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Genes to Cells (2007) 12, 1329-1338. doi:10.1111/j.1365-2443.2007.01137.x
© 2007 Blackwell Publishing or its licensors
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Structural basis of PSGL-1 binding to ERM proteins

 Yumiko Takai1, Ken Kitano1, Shin-ichi Terawaki1, Ryoko Maesaki1 and Toshio Hakoshima1,2,*

1 Structural Biology Laboratory, Nara Institute of Science and Technology, and
2 CREST, Japan Science and Technology Agency, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
P-selectin glycoprotein ligand-1 (PSGL-1), an adhesion molecule with O-glycosylated extracellular sialomucins, is involved in leukocyte inflammatory responses. On activation, ezrin–radixin–moesin (ERM) proteins mediate the redistribution of PSGL-1 on polarized cell surfaces to facilitate binding to target molecules. ERM proteins recognize a short binding motif, Motif-1, conserved in cytoplasmic tails of adhesion molecules, whereas PSGL-1 lacks Motif-1 residues important for binding to ERM proteins. The crystal structure of the complex between the radixin FERM domain and a PSGL-1 juxtamembrane peptide reveals that the peptide binds the groove of FERM subdomain C by forming a β-strand associated with strand β5C, followed by a loop flipped out towards the solvent. The Motif-1 310 helix present in the FERM–ICAM-2 complex is absent in PSGL-1 given the absence of a critical Motif-1 alanine residue, and PSGL-1 reduces its contact area with subdomain C. Non-conserved positions are occupied by large residues Met9 and His8, which stabilize peptide conformation and enhance groove binding. Non-conserved residues play an important role in compensating for loss of binding energy resulting from the absence of conserved residues important for binding.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesion molecule that belongs to the family of mucin-type molecules that possess heavily O-glycosylated extracellular sialomucins rich in serine and threonine. Leukocytes express PGSL-1 to mediate adhesion to endothelial cells and are critically involved in inflammatory responses both in brain and in peripheral tissues (McEver & Cummings 1997). Leukocytes emigrating from the bloodstream towards an inflammatory site follow different chemoattractant and chemokine signals that guide their path and induce both leukocyte activation and migration (Baggiolini 1998; Luster 1998). These include CXC chemoattractants and chemokines such as interleukin 8 (IL-8) that is released in response to inflammatory stimuli and induces the transmigration of neutrophils across the vascular endothelium. On activation, endothelial cells express P- and E-selectins that mediate the initial contact and rolling of neutrophils along the endothelium by direct binding to flowing neutrophils (Kansas 1996). These selectins bind to PSGL-1 sialylated and fucosylated oligosaccharides (Sako et al. 1993).

An early response of neutrophils to CXC stimuli involves transition from a spherical to a polarized morphology, which occurs immediately following cell movement (Shields & Haston 1985). On polarization, several adhesion molecules including PSGL-1 redistribute to the uropod, a cytoplasmic projection that forms at the rear end of the moving cell and is enriched in microvilli and microspikes (McFarland 1969; del Pozo et al. 1995; Nieto et al. 1998; Vicente-Manzanares. et al. 1998). Similarly, activation of neutrophils with platelet-activating factor initiates redistribution of PSGL-1 to the uropod and facilitates binding to platelets (Doré et al. 1996). The redistribution of PSGL-1 is mediated by direct interaction with members of the ezrin–moesin–radixin (ERM) proteins (Alonso-Lebrero et al. 2000; Serrador et al. 2002; Snapp et al. 2002).

