, G. S. (1998). A novel calciumsensitive switch revealed by the structure of human S100B in the calcium-bound form. Structure, 6, 211222. 38. Sastry, M., Ketchem, R. R., Crescenzi, O., Weber, C., Lubienski, M. J., Hidaka, H. & Chazin, W. J. (1998). The three-dimensional structure of Ca2-bound calcyclin:implications for Ca2-signal transduction by S100 proteins. Structure, 6, 223-231. 39. Itou, H., Fujita, I., Ishikawa, K., Yao, M., Watanabe, N., Suzuki, M. et al. (2001). Expression, puri®cation, crystallization, and preliminary X-ray diffraction analysis of human calcium-binding protein MRP14 (S100A9). Acta Crystallog. sect. D, 58, 1174-1176. 40. Kelly, S. E., Jones, D. B. & Fleming, S. (1989). Calgranulin expression in in¯ammatory dermatoses. J. Pathol. 49, 17-21. 41. Edgeworth, J., Gorman, M., Bennett, R., Freemont, P. & Hogg, N. (1991). Identi®cation of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. J. Biol. Chem. 266, 7706-7713. 42. Vogl, T., ProÈpper, C., Hartmann, M., Strey, A., Strupat, K., van den Bos, C. et al. (1999). S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14. J. Biol. Chem. 274, 25291-25296. 43. Hunter, M. J. & Chazin, W. J. (1998). High level expression and dimer characterization of the S100
276
44. 45. 46.
47.
48.
49.
50.
51.
52. 53. 54. 55. 56. 57.
The Crystal Structure of Human MRP14
EF-hand proteins, migration inhibitory factor-related proteins 8 and 14. J. Biol. Chem. 273, 12427-12435. Hessian, P. A. & Lorryn, F. (2001). The heterodimeric complex pf MRP-8 (S100A8) and MRP-14 (S100A9). Eur. J. Biochem. 268, 353-363. Robinson, M. J. & Hogg, N. (2000). A comparison of human S100A12 with MRP-14 (S100A9). Biochem. Biophys. Res. Commun. 175, 865-870. ProÈpper, C., Huang, X., Roth, J., Sorg, C. & Nacken, W. (1999). Analysis of the MRP8-MRP14 proteinprotein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation. J. Biol. Chem. 274, 183-188. Nacken, W., Sopalla, C., ProÈpper, C., Sorg, C. & Kerkhoff, C. (2000). Biochemical characterization of the murine S100A9 (MRP14) protein suggests that it is functionally equivalent to its human counterpart despite its low degree of sequence homology. Eur. J. Biochem. 267, 560-565. Edgeworth, J., Freemont, P. & Hogg, N. (1989). Ionomycin-regulated phosphorylation of the myeloid calcium-binding protein p14. Nature, 342, 189-192. Osterloh, D., Ivanenkov, V. V. & Gerke, V. (1998). Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding but not for dimerization. Cell. Calcium. 24, 137-151. Kilby, P. M., Van Eldik, L. J. & Roberts, G. C. (1997). Identi®cation of the binding site on S100B protein for the actin capping protein CapZ. Protein Sci. 6, 2494-2503. Kube, E., Becker, T., Weber, K. & Gerke, V. (1992). Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin IIbinding site on p11, a member of the S100 protein family. J. Biol. Chem. 267, 14175-14182. Goetzl, E. J. & Austen, K. F. (1972). A neutrophilimmobilizing factor derived from human leukocytes. J. Exp. Med. 146, 1564-1580. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. Leslie, A. G. W. (1993). Auto-indexing of rotatin diffraction images and parameter re®nement. In Proc. CCP4 Study Weekend, pp. 44-51, . Evans, P. R. (1997). Scaling of MAD data. In Proc. CCP4 Study Weekend, pp. 97-102. Collaborative Computational Project No. 4. (1994). The CCP4 suite: programs for protein crystallography. Acta. Crystallog. sect. D, 50, 760-763. Sheldrick, G. M., Dauter, Z., Wilson, K. S., Hope, H. & Sieker, L. C. (1993). The application of direct methods and Patterson interpretation to high-resol-
58.
59.
60.
61.
62.
63. 64.
65. 66. 67.
68. 69. 70.
ution native protein data. Acta. Crystallog. sect. D, 49, 18-23. La Fortelle, E. & Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter re®nement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472-494. Abrahams, J. P. & Leslie, A. G. W. (1996). Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta. Crystallog. sect. D, 52, 30-42. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta. Crystallog. sect. A, 47, 110-119. BruÈnger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system (CNS): a new software system for macromolecularstructure determination. Acta. Crystallog. sect. D, 54, 905-921. Cowtain, K. D. & Main, P. (1996). Phase combination and cross validation in iterated densitymodi®cation calculations. Acta. Crystallog. sect. D, 52, 43-48. Kleywegt, G. J. & Jones, T. A. (1998). Databases in protein crystallography. Acta. Crystallog. sect. D, 54, 1119-1131. Laskowski, R. A., Mac Arthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check stereochemical quality of protein structure. J. Appl. Crystallog. 26, 283-291. Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946-950. Merritt, E. A. & Bacon, D. J. (1997). Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505-524. Johnsson, N. & Weber, K. (1990). Alkylation of cysteine 82 of p11 abolishes the complex formation with the tyrosine-protein kinase substrate p36 (annexin 2, calpactin 2, lipocortin 2). J. Biol. Chem. 265, 14464-14468. Bairoch, A. & Apweiler, R. (2000). The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucl. Acids Res. 28, 45-48. Berman, H. M., Westblock, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H. et al. (2000). The Protein Data Bank. Nucl. Acids Res. 28, 235-242. Nicholls, A., Sharp, K. A. & Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. Struct. Funct. Genet. 11, 281-296.
Edited by R. Huber (Received 17 September 2001; received in revised form 3 December 2001; accepted 4 December 2001)
J. Mol. Biol. (2002) 316, 265±276
The Crystal Structure of Human MRP14 (S100A9), a Ca2-dependent Regulator Protein in Inflammatory Process Hiroshi Itou1, Min Yao1,2, Ikuko Fujita1, Nobuhisa Watanabe1 Masaki Suzuki3, Jun Nishihira3 and Isao Tanaka1,2* 1
Division of Biological Sciences Graduate School of Science Hokkaido University, Sapporo 060-0810, Japan 2
Division of BioCrystallography Technology RIKEN Harima Institute/ SPring-8, Kouto, Mikazuki Sayo, Hyogo 679-5148, Japan 3
Central Research Institute School of Medicine, Hokkaido University, Sapporo 060-0810, Japan
Human MRP14 (hMRP14) is a Ca2-binding protein from the S100 family of proteins. This protein is co-expressed with human MRP8 (hMRP8), a homologue protein in myeloid cells, and plays an indispensable role in Ca2-dependent functions during in¯ammation. This role includes the activation of Mac-1, the b2 integrin which is involved in neutrophil adhesion to endothelial cells. The crystal structure of the holo form of Ê resolution. hMRP14 is distinguished from hMRP14 was analyzed at 2.1 A other S100 member proteins by its long C-terminal region, and its structure shows that the region is extensively ¯exible. In this crystal structure of hMRP14, Chaps molecules bind to the hinge region that connects two EF-hand motifs, which suggests that this region is a target-binding site of this protein. Based on a structural comparison of hMRP14 with hMRP8 and human S100A12 (hS100A12) that is another homologue protein, the character of MRP8/14 hetero-complex and the functional signi®cance of the ¯exibility of the C-terminal region of hMRP14 are discussed. # 2002 Elsevier Science Ltd.
