ARTICLE |
CORRESPONDENCE Haruo Saito: h-saito{at}ims.u-tokyo.ac.jp
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Abbreviations used: BNL, Brookhaven National Laboratory; D1, domain 1; D2, domain 2; ITAM, immunoreceptor tyrosine-based activation motif; LAR, leukocyte common antigenrelated; PTP, protein tyrosine phosphatase; RPTP, receptor-like PTP; Rsym, R factor for symmetry-related reflections; Sel-Met, selenomethionine.
Protein tyrosine phosphorylation is a key mechanism for nearly every aspect of cell regulation, ranging from cell survival and proliferation to apoptotic cell death in multicellular eukaryotes. Tyrosine phosphorylation itself is regulated by a concerted action of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). The human genome encodes as many as 38 classical PTPs, which share highly homologous PTP domains (1). These PTPs are divided into two major subfamilies: 17 nonreceptor (or cytoplasmic) PTPs and 21 receptor-like (transmembrane) PTPs. CD45, also known as the leukocyte common antigen, is the prototype of the receptor-like PTP (RPTP) subfamily and is found in all nucleated hematopoietic cells (24). CD45 is essential for development and antigen-induced activation of T and B cells (59). Mutations in the human CD45 gene have been associated with SCID and multiple sclerosis (1012). CD45 controls immune response, both positively and negatively, by dephosphorylating a number of signaling molecules, including Src family kinases (Lck and Fyn), the CD3
Like many other RPTPs, CD45 consists of an extracellular receptor-like region, a short transmembrane segment, and a cytoplasmic region comprising tandem PTP domains (see Fig. 1 A). The length of the extracellular segment varies among the CD45 isoforms generated by alternative splicing (15). Mutational analyses have shown that the membrane proximal PTP domain 1 (D1), but not the membrane distal domain 2 (D2), is catalytically active (16). The entire cytoplasmic region of CD45 is conserved among vertebrates, from shark to mammalian (17). The strong conservation of the CD45 D2 primary structure implies a functional role, but this role is not yet clearly defined. Nonetheless, there are observations that suggest a regulatory role of the catalytically inert D2 domain (18). D2 perhaps influences the activity of D1 by direct intermolecular and intramolecular interaction, as shown by in vitro binding studies (19, 20). Phosphorylation of D2 by casein kinase 2 enhances the phosphatase activity of D1, consistent with a regulatory role of the D2 domain (21). It was also reported that the CD45 D2 domain could, by itself, bind one of the important substrates, Lck, facilitating its dephosphorylation by D1 domain (22). In the case of another RPTP, leukocyte common antigenrelated (LAR) protein, we observed a high degree of similarity between the crystal structures of the D1 and D2 domains (23). Perhaps consistent with the structural similarity of D2 and D1, only two amino acid substitutions were required to convert the otherwise inert D2 domains of LAR and PTP into active enzymes (2325). These findings led us to propose that D2 might function as an auxiliary catalytic site that would be activated under specific cellular conditions or in the presence of a proper substrate (23).
A generalized model, known as the dimeric inhibition model or the wedge hypothesis, for the regulation of RPTP activity has been proposed based on the crystal structure of the RPTP D1 domain (26). The RPTP
D1 domain formed a homodimeric structure in the crystal, in which the active site of one monomer was blocked by an NH2-terminal helix-turn-helix wedge motif of the other monomer, suggesting that dimer formation on the membrane negatively regulates the PTP activity. Experimental data that seem to corroborate this model have been reported. For example, ligand-induced dimerization of the epidermal growth factor receptorCD45 chimeric molecule and enforced covalent dimerization of full-length PTP
resulted in down-regulation of their PTP activities (27, 28). Impairment of dimer formation, by introducing mutations into the wedge region, led to apparent PTP activation both in vivo and in vitro (2830). Transiently expressed PTP
was shown to exist predominantly as homodimers (31). In the same study, however, mutations in the wedge region reduced but did not completely abolish PTP
dimerization, suggesting that other factors are also contributing to the dimer formation.