The ERM proteins function as linking proteins between the plasma membrane and the actin cytoskeleton (Tsukita et al. 1997; Vaheri et al. 1997; Mangeat et al. 1999; Tsukita & Yonemura 1999) and are generally associated with cell surface protrusions such as microvilli, filopodia, microspikes, retraction fibers and membrane ruffling (Amieva & Furthmayr 1995; Berryman et al. 1995; Fath & Burgess 1995; Schwartz-Albiez et al. 1995). ERM proteins possess the N-terminal FERM (four point one, ERM) domain that mediates binding to PSGL-1 and other adhesion molecules. The FERM domain also binds intracellular proteins such as Na+/H+ exchanger regulatory factor (NHERF) and Rho GDP-dissociation inhibitor. Recent structural studies have revealed how the radixin FERM domain binds target proteins (Hamada et al. 2003; Terawaki et al. 2006). Mutation studies guided by the crystal structure of the radixin–ICAM-2 complex revealed that the juxtamembrane region of ICAM-2 contains the Motif-1 sequence motif, RXXTYXVXXA (where X represents any amino acid), for binding to the FERM domain (Hamada et al. 2003). Interestingly, PSGL-1 possesses the sequence RXXMYXVXXY, comprising a substitution of T with M and A with Y. The Ala residue of Motif-1 is essential for formation and docking of a 310-helix (VXXA) into the hydrophobic pocket of the FERM domain, whereas the bulky Tyr residue is unable to be accommodated in the pocket. The Thr residue of Motif-1 forms a hydrogen bond to Asn247 of subdomain C in the FERM–ICAM-2 complex. This hydrogen bond is not formed in PSGL-1. Moreover, PSGL-1 lacks a C-terminal basic region that is critical for strong binding of ICAM-2 to ERM proteins (Hamada et al. 2003) (Fig. 1). Detailed knowledge concerning how ERM proteins recognize the PGSL-1 cytoplasmic region remains unknown. Here, we report on the crystal structure of the radixin FERM domain complexed with a PGSL-1 cytoplasmic peptide. We reveal that the PGSL-1 cytoplasmic peptide binds subdomain C of the FERM domain in a similar manner to that displayed by Motif-1 binding, notwithstanding the absence of Motif-1 residues A and T.


Figure 1
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  		Figure 1  Comparison of FERM-binding sequences in PSGL-1 and ICAM-2. The juxtamembrane region of the cytoplasmic tail of PSGL-1 is compared with that of ICAM-2, a representative of the immunoglobulin superfamily of adhesion molecules that bind the FERM domain of ERM proteins. Basic and acidic residues are in blue and red, respectively. Compared with the ICAM-2 tail that displays three characteristic regions that include the N-terminal basic region, the middle nonpolar region and the C-terminal basic region, the PSGL-1 juxtamembrane region lacks the corresponding C-terminal basic region and instead contains acidic residues. PSGL-1-specific residues that are important in stabilizing conformation and binding are highlighted in green. The residue numbering for PSGL-1 follows that of ICAM-2. Part of the nonpolar region forms a short β-strand (residues 8–11; a red arrow) in our FERM–PSGL-1 complex. Of the 18 PSGL-1 peptide residues, 17 (residues 2–18; indicated with bold lines) were defined in the current electron density map. The secondary structure of the mouse ICAM-2 cytoplasmic tail is shown at the bottom of the alignment. Conserved Motif-1 residues (RXXTYXVXXA) present in ICAM-2 are highlighted in yellow and the most important residues for binding to the FERM domain are boxed. PSGL-1 lacks Thr and Ala of Motif-1.

 

   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Overall structure

The PSGL-1 peptide corresponding to the 18-residue juxtamembrane region from the mouse PSGL-1 cytoplasmic tail (residues 2–19 in Fig. 1) binds to the radixin FERM domain with a dissociation constant Kd of 201 nM, as determined from surface plasmon resonance measurements (Takai et al. 2007). The crystal structure of the FERM–PGSL-1 complex was determined by molecular replacement. The model of the FERM–PSGL-1 complex was refined to an R value of 23.2% for intensity data at 2.8 Å resolution.

Like the previously reported structures in the free, IP3-bound, ICAM-2-bound and NHERF-bound forms (Hamada et al. 2000, 2003; Terawaki et al. 2006), the radixin FERM domain in the PSGL-1-bound form consists of three subdomains, A, B and C (Fig. 2). The PSGL-1 peptide binds subdomain C, which is folded into a seven-stranded β-sandwich enclosing a hydrophobic core with the C-terminal {alpha}-helix packed between strands β5C and β1C. The structure of the radixin FERM domain in our complex resembles that of the free form rather than other forms: the PSGL-1-bound and free forms gave a small overall root mean square (r.m.s) deviation of 0.77 Å (Supplementary Fig. S1). Contrary to this small deviation, the PSGL-1- and ICAM-2-bound forms yielded a large overall r.m.s deviation of 1.28 Å. This deviation is caused by a difference in the torsion of the linker between subdomains A and B, which may result from crystal packing.