*Corresponding author
Keywords: calcium-binding protein; crystal structure analysis; human MRP14; human S100A9; S100 family of proteins
Introduction Calcium has been implicated as a regulatory ion in a variety of cellular processes such as muscle contraction, secretion, adhesion, synaptic transmission, cell-cycle progression and cell differentiation. In such processes, the change of intracellular Ca2 concentration acts as a signal mediator, and the signal is transduced as activation or inactivation of the Ca2-binding protein. There is a multigenic family of Ca2-binding proteins that are involved in such signal mediation called the S100 family of proteins.1 The S100 family proteins were found to be expressed in a cell and tissue-speci®c manner, and were associated with different human pathological states such as cancer, neurodegeneration and cardiomyopathies.2 The members of the S100 family of proteins are small Abbreviations used: hMRP14, human MRP14; NIF, neutophil immobilizing factor; NCS, non-crystallographic symmetry; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate. E-mail address of the corresponding author: [email protected] 0022-2836/02/020265±12 $35.00/0
(their molecular weights are in the range of 10-13 kDa), acidic, and have a strong tendency to dimerize. They have two EF-hand motifs as Ca2-binding sites, and are implicated in Ca2-dependent regulation of a variety of intracellular activities such as protein phosphorylation, cell proliferation and differentiation, the dynamics of cytoskeleton constituents, and intracellular Ca2 homeostasis.2 The in®ltrating macrophages express speci®c S100 member proteins MRP14 and MRP8 in the event of acute or chronic in¯ammation.3,4 These proteins were originally isolated from human peripheral blood mononuclear cell cultures as a part of a complex using a monoclonal antibody directed against human macrophage migration inhibitory factor (MIF), and were thus named MIF-related proteins.3,5 MRP14 and MRP8 are expressed with tight regulation in granulocytes, monocytes, neutrophils, and keratinocytes during their differentiation.6,7 Phagocytes expressing these proteins were found in a variety of in¯ammatory conditions including rheumatoid arthritis, allograft rejections, in¯ammatory bowel, and lung diseases.8,9 In¯ammatory disorders such as chronic # 2002 Elsevier Science Ltd.
266 bronchitis, cystic ®brosis, and rheumatoid arthritis are associated with elevated plasma levels of MRP14 and MRP8.10,11 These proteins are coexpressed and associate to form a non-covalent hetero-dimer in a Ca2-dependent manner.12 This fact distinguishes MRP14 and MRP8 from other members of the S100 family that tend to act as homo-dimers. The MRP8/14 hetero-complex exhibits speci®c biological functions such as inhibiting casein kinase,13 exhibiting fatty acid-binding ability,14 ± 18 and changing its localization (translocating to the cytoskeleton and plasma membrane19,20) in phagocytes during the in¯ammatory process. Independent expression and functioning of MRP14 protein have also been observed.3,8,21 ± 25 A recent report has also shown that this protein selectively activates the b2 integrin, Mac-1,26 suggesting that MRP14 plays a direct role in immobilizing leukocytes at the endothelial surface. Human MRP14 (hMRP14) has a molecular mass of about 13 kDa and is composed of 114 amino acid residues. A distinguishing feature of this protein is its extended C terminus, rendering it considerably larger than other S100 family members. This C-terminal region of hMRP14 is homologous in sequence with a region of the plasma glycoprotein known as high molecular weight kininogen (HMWK), and functions similarly.27 It has also been reported that the C-terminal region possesses complete identity with the N terminus of the neutrophil immobilizing factor (NIF).28,29 As mentioned above, MRP14 protein plays an indispensable role in Ca2-dependent functions during in¯ammation, but the functional details of this protein are still unknown. Structural analysis of hMRP14 protein is expected to accelerate the understanding of such functions at the molecular level. Here we report the three-dimensional structure of the Ca2-bound (holo) form of hMRP14 as Ê resolution. A number of crysdetermined at 2.1 A tal structures of the holo form S100 family of proteins have been reported: bovine S100B,30 calbindin D9 K,31 human S100A7,32 S100A10,33 S100A11,34 S100A12,35 and MRP8.36 Investigating the hMRP14 molecule and comparing it with structures of the S100 family of proteins has revealed a possible target interaction site and the functional signi®cance of its C-terminal region.
Results and Discussion Structure description Overall structure A view of the overall structure of the holo form hMRP14 protein is shown in Figure 1(a). The hMRP14 monomer consists of four a-helices (H1H4), and these helices form a pair of helix-loophelix motifs (EF-hand motifs). The N-terminal EFhand (EF-1) consists of helix H1 (Gln7-Ser23), a Ca2-binding loop (Val24-Asn33; loop 1) and helix H2 (Gln34-Asp44). Following H2, there is a hinge
The Crystal Structure of Human MRP14
Figure 1. (a): A ribbon representation of the hMRP14 dimer, viewed parallel to the 2-fold symmetry axis. The two monomers are drawn in different colors (blue and red) and calcium atoms are shown in yellow. Dotted lines show the direction of the C-terminal loop region. All of the ribbon models representing three-dimensional structures in this paper were drawn using the programs MOLSCRIPT65 and Raster3D.66 (b) A presentation of the arrangement of four independent pairs of hMRP14 dimers (DIM1, DIM2, DIM3, DIM4) and Chaps molecules. hMRP14 and Chaps molecules are drawn in a ribbon model and ball-and-stick model, respectively. Two Chaps molecules interact with each other and bridge two hMRP14 dimers.
