Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.     

Structural basis for recognition of S-adenosylhomocysteine by riboswitches

S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.     

Structural basis of SARM1 activation,substrate recognition,and inhibition by small molecules

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Structural basis for the recognition of Asef by adenomatous polyposis coli

Adenomatous polyposis coli (APC) regulates cell-cell adhesion and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (GEF; Asef), which is usually autoinhibited through the binding between its Src homology 3 (SH3) and Dbl homology (DH) domains. The APC-activated Asef stimulates the small GTPase Cdc42, which leads to decreased cell-cell adherence and enhanced cell migration. In colorectal cancers, truncated APC constitutively activates Asef and promotes cancer cell migration and angiogenesis. Here, we report crystal structures of the human APC/Asef complex. We find that the armadillo repeat domain of APC uses a highly conserved surface groove to recognize the APC-binding region (ABR) of Asef, conformation of which changes dramatically upon binding to APC. Key residues on APC and Asef for the complex formation were mutated and their importance was demonstrated by binding and activity assays. Structural superimposition of the APC/Asef complex with autoinhibited Asef suggests that the binding between APC and Asef might create a steric clash between Asef-DH domain and APC, which possibly leads to a conformational change in Asef that stimulates its GEF activity. Our structures thus elucidate the molecular mechanism of Asef recognition by APC, as well as provide a potential target for pharmaceutical intervention against cancers.     

Structural basis of CoA recognition by the Pyrococcus single-domain CoA-binding proteins

The single-domain coenzyme A (CoA)-binding protein is conserved in bacteria, archaea, and a few eukaryal taxa. It consists of a Rossmann-fold domain, belonging to the FAD/NAD(P)-binding ;superfamily. The crystal structure of the Thermus thermophilus single-domain CoA-binding protein, TTHA1899, has been determined and it has been demonstrated, by isothermal titration calorimetry, that the protein interacts with CoA [Wada T. et al. Acta Crystallogr D Biol Crystallogr 59 (2003) 1213]. In the present study, we determined the crystal structures of an orthologous protein from the archaeon Pyrococcus horikoshii (PH1109), alone and complexed with CoA, at 1.65 A and 1.70 A resolutions, respectively, and that of P. furiosus protein (PF0725) in the CoA-bound form at 1.70 A. The CoA-bound structures are very similar to each other, revealing that the Pyrococcus proteins bind CoA in a 1:1 stoichiometry. Five loop-containing regions form the CoA-binding groove, to which the CoA molecule is docked. A comparison of the structures and the sequences of the Pyrococcus proteins with those of the T. theromphilus orthologue TTHA1899 indicated that archaeal and bacterial single-domain CoA-binding proteins share the same CoA-binding mode. Nevertheless, many of the peripheral residues involved in the hydrogen-bonding/electrostatic interactions with CoA are not strictly conserved in the family. The CoA interaction of the single-domain CoA-binding proteins is significantly different and much more extensive than that of the multi-subunit/multi-domain CoA-binding protein succinyl-CoA synthetase.     

Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids

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Structural basis for the specific recognition of IL-18 by its alpha receptor

Interleukin 18 (IL-18), a member of the IL-1 family of cytokines, is an important regulator of innate and acquired immune responses. It signals through its ligand-binding primary receptor IL-18Rα and accessory receptor IL-18Rβ. Here we report the crystal structure of IL-18 with the ectodomain of IL-18Rα, which reveals the structural basis for their specific recognition. It confirms that surface charge complementarity determines the ligand-binding specificity of primary receptors in the IL-1 receptor family. We suggest that IL-18 signaling complex adopts an architecture similar to other agonistic cytokines and propose a general ligand-receptor assembly and activation model for the IL-1 family.     