Figure 2
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  		Figure 2  Overall structure of the FERM–PSGL-1 complex. Ribbon representation of the radixin FERM domain bound to the PSGL-1 peptide (cyan). The radixin FERM domain consists of subdomains A (wheat), B (light blue) and C (pale green). Linkers A–B (residues 83–95), B–C (residues 196–203) and the C-terminal linker are colored gray. The asymmetric unit of the crystal contains two complexes with no significant structural differences between the two complexes. We show here and discuss the complex structure using molecule B bound to molecule D.

 
Structural similarity among the subdomains was much higher. In particular, the structure of subdomain C bound to PSGL-1 was very similar to that of the ICAM-2-bound form (with r.m.s. deviation of 0.48 Å), whereas the r.m.s. deviation of the free form was relatively large (0.84 Å). This clearly suggests that induced-fit structural changes in subdomain C occur with PSGL-1 binding, as previously observed with ICAM-2 binding (Hamada et al. 2003).

Peptide structure

The juxtamembrane region of the PSGL-1 cytoplasmic tail contains two characteristic regions, the N-terminal basic and nonpolar regions (Fig. 1). This characteristic feature of the tail is distinct from ICAM-2 which possesses an additional C-terminal basic region (Hamada et al. 2003). In PSGL-1, the region corresponding to the ICAM-2 C-terminal basic region contains acidic and hydrophobic residues. At present, the PSGL-1 peptide model includes 17 residues (2–18) out of the 18. The N-terminal basic region of the PSGL-1 peptide (residues 2–7) forms a loop followed by a short β-strand comprising the N-terminal half of the nonpolar region (residues 8–11). The C-terminal half of the nonpolar region of PSGL-1 forms a loop, which is exposed to solvent. This displays marked contrast with the ICAM-2 peptide that forms a 310-helix following a short β-strand (Hamada et al. 2003) as described below.

Peptide recognition

The PSGL-1 peptide binds subdomain C by docking into the hydrophobic groove formed by helix {alpha}1C and strand β5C in subdomain C (Fig. 3A). The binding site of PSGL-1 in the radixin FERM domain overlaps with that of ICAM-2 (Fig. 3B). Like ICAM-2, the PSGL-1 β-strand forms an antiparallel β–β association with strand β5C from subdomain C. The association is mediated by four main chain–main chain hydrogen bonds and two additional hydrogen bonds between the loop and strand β5C (Fig. 4A). These hydrogen bonding interactions correspond well with those present in the FERM–ICAM-2 complex, whereas the ICAM-2 peptide forms two additional main chain–main chain hydrogen bonds, one at the N-terminal and the other at the C-terminal flanking regions of the β-strand (Fig. 4B). The former is caused by a difference in the N-terminal loop conformation and the latter by the absence or presence of the 310-helix following the β-strand as described below.


Figure 3
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  		Figure 3  PSGL-1 peptide binding to subdomain C of the radixin FERM domain. (A) Surface electrostatic potential of subdomain C with a stick model of the bound PSGL-1 peptide (cyan). Positive (blue) and negative (red) potentials are mapped on the van der Waals surface. The PSGL-1 peptide binds along the hydrophobic groove of subdomain C. The hydrophobic pocket of subdomain C is indicated with a broken line circle. (B) Surface electrostatic potential of subdomain C with bound ICAM-2 peptide (yellow) in the FERM–ICAM-2 complex for comparison with (A). (C) Comparison of peptide conformations in the FERM–PSGL-1 and FERM–ICAM-2 complexes. The PSGL-1 peptide (cyan) is superimposed on the ICAM-2 peptide (yellow) bound to subdomain C (gray). Side chains that represent key residues for FERM binding are shown as stick models. Two arrows (pink) indicate the shift of the PSGL-1 peptide towards strand β5C.

 

Figure 4
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  		Figure 4  Comparison of main chain–main chain interactions in the FERM–PSGL-1 and the FERM–ICAM-2 complexes. The main chain–main chain hydrogen bonds are shown as red broken lines. (A) Main chain–main chain interactions between the PSGL-1 peptide (cyan) and strand β5C of subdomain C. (B) Main chain–main chain interactions between the ICAM-2 peptide (yellow) and strand β5C of subdomain C.

