Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation.

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

MT1-MMP recognition by ERM proteins and its implication in CD44 shedding.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a key enzyme involved in tumor cell invasion by shedding their cell-surface receptor CD44 anchored with F-actin through ezrin/radixin/moesin (ERM) proteins. We found the cytoplasmic tail of MT1-MMP directly binds the FERM domain of radixin, suggesting F-actin-based recruitment of MT1-MMP to CD44 for invasion. Our crystal structure shows that the central region of the MT1-MMP cytoplasmic tail binds subdomain A of the FERM domain, and makes an antiparallel ß-ß interaction with ß2A-strand. This binding mode is distinct from the previously determined binding mode of CD44 to subdomain C. We showed that radixin simultaneously binds both MT1-MMP and CD44, indicating ERM protein-mediated colocalization of MT1-MMP and its substrate CD44 and anchoring to F-actin. Our study implies that ERM proteins contribute toward accelerated CD44 shedding by MT1-MMP through ERM protein-mediated interactions between their cytoplasmic tails.

Protein stabilizer, NDSB-195, enhances the dynamics of the ß4 -α2 loop of ubiquitin.

Non-detergent sulfobetaines (NDSBs) are a new group of small, synthetic protein stabilizers, which have advantages over classical compatible osmolytes, such as polyol, amines, and amino acids: they do not increase solution viscosity, unlike polyols, and they are zwitterionic at all pH ranges, unlike amines and amino acids. NDSBs also facilitate the crystallization and refolding of proteins. The mechanism whereby NDSBs exhibit such activities, however, remains elusive. To gain insight into this mechanism, we studied, using nuclear magnetic resonance (NMR), the effects of dimethylethylammonium propane sulfonate (NDSB-195) on the dynamics of ubiquitin, on which a wealth of information has been accumulated. By analyzing the line width of amide proton resonances and the transverse relaxation rates of nitrogen atoms, we found that NDSB-195 enhances the microsecond-millisecond dynamics of a ß4 -α2 loop of ubiquitin. Although those compounds that enhance protein dynamics are generally considered to destabilize protein molecules, NDSB-195 enhanced the stability of ubiquitin against guanidium chloride denaturation. Thus, the simultaneous enhancement of stability and flexibility by a single compound can be attained.

Structural basis for cargo binding and autoinhibition of Bicaudal-D1 by a parallel coiled-coil with homotypic registry.

Bicaudal-D1 (BICD1) is an α-helical coiled-coil protein mediating the attachment of specific cargo to cytoplasmic dynein. It plays an essential role in minus end-directed intracellular transport along microtubules. The third C-terminal coiled-coil region of BICD1 (BICD1 CC3) has an important role in cargo sorting, including intracellular vesicles associating with the small GTPase Rab6 and the nuclear pore complex Ran binding protein 2 (RanBP2), and inhibiting the association with cytoplasmic dynein by binding to the first N-terminal coiled-coil region (CC1). The crystal structure of BICD1 CC3 revealed a parallel homodimeric coiled-coil with asymmetry and complementary knobs-into-holes interactions, differing from Drosophila BicD CC3. Furthermore, our binding study indicated that BICD1 CC3 possesses a binding surface for two distinct cargos, Rab6 and RanBP2, and that the CC1-binding site overlaps with the Rab6-binding site. These findings suggest a molecular basis for cargo recognition and autoinhibition of BICD proteins during dynein-dependent intracellular retrograde transport.

Biochemical characterization of a heterotrimeric G(i)-protein activator peptide designed from the junction between the intracellular third loop and sixth transmembrane helix in the m4 muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors (GPCRs) that are activated by acetylcholine released from parasympathetic nerves. The mAChR family comprises 5 subtypes, m1-m5, each of which has a different coupling selectivity for heterotrimeric GTP-binding proteins (G-proteins). m4 mAChR specifically activates the Gi/o family by enhancing the guanine nucleotide exchange factor (GEF) reaction with the Gα subunit through an interaction that occurs via intracellular segments. Here, we report that the m4 mAChR mimetic peptide m4i3c(14)Gly, comprising 14 residues in the junction between the intracellular third loop (i3c) and transmembrane helix VI (TM-VI) extended with a C-terminal glycine residue, presents GEF activity toward the Gi1 α subunit (Gαi1). The m4i3c(14)Gly forms a stable complex with guanine nucleotide-free Gαi1 via three residues in the VTI(L/F) motif, which is conserved within the m2/4 mAChRs. These results suggest that this m4 mAChR mimetic peptide, which comprises the amino acid of the mAChR intracellular segments, is a useful tool for understanding the interaction between GPCRs and G-proteins.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module.