region that joins the two EF-hand motifs. The hinge region consists of 11 residues (Leu45-Asn55). The C-terminal EF-hand (EF-2) begins with helix H3 (Glu56-Leu66) followed by the Ca2-binding loop (Asp67-Ser75; loop 2) and ®nally helix H4 (Phe76-Arg85). As shown in Figure 2, hMRP14 has the longest C-terminal region in its sequence among the S100 family of proteins. Here, we could not build a model of the C-terminal 28 residues from residue Thr87 because the electron density of this region was poor. Inspection of the amino acid sequence of hMRP14 suggests that its C-terminal region is not likely to form a rigid secondary structure such as an a-helix because the region mainly consists of
267
The Crystal Structure of Human MRP14
Figure 2. Amino acid sequence alignment of the human S100 family of proteins (S100 beta chain and calbindin D9 K are bovine). Proteins whose three-dimensional structures have already been analyzed are marked by an asterisk. Secondary structure elements of hMRP14 are given. Residues that coordinate calcium ions are marked as follows: m, main-chain carbonyl group; s, mono-dentate side-chain of Asp or Asn; b, bi-dentate side-chain of Glu. The residues highlighted in blue are well-conserved residues the side-chains of which coordinate calcium ions (S100A10 is a protein of a permanently calcium-loaded state though the protein cannot bind calcium ions67). The residues highlighted in yellow represent highly conserved hydrophobic residues forming an intra-monomer hydrophobic cluster. Residues that interact with target molecules are marked with $. The residues highlighted in pink are other conserved hydrophobic residues that form an inter-monomer hydrophobic cluster (calbindin D9 K exists as a monomer). All sequences were obtained from the SWISS-PROT protein sequence database.68
hydrophilic residues and has many Gly and Pro residues (Figure 2). The mass spectrometry analysis showed that the molecular mass of hMRP14 protein in the native crystal is slightly smaller than that calculated (experimental, 13,115 Da; calculated, 13,242 Da). This difference is equivalent to the mass of a methionine residue. This ®nding con®rms the presence of the C-terminal region, while indicating the lack of Met at position 1 due to the post-translational processing. These results suggest that the C-terminal 28 residues (Thr87-Pro114) of hMRP14 are ¯exible and disordered in the crystal. The electron density of the N terminus of this protein (Thr2-Lys4) was also poor.
Two EF-hands The hMRP14 protein has two EF-hand motifs per monomer: N-terminal EF-1 and C-terminal EF2. The EF-2 of the S100 family of proteins is usually composed of 12 amino acid residues and is referred to as the canonical EF-hand. In this EF-hand, the Ca2-ligands are mostly carboxyl groups of acidic residues that are well-conserved among the S100 family of proteins (Figure 2). In the case of hMRP14, seven O atoms (the side-chain O atoms of Asp67, Asn69, Asp71 and Glu78, the main-chain carbonyl O atom, and a water molecule) bind to Ca2 (Figure 3(a)). The water molecule that ligates
268 to Ca2 forms a hydrogen bond with the sidechain of Asp71. On the other hand, the EF-1 is usually composed of 14 amino acid residues. The Ca2-ligands of this EF-hand are mostly mainchain carbonyl O atoms, and the residues are relatively divergent as shown in Figure 2. In the present case, seven O atoms (the main-chain carbonyl groups of Ser23, Leu26, His28 and Thr31, the carboxyl group of Glu36, and a water molecule) bind to Ca2 (Figure 3(b)). The water molecule as a seventh ligand also forms a hydrogen bond with the side-chain of Thr31. MRP14 dimer and its crystal packing Human MRP14 forms a dimeric structure. Two monomers are related with 2-fold symmetry, in the same way as in other members of the S100 family of proteins.30,32 ± 38 Dimerization is achieved by anti-parallel interactions between helices H1 and H4 of each monomer (Figure 1(a)). The present crystal contains four hMRP14 dimers (DIM1, DIM2, DIM3 and DIM4) in an asymmetric unit (Figure 1(b)). As previously reported, the quality of the hMRP14 crystal was highly improved by the addition of the detergent compound Chaps,39 suggesting that there were some interactions between Chaps and the hMRP14 molecule. The
Figure 3. Stereo representation of two EF-hand motifs of hMRP14 protein. Calcium ions (yellow) in both EFhands are ligated by seven ligands. (a) Canonical EFhand (EF-2) ligates the calcium ion mainly though the carboxyl group of the side-chain. (b) Non-canonical EFhand (EF-1) ligates the ion by main-chain carbonyl O atoms.
The Crystal Structure of Human MRP14
electron density of four Chaps molecules was clearly observed in the asymmetric unit. Each Chaps molecule binds to a hydrophobic patch at the surface of the hinge region of hMRP14. In the asymmetric unit, two hMRP14 dimers are arranged by electrostatic interaction along the crystallographic b-axis. Along the a-axis, two hMRP14 molecules are stabilized by hydrophobic interaction between two Chaps molecules, and as a consequence of this, as shown in Figure 1(b), four dimers of hMRP14 and Chaps molecules are arranged in 222 symmetry in the asymmetric unit. The electron density corresponding to one monomer of DIM3 was poor. This poor density results in rather high temperature factors for this monomer (Table 3). Intra-monomer hydrophobic interactions; predicted target interaction site of hMRP14 The hMRP14 monomer has four a-helices, and spatial arrangements of these helices are maintained by a hydrophobic cluster that is formed in the interior of the monomer (Figure 4: residues colored in yellow). As shown in Figure 2, these hydrophobic residues (Phe19, Tyr22, Leu32, Phe37, Leu40, Val41, Phe48, Leu49, Val58, Ile59, Ile62, Met63, Leu66, Leu74, Phe79, and Leu82 in hMRP14) are highly conserved among the S100 family of proteins. Inspection of the three-dimensional structures of hMRP14 and other members of the S100 family such as calbindin D9 K,31 S100B,30,37 S100A10,33 S100A11,34 and S100A1235 shows that this intra-molecular hydrophobic cluster is partly exposed to solvent at the hinge region and the inter-helix area between helices H3 and H4, and the cluster forms a hydrophobic patch at the molecular surface. Recently, the three-dimensional structures of S100A10 and S100A11 proteins bound to their target molecule annexin fragment were reported (Figure 5(b) and (c)).33,34 In both cases, the target molecules are bound to this hydrophobic patch. These facts indicate the importance of this hydrophobic patch as a target-binding site for the S100 family of proteins. In the present analysis of hMRP14, Chaps molecules are bound to this hydrophobic patch formed among the hinge region and helices H3 and H4 (Figure 5(a)). The steroid group and hydrophobic carbon-tail of the Chaps molecule adhere by hydrophobic interactions to the exposed hydrophobic residues Leu45, Phe48, and Leu49 in the hinge region, Val58 and Ile 62 in helix H3, and Leu86 at the end of helix H4 of hMRP14 (Figure 5(a)). In the case of S100A11, the solventexposed hydrophobic residues Leu45 and Phe48 in the hinge region, Val57 and Met 61 in helix H3, and Leu85 in helix H4 interact with hydrophobic residues of annexin I (Figure 5(b)). In the case of S100A10, Phe38 and Phe41 in the hinge region and Leu78 in helix H4 are involved in target-binding by hydrophobic interactions (Figure 5(c)). In the complex structure of S100A10, Ala50 and Ile54,
The Crystal Structure of Human MRP14
269
Figure 4. Stereo representation of hydrophobic clusters in hMRP14 dimer, from the direction of the hinge region (viewed from the bottom in the direction of Figure 1(a)). The residue color is presented as in Figure 2. Dotted lines are the direction of the C-terminal regions.