 
Nine residues of the PSGL-1 peptide encompassing the nonpolar region directly contact subdomain C. Like ICAM-2, the conserved representative nonpolar residues Tyr10 and Val12 of PSGL-1 dock into a large hydrophobic pocket formed by Ile245, Ile248, Leu281, His288 and Met285 (Fig. 5). These two PSGL-1 residues correspond to those present in ICAM-2 (Fig. 3C). Moreover, the Tyr10 side chain of both PSGL-1 and ICAM-2 form a similar hydrogen bond to His288 (Fig. 5).


Figure 5
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  		Figure 5  Comparison of side chain–side chain interactions in the FERM–PSGL-1 and the FERM–ICAM-2 complexes. (A) Side chain–side chain interactions between the PSGL-1 peptide (cyan) and strand β5C of subdomain C. Two arrows (pink) indicate the shift of the PSGL-1 peptide towards strand β5C. (B) Side chain–side chain interactions between the ICAM-2 peptide (yellow) and strand β5C of subdomain C.

 
In addition to these interactions, ICAM-2 forms a 310-helix (residues 12–15) that docks into the hydrophobic pocket and places the side chain methyl group of Ala15 into the pocket (Fig. 3B). PSGL-1 possesses Tyr15 in lieu of Ala15 in ICAM-2, and the aromatic side chain is too large to dock into the pocket. Consequently, the PSGL-1 peptide region following the β-strand is that of a loop flipped out towards the solvent. Thus, in the FERM–ICAM-2 complex, the hydrophobic pocket of subdomain C is packed well within ICAM-2 side chains, whereas that in the FERM–PSGL-1 complex constitutes an unoccupied space. This causes a significant difference in the buried accessible surface area: the PSGL-1 peptide buries 1240 Å2 of the total accessible surface area, which is smaller than that associated with the ICAM-2 peptide (1761 Å2). This difference is correlated with the difference in binding affinity: the ICAM-2 peptide exhibits strong binding to the FERM domain with a Kd value of 16.4 nM.

Unlike ICAM-2, PSGL-1 possesses two large side chains, His8 and Met9, at positions corresponding to the smaller residues Gly8 and Thr9 of ICAM-2. The large nonpolar side chain of Met9 makes contact with two residues (Ser249 and Asn247) from strand β5C but also packs onto PSGL-1 Pro11 and Tyr15, which may stabilize peptide conformation and binding to the groove of subdomain C. Indeed, mutation studies showed that Met9 is a key residue for FERM binding (Fig. 6). These interactions seem to cause a small movement of the peptide chain towards strand β5C (Figs 3C and 5A). In the FERM–ICAM-2 complex, Thr9 of ICAM-2 forms a hydrogen bond with Asn247 from strand β5C, which may help to maintain distance between the ICAM-2 peptide and strand β5C, and thus prevents movement of the peptide towards strand β5C in the complex. PSGL-1 peptide movement enables formation of a hydrogen bond between Thr7 and Asn251 from strand β5C (Fig. 5A). This hydrogen bond contributes to the binding affinity (Fig. 6). In the FERM–ICAM-2 complex, Thr7 of ICAM-2 fails to form the corresponding hydrogen bond with Asn251 given the greater distance between these two residues. Instead, N-terminal Arg6 of ICAM-2 forms hydrogen bonds with Asp252 from strand β5C, an interaction absent in PSGL-1. In our complex, the side chain of Lys6 is not positioned towards Asp 252 (Fig. 3A). This basic residue, however, also contributes to the binding affinity (Fig. 6). The long side chain of this solvent-exposed residue is likely to be in a more dynamic state in solution and thus is able to contribute to the binding affinity through electrostatic interactions with Asp252 from strand β5C. The position of Arg5 in the PSGL-1 peptide is more than likely too close to Asp252 to allow for proper orientation of the long side chain for hydrogen bond formation. However, these two charged residues form a salt bridge, which is supported by mutation studies that indicated a contribution by Arg5 to the binding affinity (Fig. 6). Peptide chain movement enables the aromatic side chain of His8 to stack onto Phe250 from strand β5C of subdomain C, although mutation of this residue to alanine failed to produce a significant reduction in the binding affinity. It is likely that replacement with alanine is insufficient to abolish nonpolar interaction with Phe250 of strand β5C.