Here, we report crystal structures of the CD45 cytoplasmic region containing both the D1 and D2 domains. We also determined crystal structures of the CD45 cytoplasmic region bound to phosphopeptide substrates; one bound to a nonspecific short phosphopeptide, and the other bound to the membrane-proximal immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 chain. We will describe the key features of CD45 and its substrate interactions and discuss the implications of these structures as they impact on our understanding of the mechanism of D1 and the potential function of D2. In these structures, CD45 exists as a monomer with the D1 active site cleft, as well as the D2 site, clearly unobstructed by the rest of the protein chain. The observed intramolecular orientation of the D1 and D2 domains precludes the formation of a dimer of the type predicted by the dimeric inhibition model. Such a D1 dimer interaction is impossible because of significant steric hindrance generated from the overlap of the attached D2 domains.
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Results |
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The best quality data have been collected from monoclinic crystals of selenomethionine (Sel-Met)substituted CD45-D1D2 (5971213) (C828S) protein with phosphotyrosine-containing peptides (derived from polyoma middle T antigen and CD3 ITAM motif; see below for more details). The crystal structures for both complexes were solved to 2.9 Å resolution by the molecular replacement method. Data collection and the final refinement statistics for these complexes are summarized in Table I. Both datasets are of similar quality with respect to resolution and agreement of R factor for symmetry-related reflections (Rsym). The corresponding protein structures demonstrate correct geometric parameters as well as the desired lack of model bias. The current CD45 model includes 589 residues (6011205), excluding the disordered acidic loop segment of D2.
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The first phosphopeptide (QQQQNQLpYNELNLGRREEpYDVLDKRRG, where pY is phosphotyrosine) is the membrane proximal ITAM of the CD3 chain (
ITAM-1) with both the tyrosine residues phosphorylated. CD3
is an important CD45 substrate and contains three ITAMs in its cytoplasmic domain. An ITAM is defined as a 2025amino acid sequence containing two YxxL/I segments separated by six to eight amino acid residues (36). Each of the three ITAMs in CD3
is tyrosine phosphorylated when TCR is activated. The doubly phosphorylated ITAMs serve as docking sites for the tandem SH2-containing ZAP-70, which initiates a cascade of signal transduction events (37, 38). It has been demonstrated that CD3
and CD45 physically associate in vivo and that CD45 efficiently dephosphorylates CD3
in vitro (39, 40). Mutational analyses of the tyrosine residues in the membrane-proximal ITAM of CD3
have shown that the two YxxL segments in the ITAM are functionally distinct (41), indicating the critical importance of regulating ITAM phosphorylation states.
The second phosphopeptide (NPTpYS) is derived from the polyoma middle T antigen. Although this peptide is probably not a physiologically meaningful substrate of CD45, it has the advantage of being a well-known short phospho-substrate.
When CD45 was crystallized in the presence of monomeric phosphotyrosine, there was a well-defined density corresponding to this moiety in the D1 active site pocket, but no comparable density in the D2 domain that would suggest the presence of phosphotyrosine (not depicted). In the crystal structure of the CD45 D1D2 complexed with ITAM-1, although there are two phosphotyrosyl residues in
ITAM-1, assignable extra densities were visible only at the D1 active site. No extra densities are discernible at the D2 active site, or in fact anywhere else associated with the protein. Electron densities for six amino acids surrounding the second phosphotyrosyl residue (REEpYDV) are visible at the D1 active site (Fig. 3, A and B). This result appears consistent with the previous in vitro analyses showing that the CD45 phosphatase prefers the second phosphotyrosine in
ITAM-1 over the first phosphotyrosine (42). Similarly, in the structure of CD45 bound with the polyoma phosphopeptide (NPTpYS), electron density corresponding to four residues (PTpYS) was visible only at the D1 active site (Fig. 4, A and B).
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Fig. 3 A shows detailed interactions between six amino acids (REEpYDV) of the ITAM-1 substrate and the CD45 protein. On the NH2-terminal side of the phosphotyrosyl residue, the Glu (pY-1) side chain is pointing in the direction opposite that of the phosphotyrosine. The Glu (pY-1) side chain is exposed to solvent without any interaction with the protein; thus, the presence of a hydrophilic residue at this position might be favored for binding. The carboxyl group of the Glu (pY-2) makes a hydrogen bond contact with the guanidium group of Arg734. The surface charge distribution of the CD45 protein at this region shows an extended positively charged surface contributed by residues Arg657, Arg704, Arg734, and Lys736 (Fig. 3, A and C). The negatively charged Glu (pY-2) fits nicely into this positively charged pocket and seems to be important for binding to the CD45 PTP. The positive charges of the protein at this region may repel the basic side chain of Arg (pY-3), thus allowing no direct interactions with the protein.