which are other hydrophobic residues that correspond to Val58 and Ile62 in hMRP14 (Val57 and Met61 in S100A11), do not participate in targetbinding, though these hydrophobic residues are
also exposed and form the hydrophobic area at the molecular surface (Figure 5(c)). Inspection of the three-dimensional structures of S100 family proteins reveals that there is variety in this patch, both
Figure 5. Ribbon and surface representations of the ligand-binding site of (a) hMRP14, (b) S100A11 and (c) S100A10 in the hinge region. The color coordinates are as follows; green, ligand molecule; yellow, solvent-exposed conserved hydrophobic residues. The coordinates for S100A10 (1bt6) and S100A11 (1qls) were obtained from the Protein Data Bank.69 All of the protein surface presentations in this paper were generated by the program GRASP.70
270 in shape and in size. Figure 2 also shows the variety in the hinge region in terms of amino acid sequences. Members of the S100 family of proteins have their own target molecules. The differences observed in this region may explain the divergence of targets and the way in which the protein interacts with them. These results suggest that the hydrophobic patch formed among the hinge region and helices H3 and H4 of hMRP14 protein works as a target-binding site. The Chaps binding structure indicates the target interaction residues of hMRP14 protein, though the true ligand for hMRP14 has not been identi®ed yet. Comparison with other calgranulin proteins and its implications for the complex formation of MRP proteins MRP14 (S100A9) is a protein that is expressed in myeloid cells. There are two other S100 family proteins called MRP8 (S100A8) and S100A12 that are co-expressed in the cell with this protein. These proteins are also called calgranulin A (MRP8), calgranulin B (MRP14), and calgranulin C (S100A12), and are included in the calgranulin sub-family.4,40 The amino acid sequence similarities of human MRP8 (hMRP8) and human S100A12 (hS100A12) with hMRP14 are 40 % and 46 %, respectively. The crystal structure of homo-dimeric hMRP8 has been analyzed by our group,36 and that of hS100A12 has also been reported recently.35 All of the analyzed holo form S100 family proteins exhibit a similar three-dimensional structure,30 ± 34,37,38 and the structures of hMRP14, hMRP836 and hS100A1235 are the most similar to each other (Figure 6; the r.m.s. differences between hMRP14 and hMRP8, and hS100A12 for the 66 Ca atoms excluding the hinge Ê and 0.757 A Ê , respectively). The region are 0.884 A major structural differences are localized in the
The Crystal Structure of Human MRP14
hinge region and the C-terminal helix H4 (Figure 6). As discussed in the previous section, these areas are thought to form the target-binding sites for these proteins, and these structural differences may re¯ect the divergence in both the target molecules and in the area's interactions with their target molecules. Figure 7 shows electrostatic potential and hydrophobicity distributions on the surfaces of hMRP14, hMRP8 and hS100A12. As common features of these proteins, the negatively charged nature of EF-2 Ca2-binding loops caused by the conserved acidic residues Glu and Asp is obvious (Figure 7(a)). On the opposite side of EF-2 (Figure 7(b)), positive and negative charges are scattered over the whole surface with no obvious localization. On the other hand, there were also important differences among these three proteins. For example, the side of EF-2 exhibits signi®cant differences in charge and hydrophobicity distribution around their dimeric center and hinge regions. As shown in Figure 7(a), this side of hMRP14 is strongly charged negative. This localization of negative potential around the dimeric center is due to the exposed side-chains of acidic residues in helices H3 and H4. The corresponding area in hMRP8 has larger hydrophobic surfaces (Figure 7(c)) and exhibits a neutral potential (Figure 7(a)). Human S100A12 shows a tendency similar to that of hMRP14, but its dimeric center does not show a strong negative potential like that of hMRP14 (Figure 7(a)). Figure 7(a) and (c) also shows the obvious differences in surface condition in the hinge region among these three proteins. Human MRP8 has no hydrophobic patch in this region and is the most basic, while the other two calgranulins have large hydrophobic patches that can bind other hydrophobic molecules and exhibit a rather acidic nature. All of these differences
Figure 6. The main-chain superposition of the human calgranulin proteins. The Figure is drawn in the same direction as Figure 1(a). Human MRP14 (calgranulin B) dimer is shown in red and orange, hMRP8 (calgranulin A) monomer is shown in blue, and hS100A12 (calgranulin C) is shown in yellow. The coordinates for hMRP8 (1mr8) and hS100A12 (1e8a) were obtained from the Protein Data Bank.
The Crystal Structure of Human MRP14
271
Figure 7. Electrostatic potential ((a) and (b)) and hydrophobicity (c) representations on the surface of human calgranulin proteins. (a) View from the same direction as Figure 1(a). (b) View from the opposite direction of (a). Red represents a negative charge (acidic), and blue represents a positive charge (basic). (c) View from the same direction as (a), with the exposed hydrophobic surface drawn in yellow. The Chaps molecule that binds to the hydrophobic patch of hMRP14 is indicated by the green stick-model.
among hMRP14, hMRP8 and hS100A12 proteins are assumed to re¯ect the differences in the roles they play in exhibiting biological functions. From the structural point of view, the EF-2 side is likely to play an important role as an interaction interface with target molecules for these proteins. MRP14 and MRP8 form a stable hetero-dimer complex (MRP8/14).12,41,42 It has also been reported that the hetero-dimerization of these proteins occurs more easily than does homodimerization.43 The crystal structures of the homo-dimers of hMRP836 and hS100A1235 revealed that their dimerization is achieved by hydrophobic interactions between paired monomers. Dimerization of hMRP14 is also stabilized by a hydrophobic interaction between paired monomers. The N-terminal helix H1 and the Cterminal helix H4 are respectively arranged in an anti-parallel manner to form a dimer structure (Figure 1(a)). In the case of hMRP14, residues Leu8, Ile12, Ile15, and Ile16 at helix H1, Leu45 and Phe48 in the hinge region, and Phe76, Ile80, Met81, Met83 and Ala84 at helix H4 are involved in this hydrophobic interaction between two monomers, and maintain the dimeric structure (Figure 4: residues colored in pink). As shown in Figure 2, these hydrophobic residues that take part in this inter-monomer hydrophobic interaction are highly conserved among the S100 family of proteins, suggesting that these hydrophobic residues play a key role in the dimerization of these proteins. The three-
dimensional structures of other S100 family members also show that similar hydrophobic interactions commonly take part in their dimerization.30,32 ± 34,37,38 As shown in Figure 6, the structural similarity between hMRP14 and hMRP8 monomers is likely to allow their heterodimerization to occur in the same way as their homo-dimerization. The results of some antibody recognition experiments also support the proposal that the MRP8/14 complex is formed in such a way.44 The crystal structures of homo-dimeric hMRP14 and hMRP836 provide the possible features of this biologically important MRP8/14 molecule from the structural point of view. As mentioned above, notable differences between hMRP14 and hMRP8 monomers are obvious around the hinge region and dimeric center, so the hetero-dimeric molecule may exhibit a polar nature due to the obvious differences between the hMRP14 and hMRP8 monomers. In the hetero-dimer, half of the molecule is expected to be charged negative (hMRP14 monomer) and the other half positive (hMRP8 monomer). Not only the electrostatic potential, but also the hydrophobicity distribution on the surface is also expected to be different in the two halves of the dimer. This hetero-dimer molecule may have two different target-binding sites in its hingeregions, and those of hybrid characters may play the key role in exhibiting hetero-dimer-speci®c functions.