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH-CC-Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH-CC-Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the beta-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

Crystal structure of L-2-keto-3-deoxyfuconate 4-dehydrogenase reveals a unique binding mode as a α-furanosyl hemiketal of substrates.

L-2-Keto-3-deoxyfuconate 4-dehydrogenase (L-KDFDH) catalyzes the NAD+-dependent oxidization of L-2-keto-3-deoxyfuconate (L-KDF) to L-2,4-diketo-3-deoxyfuconate (L-2,4-DKDF) in the non-phosphorylating L-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of L-KDF. On the other hand, BDH2, a mammalian homolog with L-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-L-proline (C4LHyp) with the cyclic structure. We found that L-KDFDH and BDH2 utilize C4LHyp and L-KDF, respectively. Therefore, to elucidate unique substrate specificity at the atomic level, we herein investigated for the first time the crystal structures of L-KDFDH from Herbaspirillum huttiense in the ligand-free, L-KDF and L-2,4-DKDF, D-KDP (D-2-keto-3-deoxypentonate; additional substrate), or L-2,4-DKDF and NADH bound forms. In complexed structures, L-KDF, L-2,4-DKDF, and D-KDP commonly bound as a α-furanosyl hemiketal. Furthermore, L-KDFDH showed no activity for L-KDF and D-KDP analogs without the C5 hydroxyl group, which form only the acyclic α-keto form. The C1 carboxyl and α-anomeric C2 hydroxyl groups and O5 oxygen atom of the substrate (and product) were specifically recognized by Arg148, Arg192, and Arg214. The side chain of Trp252 was important for hydrophobically recognizing the C6 methyl group of L-KDF. This is the first example showing the physiological role of the hemiketal of 2-keto-3-deoxysugar acid.

Structural insights into recognition of SL4, the UUCG stem-loop, of human U1 snRNA by the ubiquitin-like domain, including the C-terminal tail in the SF3A1 subunit of U2 snRNP.

The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704-785) adopts a typical ß-grasp fold with a topology of ß1-ß2-α1-310a-ß3-ß4-310b-ß5, closely resembling that of ubiquitin, except for the length and structure of the ß1/ß2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (ß3), Phe763 (ß4) and Lys765 (following ß4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the ß-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.

PPARα activation partially drives NAFLD development in liver-specific Hnf4a-null mice.

HNF4α regulates various genes to maintain liver function. There have been reports linking HNF4α expression to the development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis. In this study, liver-specific Hnf4a-deficient mice (Hnf4aΔHep mice) developed hepatosteatosis and liver fibrosis, and they were found to have difficulty utilizing glucose. In Hnf4aΔHep mice, the expression of fatty acid oxidation-related genes, which are PPARα target genes, was increased in contrast to the decreased expression of PPARα, suggesting that Hnf4aΔHep mice take up more lipids in the liver instead of glucose. Furthermore, Hnf4aΔHep/Ppara-/- mice, which are simultaneously deficient in HNF4α and PPARα, showed improved hepatosteatosis and fibrosis. Increased C18:1 and C18:1/C18:0 ratio was observed in the livers of Hnf4aΔHep mice, and the transactivation of PPARα target gene was induced by C18:1. When the C18:1/C18:0 ratio was close to that of Hnf4aΔHep mouse liver, a significant increase in transactivation was observed. In addition, the expression of Pgc1a, a coactivator of PPARs, was increased, suggesting that elevated C18:1 and Pgc1a expression could contribute to PPARα activation in Hnf4aΔHep mice. These insights may contribute to the development of new diagnostic and therapeutic approaches for NAFLD by focusing on the HNF4α and PPARα signaling cascade.

Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates.