Figure 6
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  		Figure 6  Interaction between FERM domains and PSGL-1 peptides. Pull-down assay of the radixin FERM domain with biotinylated 31-residue peptides of wild-type (WT) and mutated mouse PSGL-1 was performed. The mutated peptides contain alanine mutations of Arg5 (R5A), Lys6 (K6A), Thr7 (T7A), His8 (H8A) and Met9 (M9A). The sequence of the WT peptide is shown in Fig. 1.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
PSGL-1 activation

Leukocyte rolling on P-selectin through PSGL-1 requires ERM protein binding to the PSGL-1 cytoplasmic tail. Transfected HL60 and K562 cells expressing full-length PSGL-1 rolled well on P-selectin. In contrast, rolling was almost completely absent in cells transfected with PSGL-1 that lacked the cytoplasmic tail (Snapp et al. 2002). Unlike freshly isolated human neutrophils (Moore et al. 1995), however, full-length PSGL-1 on K562 transfectants showed random distribution patterns, with large numbers of PSGL-1 molecules on both the microvilli and the planar surface of the cell. Therefore, functional differences between full-length and truncated PSGL-1 expressed on these cells cannot be explained by differences in the subcellular localization of the mutant. Binding of ERM proteins to the PSGL-1 cytoplasmic tail may induce conformational changes in the extracellular domain that effect binding to P-selectins on cells. Interestingly, PSGL-1 is expressed as a homodimer and possesses a disulfide bond at the extracellular juxtamembrane region (McEver & Cummings 1997). It is tempting to hypothesize that PSGL-1 may be activated by binding to ERM proteins through a mechanism similar to that proposed for integrin activation, in which binding of the talin FERM domain to the integrin β3 tail causes a change in the position of the transmembrane helix and a packing mismatch with the {alpha}IIb-transmembrane helix, followed by separation or reorientation of the integrin tails and subsequent activation (Wegener et al. 2007). Importantly, cells expressing truncated PSGL-1 were able to bind soluble P-selectin, indicating that the P-selectin binding site on truncated PSGL-1 transfectants was intact (Snapp et al. 2002). Therefore, PSGL-1 bound to ERM proteins should effect conformational changes that facilitate binding to P-selectins on rather flat cell surfaces. However, further experimentation is required to confirm this PSGL-1 activation hypothesis.

In addition to the ‘inside–out’ signal transduction involved in activation, the ‘outside–in’ signal transduction by PSGL-1 needs to be considered. Macrophage adherence promotes signaling through a pathway involving PSGL-1, Akt and mTOR, which results in the synthesis of ROCK-1 (Fox et al. 2007). Since ROCK-1 is a protein kinase that activates ERM proteins by phosphorylation of the C-terminal tail domain, the pathway forms a positive feedback circuit that facilitates attachment of PSGL-1 to its target. While PSGL-1 is central to the trafficking of immune effector cells to areas of inflammation through direct interaction with selectins, PSGL-1 is also required for efficient homing of resting T cells to secondary lymphoid organs by mediating an enhanced chemotactic T cell response to secondary lymphoid organ chemokines CCL21 and CCL19 or to inflammatory chemokines (Veerman et al. 2007). Many adhesion molecules including CD44, syndecan and {alpha}v integrin (Kajita et al. 2001; Deryugina et al. 2002; Endo et al. 2003) possess extracellular domains that are segmented by matrix metalloproteinases (MMPs) at the juxtamembrane, and followed by events where the cytoplasmic tails are segmented by presenilin (PS)-dependent {gamma}-secretase (Itoh & Seiki 2006). CD43 is thought to be subjected to a regulated intramembrane proteolysis (RIP) pathway, while the involvement of MMP and secretase remains speculative (Andersson et al. 2004, 2005). Recently, PSGL-1 has been shown to be a substrate for the aspartyl protease BACE1, which cleaves the juxtamembrane region within the extracellular ectodomain and generates a soluble ectodomain and C-terminal transmembrane fragment (Lichtenthaler et al. 2003). However, there is no evidence to date for nuclear transport of the cytoplasmic tails. Importantly, CD44 and CD43 possess a bipartite nuclear localization signal (NLS) signal at the juxtamembrane region, which contains two clusters of positively charged amino acids typically separated by ca. 10 residues. In contrast to these adhesion molecules, there is no candidate for the NLS sequence in the PSGL-1 cytoplasmic tail. PSGL-1 may send signals by forming complexes with various cytoplasmic proteins, possibly through a mechanism involving tyrosine phosphorylation (Hidari et al. 1997; Ba et al. 2005).