On the COOH-terminal side of ITAM-1, the Asp (pY+1) side chain points toward the CD45 protein and makes a hydrogen bond with the side chain of Gln872. The main chain backbone of the Val (pY+2) runs almost perpendicular to the rest of the peptides. The overall extent of these contacts between the
ITAM-1 substrate and the D1 domain is consistent with the previous results from PTP1Bsubstrate complexes (43), indicating that residues between pY-3 and pY+2 of the substrate determine phosphatase specificities.
In the structure of CD45 bound with the polyoma phosphopeptide (NPTpYS), electron densities corresponding to four residues (PTpYS) are visible at the D1 active site (Fig. 4). In addition to the interactions between the phosphotyrosine moiety and the protein, the NH2-terminal peptide residues make contact with the KNRY loop. Thr (pY-1) is in van der Waals contact with the side chain of Asp660, and the ring of Pro (pY-2) is making a van der Waals contact with the Tyr658 ring. The terminal oxygen of Ser (pY+1) is hydrogen bonded with the side chain of residue Gln872 in the Q-rich motif.
Based on these structures, we propose that the optimal interaction of CD45 active site and phosphotyrosyl substrate occurs when a hydrophilic residue occupies the pY-1 position and negatively charged amino acids are located around the pY-2 position. The Lck and Fyn protein tyrosine kinases, two of the best known CD45 substrates, fit into these criteria. These protein kinases have two tyrosine phosphorylation sites: one in their activation loops (DENEpYTAR in Lck and EDNEpYTAR in Fyn) and another near their COOH termini (TEGQpYQPQ in Lck and TEPQpYQPG in Fyn). All of these peptides contain hydrophilic residues at the pY-1 position, as in the ITAM-1 sequence. In the activation loop peptides, the acidic amino acid at pY-3 (and perhaps at pY-4) is likely to interact with the positively charged pocket of CD45. In the COOH-terminal peptides, we can more confidently speculate that the negatively charged Glu (pY-3) interacts with the positively charged pocket of CD45, because the residues at the Y-2 position, namely Gly and Pro, are optimal at generating conformations to accommodate such interactions. The NH2-terminal phosphorylation site of
ITAM-1 (NQLpYNE) lacks these criteria. The presence of the hydrophobic Leu residue at the pY-1 position, and lack of any negatively charged amino acids at the pY-2 and pY-3 positions, explain the reduced affinity of the NH2-terminal phosphorylation site for the CD45 PTP.
The CD45 D2 domain is a totally inactive phosphatase
The strong structural conservation of the CD45 D2 domain implies that it serves a cellular function, but it is still of major mystery. Our CD45 structure suggests that the function of the CD45 D2 domain is very different from its LAR counterpart. In the LAR D1D2 structure we determined previously, the inactive D2 domain maintains an active site topology similar to that of D1. With only two substitutions of the key residues in the surrounding loops, LAR D2 could be converted to a very active enzyme (23). In contrast, the PTP signature motif in CD45 D2 deviates from the consensus sequence to the extent that it can no longer accommodate a phosphoryl group (VHCSAGVGRTG in D1 vs. IHCRDGSQQTG in D2). As a result, the CD45 D2 domain has a significantly reduced number of available ligands for oxygen binding, a significantly altered shape of the active site pocket, as well as a reduced accessibility of the Cys nucleophile. Fig. 5, A and B, show the details of the D1 and D2 active sites, respectively. In catalytically active PTP structures, including the CD45 D1 domain, the side chain of the highly conserved Arg in the PTP motif makes two hydrogen bonds with the phosphoryl group and is very important for both substrate binding and transition state stabilization (43). The fatal substitution of this Arg to Gln in CD45 D2 (Gln1150) results in a shorter side chain that is unable to interact with a phosphoryl group in the same manner as Arg834 in D1. In addition, as shown in Fig. 5 B, the bulky side chains of Asp1146 and Gln1149 are protruding toward what would have been the phosphoryl group binding site. The N1 of Asp1146 is also making a hydrogen bond with a backbone amide group, blocking the potential binding partner of the phosphoryl group. Finally, the presence of the negative charge at Asp1146, together with the lack of a positively charged amino acid at 1150, must repel any incoming phosphoryl group. Thus, it is apparent that the deviations from the consensus at three positions (Asp1146, Gln1149, and Gln1150) will abolish any affinity for an incoming phosphoryl group. Indeed, despite a significant molar excess of the potential small molecule substrates present in the crystallization trials, there is no evidence of their presence in any of the CD45 D2 domains in any of the crystal forms obtained.