272 In contrast to these hMRP14 and hMRP8 proteins, hS100A12 does not form a complex with hMRP14 or hMRP8,42,45 though the hS100A12 monomer shows a higher similarity with hMRP14 than hMRP8 monomer both in terms of amino acid sequence (Figure 2) and three-dimensional structure (Figure 6). In this sense, it may be worth noting that hMRP14 and hMRP8 monomers choose each other exclusively as partners in dimer formation, while three calgranulin proteins are coexpressed and exhibit similar localization in the cell.42,45 These facts may suggest the existence of some special mechanisms for the selection of a partner in MRP14 and MRP8 proteins. The details of hetero-dimerization of these proteins are still unclear at present. Further study from both the biological and structural perspectives is required to elucidate the functions of this biologically important hetero-dimer of two MRPs at the molecular level. Long C-terminal tail; importance of its flexibility MRP14 protein is distinguished from other S100 family members by its long C-terminal region. In the present crystal, the C-terminal region has a poor electron density, suggesting that this region has an extensively ¯exible character. The poor electron density still allows the main-chain trajectory to be traced up to a point, and this tracing shows that the C-terminal region of hMRP14 makes a turn at the end of helix H4 and turns toward the solvent region of the inter-molecular space (Figures 1(a) and 4). As shown in Figure 1(b), the crystal of hMRP14 has a relatively large solvent area around the side of helix H4 of all of the dimers. Two Chaps molecules bridge two hMRP14 dimers, thus creating enough volume of free space to allow random conformation of this C terminus of each of the monomers without interfering with any neighboring molecules. The importance of this C-terminal tail has been reported as an effector region to target molecules other than MRP8,46,47 including acting as a Ca2binding ability inducing switch,20 a regulator of translocation of MRP8/14 complex,19 and a nuetrophil immobilization inducer.48 To perform such biological functions, the C-terminal ¯exible tail may adapt its conformation for various targets. In this sense, it is assumed that the ¯exibility of the C-terminal extended region of hMRP14 is functionally indispensable. The C-terminal region including helix H4 of S100 family members is assumed to be involved in the interaction with target molecules.33,34,49 ± 51 Three-dimensional structures of several S100 family members show structural differences in the C-terminal extension region between biologically equivalent monomers of hS100A6,38 hS100A7,32 hMRP8,36 and hS100A12.35 These structural differences may also represent the ¯exible nature of this region. Human MRP14 selectively activates the b2 integrin Mac-1 that is involved in the adhesion of neu-
The Crystal Structure of Human MRP14
trophil to the endothelial surface.26 This activation of Mac-1 is achieved only by the homo-dimer of hMRP14; the hetero-dimer of hMRP8 and hMRP14 does not activate Mac-1. In this activation, hMRP8 plays a regulatory role; it suppresses hMRP14's activation of Mac-1 through the formation of the hetero-dimer.26 As shown in Figure 2, the amino acid sequence of the hMRP14 C-terminal region (Ala89-Gly107) exhibits complete identity with the N-terminal region of NIF,29 an inhibitor which acts directly and irreversibly on neutrophils to prevent their random migration and chemotaxis in a noncytotoxical way.28,52 The homo-dimeric hMRP14 molecule has two NIF-identical regions, while the hMRP8/14 molecule has only one. Previous biological studies have shown that the region Arg85Met94 of hMRP14 is involved in hetero-dimer interaction between hMRP14 and hMRP8 monomers.44,46,47 This means that the former half of the unique NIF-identical sequence of the hMRP14 molecule will be involved in the interaction with hMRP8, and the region would be expected to lose its ability to interact with target molecules. Thus, hetero-dimerization is likely to inhibit free movement of the NIF-identical region of hMRP14. This may partly explain the regulation by hMRP8 of the activation of Mac-1 by hMRP14. To understand functional activities of the unique C-terminal tail region of hMRP14 in greater detail, further investigation including structural analysis of MRP8/14 hetero-dimer will be necessary.
Experimental Procedures Sample preparation and crystallization The full-length (1-114 amino acid residues) recombinant hMRP14 protein was prepared by the overexpression system using Escherichia coli. Sample preparation and crystallization of native protein have been reported.39 Se-Met-substituted hMRP14 was also prepared for the multiple wavelength anomalous diffraction method (MAD) following the previously described method with slight modi®cations. The best crystallization condition for the Se-Met-substituted hMRP14 was the same as that of native hMRP14. The dimensions of the Se-Met crystal were 0.05 mm 1.0 mm 0.4 mm, and the crystals were isomorphous with native ones. Se-Met-substituted crystal belongs to space group P21 with cell Ê , b 178.59 A Ê , c 61.24 A Ê, dimensions of a 57.62 A and b 113.45 , and an asymmetric unit contains four dimers of hMRP14. The crystal volume per protein mass Ê 3 Daÿ1, and the solvent content was (VM)53 was 2.7 A 54.8 %. To con®rm the molecular mass of the protein that was contained in the crystal, MALDI-TOF mass spectrometry analysis (Voyager DE Pro, PE biosystems) was performed on dissolved native and Se-Met-substituted crystals of hMRP14. Data collection All data collections were carried out using SPring-8 (Hyogo, Japan) under 100 K by ¯ash cooling after soaking in the cryo-protect solution with 30 % (v/v) glycerol. Native data were collected using a wavelength of
273
The Crystal Structure of Human MRP14 Table 1. Summary of data collection
Ê) Wavelength (A Ê) Resolution (A Space group Number of observed Reflections Unique reflections Completeness(%) Averaged redundancy Averaged I/s (I) Rmeas (%)b Rl (%)c
Se-MAD (peak)
native
(edge)
(remote)
0.7000 40 2.1 (2.21 2.10) P21
0.9794
0.9792 402.3 (2.422.30) P21
0.9000
251,869 (37,031) 65,554 (9,583) 99.7 (99.7) 3.8 (3.9) 6.5 (2.5) 6.6(33.9) -
239,355(20,212) 49,806(7,035) 99.4(99.4) 5.2(2.9) 11.3(2.9) 5.6(30.6) 6.9(14.5)
261,277(20,381) 49,818(7,043) 99.4(99.4) 5.2(2.9) 11.1(2.7) 6.3(32.0) 6.3(14.8)
284,544(40,391) 50,075(7,288) 99.9(99.9) 5.7(5.5) 10.5(2.4) 6.2(36.9) 0(0)
a