Structural basis of carbohydrate recognition by a Man(alpha1-2)Man-specific lectin from Bowringia milbraedii

The crystal structure of the seed lectin from the tropical legume Bowringia milbraedii was determined in complex with the disaccharide ligand Man(alpha1-2)Man. In solution, the protein exhibits a dynamic dimer-tetramer equilibrium, consistent with the concanavalin A-type tetramer observed in the crystal. Contacts between the tetramers are mediated almost exclusively through the carbohydrate ligand, resulting in a crystal lattice virtually identical to that of the concanavalin-A:Man(alpha1-2)Man complex, even though both proteins have less than 50% sequence identity. The disaccharide binds exclusively in a "downstream" binding mode, with the non-reducing mannose occupying the monosaccharide-binding site. The reducing mannose is bound in a predominantly polar subsite involving Tyr131, Gln218, and Tyr219.     

Structural and thermodynamic basis for the interaction of the Src SH2 domain with the activated form of the PDGF beta-receptor

Recruitment of the Src kinase to the activated form of the platelet-derived growth factor (PDGF) receptor involves recognition of a unique sequence motif in the juxtamembrane region of the receptor by the Src homology 2 (SH2) domain of the enzyme. This motif contains two phosphotyrosine residues separated by one residue (sequence pYIpYV where pY indicates a phosphotyrosine). Here, we provide the thermodynamic and structural basis for the binding of this motif by the Src SH2 domain. We show that the second phosphorylation event increases the free energy window for specific interaction and that the physiological target is exquisitely designed for the task of recruiting specifically an SH2 domain which otherwise demonstrates very little intrinsic ability to discriminate sequences C-terminal to the first phosphorylation event. Surprisingly, we show that water plays a role in the recognition process.     

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.     

Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates

The c-Cbl tyrosine kinase binding domain (Cbl-TKB), essentially an 'embedded' SH2 domain, has a critical role in targeting proteins for ubiquitination. To address how this domain can bind to disparate recognition mofits and to determine whether this results in variations in substrate-binding affinity, we compared crystal structures of the Cbl-TKB domain complexed with phosphorylated peptides of Sprouty2, Sprouty4, epidermal growth factor receptor, Syk, and c-Met receptors and validated the binding with point-mutational analyses using full-length proteins. An obligatory, intrapeptidyl H-bond between the phosphotyrosine and the conserved asparagine or adjacent arginine is essential for binding and orients the peptide into a positively charged pocket on c-Cbl. Surprisingly, c-Met bound to Cbl in the reverse direction, which is unprecedented for SH2 domain binding. The necessity of this intrapeptidyl H-bond was confirmed with isothermal titration calorimetry experiments that also showed Sprouty2 to have the highest binding affinity to c-Cbl; this may enable the selective sequestration of c-Cbl from other target proteins.     

Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8 Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.     

BAK1 Directly Regulates Brassinosteroid Perception and BRI1 Activation

Plants utilize plasma membrane-localized receptor-like kinases （RLKs） to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting as a co-receptor of several distinct ligand-binding RLKs. It has been debated whether BAK1 serves as an essential regulatory component or only a signal amplifier without pathway specificity. This issue has been clarified recently. Genetic and structural analyses indicated that BAK1 and its homologs play indispensible roles in mediating brassinosteroid （BR） signaling pathway by directly perceiving the ligand BR and activating the receptor of BR, BRII. The mechanism revealed by these studies now serves as a paradigm for how a pair of RLKs can function together in ligand binding and subsequent initiation of signaling.     

Structural basis of the suppressed catalytic activity of wild-type human glutathione transferase T1-1 compared to its W234R mutant

The crystal structures of wild-type human theta class glutathione-S-transferase (GST) T1-1 and its W234R mutant, where Trp234 was replaced by Arg, were solved both in the presence and absence of S-hexyl-glutathione. The W234R mutant was of interest due to its previously observed enhanced catalytic activity compared to the wild-type enzyme. GST T1-1 from rat and mouse naturally contain Arg in position 234, with correspondingly high catalytic efficiency. The overall structure of GST T1-1 is similar to that of GST T2-2, as expected from their 53% sequence identity at the protein level. Wild-type GST T1-1 has the side-chain of Trp234 occupying a significant portion of the active site. This bulky residue prevents efficient binding of both glutathione and hydrophobic substrates through steric hindrance. The wild-type GST T1-1 crystal structure, obtained from co-crystallization experiments with glutathione and its derivatives, showed no electron density for the glutathione ligand. However, the structure of GST T1-1 mutant W234R showed clear electron density for S-hexyl-glutathione after co-crystallization. In contrast to Trp234 in the wild-type structure, the side-chain of Arg234 in the mutant does not occupy any part of the substrate-binding site. Instead, Arg234 is pointing in a different direction and, in addition, interacts with the carboxylate group of glutathione. These findings explain our earlier observation that the W234R mutant has a markedly improved catalytic activity with most substrates tested to date compared to the wild-type enzyme. GST T1-1 catalyzes detoxication reactions as well as reactions that result in toxic products, and our findings therefore suggest that humans have gained an evolutionary advantage by a partially disabled active site.     