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Possible role of the CD45 D2 domain deduced from its structure
Inspection of the D2 structure, as detailed in this section, suggests that it has a role in substrate recruitment. The CD45 D2 domain has two unique features that are not found in any other PTPs, including LAR. Those are the two extra loops, referred to, respectively, as the acidic and basic loops, whose positions are shown in Fig. 2 by color. The 20amino acid acidic loop (SKESEHDSDESSDDDSDSEE [969988]), located between the ß1 and ß2 strands, is mostly disordered and flexible. Although not all of the residues in the acidic loops could be seen in the crystal structure, the location and approximate model can be visualized based on the rest of the structure and the electron density observed in this area. The 11amino acid basic loop (KNSSEGNKHHK [11261136]) is located between the 3 helix and the ß12 strand. The entire basic loop could be refined in one of the two molecules in the P2(1) space group, but the temperature factor for these residues is quite high (average main chain B factor is
20 Å2 higher than the rest of the molecule), reflecting the flexible nature of the basic loop.
It has been reported that deletion of the acidic loop, or substitution of four Ser residues to Ala in the acidic loop, diminished CD3-mediated activation of T lymphocytes (45). Furthermore, phosphorylation of Ser residues in the acidic loop by casein kinase 2 is important for CD45 functions (21). Our structure lends a mechanistic explanation to such observations. Both the acidic and basic loops in D2 are located on the same side of the surface of the molecule with respect to the D1 active site. Because both loops have significant charge biases and are located very close to the active site in D1, they could easily serve as initial substrate recruiting sites. When phosphorylated, these loops may undergo a conformational change or induce a change in the catalytic site to facilitate docking of incoming substrates.
Extensive interaction between the D1 and D2 domains
In this work, we obtained crystals of CD45-D1D2, without peptide, in two different space groups: P2(1) and P6(3)22. Although the crystallographic contacts between the neighboring molecules are totally different in the two crystal forms, the intramolecular spatial relationship between the D1 and D2 domains is essentially identical in both crystal forms. Several cocrystals of CD45-D1D2 with phosphopeptides also had very similar D1D2 domain orientation. If the atoms of the D1 domains from two different crystal forms, P2(1) and P6(3)22, are superimposed, the atoms in the D2 domains differ by an average (root-mean-square) distance of 3.2 Å between the equivalent C atoms. They differ by 2.9 Å between the two noncrystallographically related molecules in the P2(1) space group. The average distances are even smaller between the residues located at the interface of the D1 and D2 domains (
1.5 Å). In all cases, the D1 and D2 domains are connected by a four-residue linker, GETE (891894), and packed tightly against each other. This linking segment is conserved between CD45 and LAR (GETE and GHTE, respectively) and is bound to both D1 and D2 domains in a similar fashion in the two different proteins. The interdomain orientation between D1 and D2 is further stabilized by an extensive network of interactions, consisting of hydrogen bonds, van der Waals interactions, and salt bridges (Fig. 6). Hydrophobic interactions were observed at two regions of the interdomain surface. Phe890 at the end of D1
6 is making van der Waals contact with the Phe1173 in D2
5, and Pro765 (in the loop between ß9 and ß10 of D1) is in contact with Tyr1001 and Trp1002 (in the loop between ß2 and ß3 of D2). Hydrophobic interactions are further stabilized by salt bridges, observed between the basic side chains (His804, Lys808, Arg811, and Arg812) in D1
3 and acidic side chains (Glu1167 and Asp1171) in the D2 domain. Among these residues, Arg811 and Arg812 are highly conserved among most RPTP D1 domains, and Glu1167 and Asp1171 are conserved in most RPTP D2 domains. Overall, there are extensively complementary surfaces between the D1 and D2 domains, including the residues of the
3 and
6 helices of D1 and the
4 and
5 helices of D2.