Values in parentheses are for the outermost resolution shell. Rmeas h[m/(m ÿ 1)]1/2jjhIih ÿ Ih,jj/hjIh,j, where hIih is the mean intensity of symmetry-equivalent re¯ections and m is redundancy. c Rl hjjFljj ÿ jFlojj/hjFloj, where Flj is the structure factor of the data collected at lj and Flo is the structure factor of the data collected at remote wavelength. b
Ê on beamline BL44B2. MAD data were collected 0.7000 A using three different wavelengths (0.9792, 0.9794 and Ê ) from a single Se-Met-substituted crystal on 0.9000 A beamline BL41XU. The data were processed using the program MOSFLM,54 and SCALA55 in the CCP4 program suite.56 Data-collection statistics are given in Table 1. Phase calculation The full-length hMRP14 protein has six Met residues per monomer in its amino acid sequence. The results of mass-spectrometry analysis suggested that the N-terminal Met of the recombinant hMRP14 did not exist due to the post-translational processing, so the Se-Met-substituted proteins had ®ve Se-Met residues per monomer. The asymmetric unit contains four dimers of hMRP14, each of which has ten Se-Met residues. Eight of 40 Se sites were found by the program SHELXS in the SHELX97 package.57 Initial phases with the eight sites were calculated, and 20 additional sites were located by the difference-Fourier methods using the SHARP program.58 The initial electron density map was obtained after phase improvement by non-crystallographic symmetry (NCS) averaging using the program SOLOMON.59 The phasing statistics are summarized in Table 2. Model building and refinement Based on the initial electron density map, 85 residues out of 114 from one hMRP14 monomer were built using the graphic program O,60 and the other monomer that comprises the dimer was generated by NCS operation. Three other hMRP14 dimers were also generated by NCS operation from the ®rst dimer model. A total of 28 carboxyl-terminal residues of each monomer were disordered and could not be identi®ed in the electron density map. After the ®rst round of re®nement, including rigidbody re®nement, positional re®nement and temperaturefactor re®nement with the program CNS61 using MAD Ê resolution, those models were re®ned data at 2.3 A Ê resolution. To improve the using native data up to 2.1 A electron density map of the C-terminal region, a density modi®cation technique with NCS averaging was applied using the program DM62 in the CCP4 program suite.
Simulated-annealing re®nement using slow-cooling protocols, positional re®nement, and temperature-factor Ê ) with re®nement were performed using CNS (10-2.1 A NCS restraints. A manual model ®tting was carried out between re®nement rounds using the program O. At ®rst, the NCS restraints were applied to main-chain atoms of all monomers. After several rounds of re®nement, NCS restraints were used for only main-chain atoms of secondary structures among the NCS equivalent monomers, and Chaps molecules were identi®ed from the 2Fo ÿ Fc and Fo ÿ Fc maps. The initial model, topology, and parameter ®les of Chaps for structure re®nement were obtained from the HIC-up,63 and its carbon-tail was elongated to ®t the electron density map according to the progress of re®nement. Water molecules were found using the CNS program. Further positional re®nement and temperature-factor re®nement were performed without NCS restraints. Analysis of protein geometry was performed using the program PROCHECK.64 The ®nal re®nement statistics are given in Table 3.
Table 2. Summary of phase calculation Se-Met hMRP14 crystal Ê) Resolution (A Space group MAD phasing RCullis_isoa Phasing power_isob Phasing power_anoc FOMd NCS averaging CCe R-factor
Remote 2.549 0.666
202.3 P21 Edge 0.300 6.020 2.417
Peak 0.392 5.625 3.335
0.793 35.2 %
a RCullis_iso is the mean residual lack of closure error divided by dispersive difference. Values are for centric re¯ections. b Phasing power_iso is the root mean square of FH/E, where FH is the dispersive difference of FH and E is the lack of closure error. c Phasing power_ano is as for Phasing power_iso except that FH is the anomalous difference of FH. d FOM is the mean ®gure of merit. e CC is standard linear correlation coef®cient between observed and calculated structure factor amplitudes.
274
The Crystal Structure of Human MRP14
Table 3. Final re®nement statistics Ê) Resolution range (A Number of reflections Completeness(%)
10.02.1 65,101 99.8
Total number of non-hydrogen atoms Protein Others Solvent R-factor (%)a R-free-factor (%)b r.m.s. deviation from standard values Ê) Bonds(A Bond angles (deg.) Dihedral angles (deg.) Ê 2) Average B-factor (A Ramachandran Residues in Residues in Residues in Residues in a b c
plotc most favored regions (%) additional allowed regions (%) generously allowed regions (%) disallowed regions (%)
5603 140 140 24.49 27.36 DIM1 0.008 1.278 19.36 39.4
DIM2 0.007 1.174 18.94 43.9
DIM3 0.006 1.047 18.08 59.2
DIM4 0.010 1.384 19.52 38.6
Total 0.008 1.228 18.99 47.7
89.4 10.6 0 0
89.7 10.3 0 0
84.1 15.9 0 0
91.8 8.2 0 0
88.8 11.2 0 0
R-factor jFobs ÿ Fcalj/ Fobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. R-free-factor value was calculated for R-factor, using only an unre®ned subset of re¯ections data (10 %). Ramachandran plot was calculated by PROCHECK.64
Atomic coordinates The atomic coordinates of hMRP14 have been deposited in the Protein Data Bank (ID code 1irj).
Acknowledgments We thank M. Kawamoto and S. Adachi of SPring-8 (Hyogo, Japan) for their kind help during data collection. This work was partly supported by the Research for the Future Program (JSPS-RFTF 97 L0051) from the Japan Society for the Promotion of Science. H.I. is a research fellow of the Japan Society for the Promotion of Science.
References 1. Kligman, D. & Hilt, D. C. (1988). The S100 protein family. Trends Biochem. Sci. 13, 437-443. 2. Donato, R. (1999). Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type. Biochim. Biophys. Acta, 1450, 191-231. 3. Odink, K., Cerletti, N., BruÈggen, J., Clerc, R. G., Tarcsay, L., Zwadlo, G. et al. (1987). Two calciumbinding proteins in in®ltrate macrophages of rheumatoid arthritis. Nature, 330, 80-82. 4. Kerkhoff, C., Klempt, M. & Sorg, C. (1998). Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9). Biochim. Biophys. Acta, 1448, 200-211. 5. Burmeister, G., Tarcsay, L. & Sorg, C. (1986). Generation and characterization of a monoclonal antibody (1C5) to human migration inhibitory factor (MIF). Immunobiology, 171, 461-474. 6. Lagasse, E. & Clerc, R. G. (1988). Cloing and expression of two human genes encoding calciumbinding proteins that are regulated during myeloid differentiation. Mol. Cell. Biol. 8, 2402-2410.