Peptide recognition by the FERM domain

The FERM domain of ERM proteins represents a versatile protein module that is being shown to bind an ever-growing number of target proteins including adhesion molecules, ion channels and cytoplasmic proteins such as adaptor proteins (Tsukita et al. 1997; Tsukita. & Yonemura 1999). There are two distinct sequence motifs that have been shown to bind the FERM domain: Motif-1, identified in the ICAM-2 juxtamembrane region (Hamada et al. 2003), and Motif-2, MDWXXXXX(L/I)FXX(L/F), identified in the C-terminal region of NHERF-1 and -2 (Terawaki et al. 2006). The FERM-binding region of PSGL-1 is similar to Motif-1, although PSGL-1 lacks two conserved residues (T and A) present in Motif-1. This diversity is in sharp contrast to adhesion molecules of the immunoglobulin superfamily such as ICAM-1, -2 and VCAM-1, which bind strongly and possess strictly conserved determinant residues (Hamada et al. 2003). It is somewhat surprising that PSGL-1 exhibits relatively strong binding to the FERM domain, although it lacks both the 310-helix following the β-strand and the C-terminal flanking basic region. In the case of ICAM-2, truncation of the C-terminal flanking basic region resulted in a ninefold reduction in binding affinity (in terms of Kd), while disruption of the 310-helix by replacing Y with A resulted in an 11-fold reduction. Thus, assuming that these free energy changes are additive, ca. a 100-fold reduction in the binding affinity of the PSGL-1 peptide compared with that of ICAM-2 was expected given the differences in the peptide sequences. Notwithstanding this expectation, the PSGL-1 peptide displays a relatively small reduction (ca. tenfold) in the binding affinity (in terms of Kd). This unexpectedly strong affinity of the PSGL-1 peptide can be accounted for by considering the additional interactions involving the non-conserved residues (His8 and Met9) and the presence of inter-chain interactions (Met9, Pro10 and Tyr11) that stabilize the conformation. These interactions seem to compensate for the loss in binding energy associated with the absence of the 310-helix that should dock into the hydrophobic pocket. Moreover, the newly formed Thr7–Asn251 hydrogen bond in the FERM–PSGL-1 complex compensates for the absence of the corresponding Thr9–Asn247 interaction in the FERM–ICAM-2 complex. These mechanisms by which organisms preserve variation in protein sequences are reminiscent of suppressor mutations that suppress the phenotypic effect of other mutations in genetics, so that double or multiple mutations appear normal.

Key residues of the radixin FERM domain involved in direct interactions with PSGL-1 are conserved in all ERM protein members and merlin, suggesting that PSGL-1 binding to other ERM members would essentially reflect the same form as that shown for the radixin FERM domain.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Crystallization and data collection