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Because of these extensive D1D2 interactions, even subtle structural changes in D2 might affect the D1 conformation. Indeed, a mutation in D2 is known (Q1180G in mouse CD45, which is equivalent to Q1192G in our human structure) that nearly completely abolishes the D1 catalytic activity (48). Because Gln1192 is not making any direct contact with D1 residues, its effect must be propagated indirectly through the D1D2 interface. Exactly how this is effected will require the structural solution of the CD45 Q1192G mutant protein. Thus, stabilization of the D1 domain seems an important role of D2. We can hypothesize that the primordial two-domain RPTPs had two enzymatically active PTP domains, and over the history of RPTP evolution, D1 and D2 domains established extensive association to the extent that one requires another for maintenance of its proper structure. Thus, although the CD45 D2 domain has lost its catalytic activity, it may still be needed to support the D1 structure and activity.
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Discussion |
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Nonetheless, our present CD45 structure shows that the cytoplasmic region of CD45 does not dimerize even when highly concentrated, and the dimeric interaction as observed for RPTP D1 would be impossible given the D1D2 intramolecular domain orientation. The CD45 D1D2 interaction seems too tight to allow the deformation needed for D1D1 interaction, given the extensive interface and the essentially identical D1D2 orientation observed among different CD45 crystal forms and LAR. Furthermore, the CD45 wedge region (610634; refer to Fig. 2) is not involved in any intermolecular interaction. We previously reached a similar conclusion regarding lack of dimerization of the D1D2 domains based on the LAR structure (23). At that time, we could not know for sure whether what we observed for LAR represented a general phenomenon or merely an isolated case of that particular RPTP. However, now that we have solved the CD45 structure itself, we can convincingly conclude that the steric hindrance from the D2 domain prevents D1D1 dimer formation in CD45 as proposed previously (26).
Our data, therefore, indicate that the regulation of CD45 cannot be simply explained by the dimeric inhibition model mediated by the NH2-terminal wedge. Nonetheless, the wedge region (helix-turn-helix motif) is conserved, suggesting that it has a regulatory function (29, 30). If so, however, it is likely that the wedge region influences the CD45 function by a mechanism other than the dimer formation. Indeed, it was recently shown that CD45 could dimerize without the cytoplasmic domain (49). Furthermore, a CD45 wedge mutation that had significant effect on the CD45 function did not affect the dimerization. Obviously, more studies, including the structures of additional RPTPs as well as the extracellular portion of CD45, will be needed to further such understanding.
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Materials and Methods |
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Preparation of recombinant proteins
Proteins were expressed using the T7 expression system essentially as described previously (23, 50). The catalytic site mutant (C828S) of CD45-D1D2 (5971213) was tagged at the NH2 terminus with cleavable six-His, whereas the wild-type CD45-D1D2 (6031203) was tagged with noncleavable COOH-terminal six-His. The proteins were first purified using a Ni-affinity column (and in the case of the cleavable tag, six-His was removed by the TEV protease). The proteins were further purified using an anion exchange column. After desalting, the proteins were concentrated to 10 mg/ml. For expression of Sel-Metsubstituted protein, the methionine auxotrophic strain E. coli B834 (DE3) was used. The transformed cells were grown in the M9 medium supplemented with 40 mg/ml LSel-Met and 19 other amino acids. Purification was performed following the similar procedure as the native protein.
Crystallization
Two different crystal forms of CD45 D1D2 protein were obtained. Monoclinic crystals of CD45-D1D2 (5971213) C828S were grown by vapor diffusion in hanging drops in 100 mM sodium acetate, pH 5.0, 1013% (wt/vol) polyethylene glycol (PEG) 4000, 10 mM dithiothreitol (DTT), and 25% glycerol, with various additives such as ammonium sulfate, phosphotyrosine, or phosphopeptide. For cocrystallization experiments with phosphopeptides, either NPTpYS derived from polyoma middle T antigen or QQQQNQLpYNELNLGRREEpYDVLDKRRG, the membrane proximal ITAM of the CD3 chain (
ITAM-1), was added to the purified CD45-D1D2 (5971213) (C828S) protein so that the final concentration of the peptides would be
10 mM.