7. Zwadlo, G., BruÈggen, J., Gerhards, G., Schlegel, R. & Sorg, C. (1988). Two calcium-binding proteins associated with speci®c stages of myeloid cell differentiation are expressed by subsets of macrophages in in¯ammatory tissues. Clin. Exp. Immunol. 72, 510515. 8. Goebeler, M., Roth, J., Burwinkel, F., Vollmer, E., Bocker, W. & Sorg, C. (1994). Expression and complex formation of S100-like proteins MRP8 and MRP14 by macrophages during renal allograft rejection. Transplantation, 58, 355-361. 9. Rugtweit, J., Brandtzaeg, P., Halstensen, T. S., Fausa, O. & Scott, H. (1994). Increased macrophage subset in in¯ammatory bowel disease: apparent recruitment from peripheral blood monocytes. Gut, 35, 669-674. 10. Roth, J., Teigelkamp, S., Wilke, M., Grun, L., Tummler, B. & Sorg, C. (1992). Complex pattern of the myelo-monocytic differentiation antigens MRP8 and MRP14 during chronic airway in¯ammation. Immunobiology, 186, 304-314. 11. Brun, J. G., Jonsson, R. & Haga, H. J. (1994). Measurement of plasma calprotectin as an indicator of arthritis and disease activity in patients with in¯ammatory rheumatic diseases. J. Rheumatol. 21, 733-738. 12. Teigelkamp, S., Bhardwaj, R. S., Roth, J., MeinardusHager, G., Karas, M. & Sorg, C. (1991). Calciumdependent complex assembly of the myeloic differentiation proteins MRP-8 and MRP-14. J. Biol. Chem. 266, 13462-13467. 13. Murao, S., Collart, F. R. & Huberman, E. (1989). A protein containing the cystic ®brosis antigen is an inhibitor of protein kinases. J. Biol. Chem. 264, 83568360. 14. Siegenthaler, G., Roulin, K., Chatellard-Gruaz, D., Hotz, R., Saurat, J. H., Hellman, U. & Hagens, G. (1997). A heterocomplex, formed by the calciumbinding proteins MRP8 (S100A8) and MRP14
The Crystal Structure of Human MRP14
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26. 27.
(S100A9) binds unsaturated fatty acids with high af®nity. J. Biol. Chem. 272, 9371-9377. Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U. & Sorg, C. (1997). The heterodimer of the Ca2-binding proteins MRP8 and MRP14 binds arachidonic acid. FEBS Letters, 408, 81-84. Kerkhoff, C., Klempt, M., Kaever, V. & Sorg, C. (1999). The two calcium-binding proteins, S100A8 and S100A9, are involved in the methabolism of arachidonic acid in human neutrophils. J. Biol. Chem. 274, 32672-32679. Roulin, K., Hagens, G., Hotz, R., Saurat, J. H., Veerkamp, J. H. & Siegenthaler, G. (1999). The fatty acid-binding heterocomplex FA-p34 formed by S100A8 and S100A9 is the major fatty acid carrier in neutrophils and translocates from the cytosol to the membrane upon stimulation. Exp. Cell Res. 247, 410421. Kerkhoff, C., Sorg, C., Tandon, N. N. & Nacken, W. (2001). Interaction of S100A8/S100A9-arachidnic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells. Biochemistry, 40, 241-248. Roth, J., Burwinkel, F., van den Bos, C., Goebeler, M., Vollmer, E. & Sorg, C. (1993). MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to plasma membrane and intermediate ®laments in a calcium-dependent manner. Blood, 82, 1875-1883. van den Bos, C., Roth, J., Koch, H. G., Hartmann, M. & Sorg, C. (1996). Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca2-dependent translocation from cytoplasm to membranes and cytoskelton. J. Immunol. 156, 1247-1254. Sunderkotter, C., Beil, W., Roth, J. & Sorg, C. (1991). Cellular events associated with in¯ammatory angiogenesis in the mouse cornea. Am. J. Pathol. 138, 931939. Delabie, J., de Wolf-Peeters, C., van den Oord, J. J. & Desmet, V. J. (1990). Differential expression of the calcium-binding proteins MRP8 and MRP14 in granulomatous conditions: an immunohistochemical study. Clin. Exp. Immunol. 81, 123-126. Bhardwaj, R. S., Zotz, C., Zwadlo, G., Roth, J., Goebeler, M., Mahnke, K. et al. (1992). The calciumbinding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic in¯ammatory lesions. Eur. J. Immunol. 22, 1891-1897. Akiyama, H., Ikeda, K., Katoh, M., McGeer, E. G. & McGeer, P. L. (1994). Expression of MRP14, 27E10, interferon-alpha and leukocyte common antigen by reactive microglia in postmortem human brain tissue. J. Neuroimmunol. 50, 195-201. Aguiar-Passeti, T., Postol, E., Sorg, C. & Mariano, M. (1997). Epithelioid cells from foreign-body granuloma selectively express the calcium-binding protein MRP-14, a novel down-regulatory molecule of macrophage activation. J. Leuk. Biol. 62, 852-858. Newton, R. A. & Hogg, N. (1998). The human S100 protein MRP-14 is a novel activator of the b2 integrin Mac-1 on neutrophils. J. Immunol. 160, 1427-1435. Hessian, P. A., Wilkinson, L. & Hogg, N. (1995). The S100 family protein MRP-14 (S100A9) has homology with the contact domain of high molecular weight kininogen. FEBS Letters, 371, 271-275.