N-terminal truncation of the Escherichia coli ethanolamine ammonia-lyase beta-subunit does not affect the catalytic properties of the enzyme (Akita, K., Hieda, N., Baba, N., Kawaguchi, S., Sakamoto, H., Nakanishi, Y., Yamanishi, M., Mori, K., and Toraya, T. (2010) J. Biochem. 147, 83-93). The binary complex of the truncated enzyme with cyanocobalamin and the ternary complex with cyanocobalamin or adeninylpentylcobalamin and substrates were crystallized, and their x-ray structures were analyzed. The enzyme exists as a trimer of the (alphabeta)(2) dimer. The active site is in the (beta/alpha)(8) barrel of the alpha-subunit; the beta-subunit covers the lower part of the cobalamin that is bound in the interface of the alpha- and beta-subunits. The structure complexed with adeninylpentylcobalamin revealed the presence of an adenine ring-binding pocket in the enzyme that accommodates the adenine moiety through a hydrogen bond network. The substrate is bound by six hydrogen bonds with active-site residues. Argalpha(160) contributes to substrate binding most likely by hydrogen bonding with the O1 atom. The modeling study implies that marked angular strains and tensile forces induced by tight enzyme-coenzyme interactions are responsible for breaking the coenzyme Co-C bond. The coenzyme adenosyl radical in the productive conformation was modeled by superimposing its adenine ring on the adenine ring-binding site followed by ribosyl rotation around the N-glycosidic bond. A major structural change upon substrate binding was not observed with this particular enzyme. Glualpha(287), one of the substrate-binding residues, has a direct contact with the ribose group of the modeled adenosylcobalamin, which may contribute to the substrate-induced additional labilization of the Co-C bond.

Crystallographic characterization of the DIX domain of the Wnt signalling positive regulator Ccd1.

Coiled-coil DIX1 (Ccd1) is a positive regulator that activates the canonical Wnt signalling pathway by inhibiting the degradation of the key signal transducer ß-catenin. The C-terminal DIX domain of Ccd1 plays an important role in the regulation of signal transduction through homo-oligomerization and protein complex formation with other DIX domain-containing proteins, i.e. axin and dishevelled proteins. Here, the expression, purification, crystallization and X-ray data collection of the Ccd1 DIX domain are reported. The crystals of the Ccd1 DIX domain belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=72.9, b=75.7, c=125.6 Å. An X-ray diffraction data set was collected at 3.0 Šresolution.

Expression, crystallization and preliminary X-ray crystallographic study of ethanolamine ammonia-lyase from Escherichia coli.

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. The wild-type enzyme shows a very low solubility. N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties. Two deletion mutants of the enzyme [EAL(betaDelta4-30) and EAL(betaDelta4-43)] have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Crystals of EAL(betaDelta4-30) and EAL(betaDelta4-43) diffracted to approximately 8.0 and 2.1 A resolution, respectively.

A direct heterotypic interaction between the DIX domains of Dishevelled and Axin mediates signaling to ß-catenin.

The Wnt-ß-catenin signaling pathway regulates embryonic development and tissue homeostasis throughout the animal kingdom. Signaling through this pathway crucially depends on the opposing activities of two cytoplasmic multiprotein complexes: the Axin destruction complex, which destabilizes the downstream effector ß-catenin, and the Dishevelled signalosome, which inactivates the Axin complex and thus enables ß-catenin to accumulate and operate a transcriptional switch in the nucleus. These complexes are assembled by dynamic head-to-tail polymerization of the DIX domains of Axin or Dishevelled, respectively, which increases their avidity for signaling effectors. Axin also binds to Dishevelled through its DIX domain. Here, we report the crystal structure of the heterodimeric complex between the two DIX domains of Axin and Dishevelled. This heterotypic interface resembles the interfaces observed in the individual homopolymers, albeit exhibiting a slight rearrangement of electrostatic interactions and hydrogen bonds, consistent with the heterotypic interaction being favored over the homotypic Axin DIX interaction. Last, cell-based signaling assays showed that heterologous polymerizing domains functionally substituted for the DIX domain of Dishevelled provided that these Dishevelled chimeras retained a DIX head or tail surface capable of binding to Axin. These findings indicate that the interaction between Dishevelled and Axin through their DIX domains is crucial for signaling to ß-catenin.