Hexagonal crystals of CD45-D1D2 (6031203) with noncleavable COOH-terminal six-His tag were grown by vapor diffusion in hanging drops in 100 mM MOPS/NaOH, pH 6.5, 1315% (wt/vol) PEG 8000, 0.5 M ammonium sulfate, 10 mM DTT, and 15% glycerol.
X-ray data collection
Crystals were flash-frozen with the crystallization solution. Diffraction data were collected at various synchrotron sources including the F1 station of the Cornell High Energy Synchrotron Source, the SBC-CAT of Advanced Photon Source in Argonne National Laboratory, and the X12 station of Brookhaven National Laboratory (BNL). Images were processed with DENZO, and data were scaled and processed with SCALEPACK (51).
Structure determination
Initially, the structure of CD45-D1D2 (5971213) C828S bound with sulfate ion was determined. In this crystal form, two molecules were expected in an asymmetric unit. The structure was determined by molecular replacement using an unaltered model of the D2 domain of RPTP LAR as a search model with the program AmoRe (52). An initial search revealed four unambiguous positions of phosphatase domains. After rigid body refinement, the identity of the PTP domains was determined based on the locations of the N and COOH termini, and amino acid sequence was changed to reflect the CD45 molecule. Several iterative cycles of refinement were performed using minimization and anisotropic temperature factor refinement options of the crystallography & NMR system (CNS) software packages, followed by manual rebuilding (53). Noncrystallographic symmetry restraints were used throughout refinement. Composite omit map in CNS was used for most of the refinement steps.
A refined model of the CD45-D1D2 (5971213) C828Ssulfate complex was then used to determine other crystal structures. A Sel-Met dataset was collected with the intention of confirming the refined structure. Because of the anisomorphism, the Sel-Met data could not be used for phase determination. Instead, an Fobs-Fcalc map was generated using the CD45-D1D2 (5971213) C828S model and Sel-Met data. The map showed strong (>3.5) corresponding densities at most of the Met positions, validating the structure. For structure determination of Sel-Met CD45-D1D2 (5971213) C828Sphosphopeptide complexes, a refined model of Sel-Met CD45-D1D2 (5971213) C828S was used to generate an Fobs-Fcalc map. For both phosphopeptides,
ITAM-1 and polyoma middle T antigen, initial maps revealed clear peptide density at each D1 active site. Refinement procedures include minimization and individual temperature factor refinement using the CNS software package.
In the hexagonal crystal of CD45-D1D2 (6031203), one molecule was expected in an asymmetric unit. For the structure determination, the refined model of the CD45-D1D2 (5971213) C828S was used for molecular replacement. The CD45 D1 and D2 domains were used either together or separately for the search. In both cases, the highest solutions were the same, and the relative orientation of the D1 and D2 domains was basically identical as in monoclinic crystals.
Calculation of the free energy required to separate the D1 and D2 domains
Environment-free energies of the CD45 D1D2 proteins have been calculated using the program ENVIRON (54). The atomic model of the protein was manually modified so that there were no interdomain interactions between the D1 and D2 domains, with the linker region still attached. The environment-free energies of the several modified models were calculated, and the energy required for separation of the D1 and D2 domains was calculated by subtracting the free energy of the native protein from that of the modified proteins.
Coordinates of the CD45/CD3 ITAM-1 and the CD45/polyoma middle T antigen complex have been deposited into the Protein Data Bank with the ID clones IYGR and IYGU, respectively.
Graphics
Molecular models were generated using the programs ALSCRIPT (55), MOLSCRIPT (56), RASTER3D (57), BOBSCRIPT (56, 58), and GRASP (59).
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Acknowledgments |
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This work was supported in part by a grant from National Institutes of Health (NIH; R01-GM53415) to H. Saito and C.A. Frederick and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H. Saito. H.-J. Nam was a recipient of an NIH training grant (T32-CA09361).
The authors have no conflicting financial interests.
Submitted: 13 September 2004
Accepted: 3 December 2004
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References |
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