The peptide PSGL-1 regions selected for crystallization were determined according to a previous study, which showed that the juxtamembrane region encompassing the N-terminal 18 residues out of 69 residues of the full-length cytoplasmic peptide were critical for ERM binding (Serrador et al. 2002). The 18-residue PSGL-1 peptide used in this study corresponds to the juxtamembrane region of the mouse PSGL-1 cytoplasmic tail (331 RLSRKTHMYPVRNYSPTE 348). For convenience, residues are numbered according to that employed for ICAM-2. In this scheme, our PSGL-1 peptide encompasses residues 2–19 (Fig. 1). Crystallization and data collection of the FERM–PSGL-1 complex were performed as previously described (Takai et al. 2007). In brief, 1 mM purified radixin FERM domain was mixed with 10 mM of the 18-residue PSGL-1 peptide at 1 : 1 volume ratio (mol ratio, protein : peptide = 1 : 10). The final protein concentration was 31 mg/mL. Crystals with bound PSGL-1 were obtained from a solution containing 100 mM Tris–HCl (pH 8.2) and 8% polyethylene glycol 8000 (PEG8K) at 4 °C. FERM–PSGL-1 complex crystals were found to belong to space group P212121, with unit-cell parameters a = 80.74, b = 85.73 and c = 117.75 Å. All diffraction data were collected from crystals flash-frozen to 100 K following cryoprotection in a solution containing 8% PEG8K, 100 mM Tris buffer and 20% PEG400. Data sets were collected at beam line BL41XU at the SPring-8 synchrotron facility, Harima, Japan. The wavelength was set to 1.00 Å with a crystal-to-detector distance of 170 mm. Data were collected from two crystals with angular ranges of 180° and step sizes of 1° with an exposure time of 7 s. Two data sets were merged into one set for structure determination (Table 1).


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  		Table 1  Crystallographic analysis of the radixin FERM–PSGL-1 complex
 
Structure determination

The crystal structure of the FERM–PGSL-1 complex was determined by molecular replacement using the program MOLREP in the CCP4 program suite (CCP4 1994) with the ICAM-2-bound structure of the radixin FERM domain (PDB accession code 1J19) as a search model. Structural refinement was performed with CNS (Jones et al. 1991; Brünger et al. 1998) using a rigid-body, simulated annealing and individual B factor refinement. The resultant initial map showed clear electron densities for most of the FERM domain and the peptide. An initial model of the peptide was built into the electron density map using the graphics program O (Jones et al. 1991) in addition to rebuilding part of the FERM domain. Following several cycles of rebuilding and refinement, the model of the FERM–PSGL-1 complex was refined to an R value of 23.2% for intensity data at 2.8 Å resolution. The asymmetric unit of the crystal contains two complexes with no significant structural differences between the two complexes. We discuss the complex structure using molecule B (FERM) bound to molecule D (PSGL-1). At present, the PSGL-1 peptide model contains 17 residues (residues 2–18) out of 18 (Supplementary Fig. S2). We observed residual density around the crystallographic twofold axis close to strand β2A of molecule A, whereas we could not fully interpret the density at the present resolution. Probably, the residual density is produced by disordered molecule(s) such as the peptide or other organic compounds from the crystallization solution. The refinement statistics are summarized in Table 1. There were no residues in disallowed main-chain torsion angle regions as determined by PROCHECK (Laskowski et al. 1993). Figures were generated using PYMOL <http://www.pymol.org>. Atomic coordinates and structure factors (code 2EMT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ <http://www.rcsb.org/>.

Binding assay

Pull-down assays to detect peptide–protein interactions were performed using streptavidin-conjugated resin coupled with biotinylated PSGL-1 polypeptides. Following addition of the sample solution (50 µL) containing the radixin FERM domain (100 µM) to the bead suspension, the beads were incubated for 30 min at room temperature, washed 2 times with a buffer solution containing 10 mM HEPES (pH 7.5), 70 mM KCl and 1 mM DTT, and then centrifuged. The amount of biotinylated PSGL-1 peptide bound to the beads was monitored by SDS-PAGE. An appropriate amount of bead slurry containing the same amount of biotinylated PSGL-1 peptide was also subjected to SDS-PAGE. Proteins were stained using Coomassie Blue.


   Acknowledgements
 
We would like to thank J. Tsukamoto for technical support in performing the MALDI-TOF MS analysis and S. Sakurai for calculation of the rotation function. We gratefully acknowledge Sachiko Tsukita and Shoichiro Tsukita for providing mouse radixin cDNA. This work was supported in part by a Protein 3000 project on Signal Transduction from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to T. H.). S. T. was supported by a Center of Excellence (COE) postdoctoral research fellowship of a Grant-in-Aid for the 21st Century COE Research from MEXT. R. M. was supported by a postdoctoral research fellowship for Young Scientists from the Japan Society for the Promotion of Science.


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: hakosima{at}bs.naist.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 20 August 2007




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