Head-to-Tail Complex of Dishevelled and Axin-DIX Domains: Expression, Purification, Crystallographic Studies and Packing Analysis.

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/ß- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.

High-resolution structure of a Y27W mutant of the Dishevelled2 DIX domain.

Dishevelled (Dvl) is a positive regulator of the canonical Wnt pathway that downregulates the phosphorylation of ß-catenin and its subsequent degradation. Dvl contains an N-terminal DIX domain, which is involved in its homooligomerization and interactions with regulators of the Wnt pathway. The crystal structure of a Y27W mutant of the Dishevelled2 DIX domain (DIX-Y27W) has been determined at 1.64 Šresolution. DIX-Y27W has a compact ubiquitin-like fold and self-associates with neighbouring molecules through ß-bridges, resulting in a head-to-tail helical molecular arrangement similar to previously reported structures of DIX domains. Glu23 of DIX-Y27W forms a hydrogen bond to the side chain of Trp27, corresponding to the Glu762...Trp766 hydrogen bond of the rat Axin DIX domain, whereas Glu23 in the Y27D mutant of the Dishevelled2 DIX domain forms a salt bridge to Lys68 of the adjacent molecule. The high-resolution DIX-Y27W structure provides details of the head-to-tail interaction, including solvent molecules, and also the plausibly wild-type-like structure of the self-association surface compared with previously published Dvl DIX-domain mutants.

Crystallographic characterization of the membrane-targeting domains of the Rac-specific guanine nucleotide-exchange factors Tiam1 and Tiam2.

T-lymphoma invasion and metastasis 1 and 2 (Tiam1 and Tiam2) are guanine nucleotide-exchange factors that specifically activate Rac GTPase by facilitating the dissociation of GDP. Translocation of Tiam1 and Tiam2 from the cytoplasm to the plasma membrane is an essential step in Rac activation and is mediated by the conserved PH-CC-Ex (pleckstrin-homology, coiled-coil and extra region) region in the N-terminal region. Here, the purification, crystallization and X-ray data collection of the Tiam1 and Tiam2 PH-CC-Ex regions are reported. The regions are shown to exist as a monomer in solution as a folded globular domain. The Tiam2 PH-CC-Ex domain crystallizes in space group P4(1)2(1)2 or P4(3)2(1)2 with four molecules in the asymmetric unit. An X-ray diffraction data set has been collected to 3.2 A resolution.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP).

ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6(1)22, with unit-cell parameters a = b = 122.7, c = 128.3 A, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 A.

Structural basis for NHERF recognition by ERM proteins.

The Na+/H+ exchanger regulatory factor (NHERF) is a key adaptor protein involved in the anchoring of ion channels and receptors to the actin cytoskeleton through binding to ERM (ezrin/radixin/moesin) proteins. NHERF binds the FERM domain of ERM proteins, although NHERF has no signature Motif-1 sequence for FERM binding found in adhesion molecules. The crystal structures of the radixin FERM domain complexed with the NHERF-1 and NHERF-2 C-terminal peptides revealed a peptide binding site of the FERM domain specific for the 13 residue motif MDWxxxxx(L/I)Fxx(L/F) (Motif-2), which is distinct from Motif-1. This Motif-2 forms an amphipathic alpha helix for hydrophobic docking to subdomain C of the FERM domain. This docking causes induced-fit conformational changes in subdomain C and affects binding to adhesion molecule peptides, while the two binding sites are not overlapped. Our studies provide structural paradigms for versatile ERM linkages between membrane proteins and the cytoskeleton.

In-frame Val216-Ser217 deletion of KIT in mild piebaldism causes aberrant secretion and SCF response.

BACKGROUND: Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. OBJECTIVE: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. METHODS: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. RESULTS: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. CONCLUSION: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1.

Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4(3)22, with unit-cell parameters a = b = 68.72, c = 201.39 A, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 80.74, b = 85.73, c = 117.75 A, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9 A for the FERM-CD43 complex and 2.8 A for the FERM-PSGL-1 complex.