275 28. Watt, K. W., Brightman, I. L. & Goetzl, E. J. (1983). Isolation of two polypeptides comprising the neutrophil-immobilizing factor of human leucocytes. Immunobiology, 48, 79-86. 29. Freemont, P., Hogg, N. & Edgeworth, J. (1989). Sequence identity. Nature, 339, 516. 30. Matsumura, H., Shiba, T., Inoue, T., Harada, S. & Kai, Y. (1998). A novel mode of target recognition suggested by the 2.0 angstrom structure of holo S100B from bovine brain. Structure, 6, 233-241. 31. Szebenyi, D. M. & Moffat, K. (1986). The re®ned structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins. J. Biol. Chem. 261, 87618777. 32. Brodersen, D. E., Etzerodt, M., Madsen, P., Celis, J. E., Thùgersen, H. C., Nyborg, J. & Kjeldgaard, M. (1998). EF-hands at atmic resolution: the structure of human psoriasin (S100A7) solved by MAD phasing. Structure, 6, 477-489. 33. ReÂty, S., Sopkova, J., Renouard, M., Osterloh, D., Gerke, V., Tabaries, S. et al. (1999). The crystal structure of a complex pf p11 with the annexin II Nterminal peptide. Nature Struct. Biol. 6, 89-95. 34. ReÂty, S., Osterloh, D., Arie, J., Tabaries, S., Seeman, J., Russo-Marie, F. et al. (2000). Structural basis of the Ca2-dependent association between S100C (S100A11) and its target, the N-terminal part of annexin I. Structure, 8, 175-184. 35. Moroz, O. V., Antson, A. A., Murshudov, G. N., Maitland, N. J., Dodson, G. G., Wilson, K. S. et al. (2001). The three-dimensional structure of human S100A12. Acta Crystallog. sect. D, 57, 20-29. 36. Ishikawa, K., Nakagawa, A., Tanaka, I., Suzuki, M. & Nishihira, J. (2000). The structure of human MRP8, a member of the S100 calcium-binding protein family, by MAD phasing at 1.9 angstrom resolution. Acta Crystallog. sect. D, 56, 559-566. 37. Smith, S. P. & Shaw, G. S. (1998). A novel calciumsensitive switch revealed by the structure of human S100B in the calcium-bound form. Structure, 6, 211222. 38. Sastry, M., Ketchem, R. R., Crescenzi, O., Weber, C., Lubienski, M. J., Hidaka, H. & Chazin, W. J. (1998). The three-dimensional structure of Ca2-bound calcyclin:implications for Ca2-signal transduction by S100 proteins. Structure, 6, 223-231. 39. Itou, H., Fujita, I., Ishikawa, K., Yao, M., Watanabe, N., Suzuki, M. et al. (2001). Expression, puri®cation, crystallization, and preliminary X-ray diffraction analysis of human calcium-binding protein MRP14 (S100A9). Acta Crystallog. sect. D, 58, 1174-1176. 40. Kelly, S. E., Jones, D. B. & Fleming, S. (1989). Calgranulin expression in in¯ammatory dermatoses. J. Pathol. 49, 17-21. 41. Edgeworth, J., Gorman, M., Bennett, R., Freemont, P. & Hogg, N. (1991). Identi®cation of p8,14 as a highly abundant heterodimeric calcium binding protein complex of myeloid cells. J. Biol. Chem. 266, 7706-7713. 42. Vogl, T., ProÈpper, C., Hartmann, M., Strey, A., Strupat, K., van den Bos, C. et al. (1999). S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14. J. Biol. Chem. 274, 25291-25296. 43. Hunter, M. J. & Chazin, W. J. (1998). High level expression and dimer characterization of the S100
276
44. 45. 46.
47.
48.
49.
50.
51.
52. 53. 54. 55. 56. 57.
The Crystal Structure of Human MRP14
EF-hand proteins, migration inhibitory factor-related proteins 8 and 14. J. Biol. Chem. 273, 12427-12435. Hessian, P. A. & Lorryn, F. (2001). The heterodimeric complex pf MRP-8 (S100A8) and MRP-14 (S100A9). Eur. J. Biochem. 268, 353-363. Robinson, M. J. & Hogg, N. (2000). A comparison of human S100A12 with MRP-14 (S100A9). Biochem. Biophys. Res. Commun. 175, 865-870. ProÈpper, C., Huang, X., Roth, J., Sorg, C. & Nacken, W. (1999). Analysis of the MRP8-MRP14 proteinprotein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation. J. Biol. Chem. 274, 183-188. Nacken, W., Sopalla, C., ProÈpper, C., Sorg, C. & Kerkhoff, C. (2000). Biochemical characterization of the murine S100A9 (MRP14) protein suggests that it is functionally equivalent to its human counterpart despite its low degree of sequence homology. Eur. J. Biochem. 267, 560-565. Edgeworth, J., Freemont, P. & Hogg, N. (1989). Ionomycin-regulated phosphorylation of the myeloid calcium-binding protein p14. Nature, 342, 189-192. Osterloh, D., Ivanenkov, V. V. & Gerke, V. (1998). Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding but not for dimerization. Cell. Calcium. 24, 137-151. Kilby, P. M., Van Eldik, L. J. & Roberts, G. C. (1997). Identi®cation of the binding site on S100B protein for the actin capping protein CapZ. Protein Sci. 6, 2494-2503. Kube, E., Becker, T., Weber, K. & Gerke, V. (1992). Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin IIbinding site on p11, a member of the S100 protein family. J. Biol. Chem. 267, 14175-14182. Goetzl, E. J. & Austen, K. F. (1972). A neutrophilimmobilizing factor derived from human leukocytes. J. Exp. Med. 146, 1564-1580. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. Leslie, A. G. W. (1993). Auto-indexing of rotatin diffraction images and parameter re®nement. In Proc. CCP4 Study Weekend, pp. 44-51, . Evans, P. R. (1997). Scaling of MAD data. In Proc. CCP4 Study Weekend, pp. 97-102. Collaborative Computational Project No. 4. (1994). The CCP4 suite: programs for protein crystallography. Acta. Crystallog. sect. D, 50, 760-763. Sheldrick, G. M., Dauter, Z., Wilson, K. S., Hope, H. & Sieker, L. C. (1993). The application of direct methods and Patterson interpretation to high-resol-
58.
59.
60.
61.
62.
63. 64.
65. 66. 67.
68. 69. 70.
ution native protein data. Acta. Crystallog. sect. D, 49, 18-23. La Fortelle, E. & Bricogne, G. (1997). Maximum-likelihood heavy-atom parameter re®nement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472-494. Abrahams, J. P. & Leslie, A. G. W. (1996). Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta. Crystallog. sect. D, 52, 30-42. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta. Crystallog. sect. A, 47, 110-119. BruÈnger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, R. W. et al. (1998). Crystallography and NMR system (CNS): a new software system for macromolecularstructure determination. Acta. Crystallog. sect. D, 54, 905-921. Cowtain, K. D. & Main, P. (1996). Phase combination and cross validation in iterated densitymodi®cation calculations. Acta. Crystallog. sect. D, 52, 43-48. Kleywegt, G. J. & Jones, T. A. (1998). Databases in protein crystallography. Acta. Crystallog. sect. D, 54, 1119-1131. Laskowski, R. A., Mac Arthur, M. W., Moss, D. S. & Thornton, J. M. (1993). PROCHECK: a program to check stereochemical quality of protein structure. J. Appl. Crystallog. 26, 283-291. Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946-950. Merritt, E. A. & Bacon, D. J. (1997). Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505-524. Johnsson, N. & Weber, K. (1990). Alkylation of cysteine 82 of p11 abolishes the complex formation with the tyrosine-protein kinase substrate p36 (annexin 2, calpactin 2, lipocortin 2). J. Biol. Chem. 265, 14464-14468. Bairoch, A. & Apweiler, R. (2000). The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucl. Acids Res. 28, 45-48. Berman, H. M., Westblock, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H. et al. (2000). The Protein Data Bank. Nucl. Acids Res. 28, 235-242. Nicholls, A., Sharp, K. A. & Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. Struct. Funct. Genet. 11, 281-296.
Edited by R. Huber (Received 17 September 2001; received in revised form 3 December 2001; accepted 4 December 2001)