Structural basis of carbohydrate recognition by calreticulin

The calnexin cycle is a process by which glycosylated proteins are subjected to folding cycles in the endoplasmic reticulum lumen via binding to the membrane protein calnexin (CNX) or to its soluble homolog calreticulin (CRT). CNX and CRT specifically recognize monoglucosylated Glc₁Man₉GlcNAc₂ glycans, but the structural determinants underlying this specificity are unknown. Here, we report a 1.95-Å crystal structure of the CRT lectin domain in complex with the tetrasaccharide α-Glc-(1→3)-α-Man-(1→2)-α-Man-(1→2)-Man. The tetrasaccharide binds to a long channel on CRT formed by a concave β-sheet. All four sugar moieties are engaged in the protein binding via an extensive network of hydrogen bonds and hydrophobic contacts. The structure explains the requirement for glucose at the nonreducing end of the carbohydrate; the oxygen O₂ of glucose perfectly fits to a pocket formed by CRT side chains while forming direct hydrogen bonds with the carbonyl of Gly¹²⁴ and the side chain of Lys¹¹¹. The structure also explains a requirement for the Cys¹⁰⁵–Cys¹³⁷ disulfide bond in CRT/CNX for efficient carbohydrate binding. The Cys¹⁰⁵–Cys¹³⁷ disulfide bond is involved in intimate contacts with the third and fourth sugar moieties of the Glc₁Man₃ tetrasaccharide. Finally, the structure rationalizes previous mutagenesis of CRT and lays a structural groundwork for future studies of the role of CNX/CRT in diverse biological pathways.     

Structural basis for recognition of ubiquitinated cargo by Tom1-GAT domain

Tom1 (Target of Myb1) is suggested to be involved in the transport of ubiquitinated proteins, through the interaction of its GAT (GGA and Tom1) domain with ubiquitin. Here, we demonstrate that the three-helix bundle of Tom1-GAT has two ubiquitin-binding sites recognizing the hydrophobic Ile44 surface of ubiquitin. The complex crystal structure demonstrates that the first site is a hydrophobic patch on helices alpha1 and alpha2. NMR and biochemical data revealed that the N-terminal half of helix alpha3 of Tom1-GAT constitutes the second, stronger binding site. The double-sided ubiquitin binding enhances the efficiency of recognition of ubiquitinated proteins by Tom1.     

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β-β associations found in radixin-adhesion molecule complexes. We also revealed direct interactions between the MyTH4-FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.     

Structural basis for monoubiquitin recognition by the Ede1 UBA domain

Monoubiquitination is a general mechanism for downregulating the activity of cell surface receptors by consigning these proteins for lysosome-mediated degradation through the endocytic pathway. The yeast Ede1 protein functions at the internalization step of endocytosis and binds monoubiquitinated proteins through a ubiquitin associated (UBA) domain. UBA domains are found in a broad range of cellular proteins but previous studies have suggested that the mode of ubiquitin recognition might not be universally conserved. Here we present the solution structure of the Ede1 UBA domain in complex with monoubiquitin. The Ede1 UBA domain forms a three-helix bundle structure and binds ubiquitin through a largely hydrophobic surface in a manner reminiscent of the Dsk2 UBA and the remotely homologous Cue2 CUE domains, for which high-resolution structures have been described. However, the interaction is dissimilar to the molecular models proposed for the hHR23A UBA domains bound to either monoubiquitin or Lys48-linked diubiquitin. Our mutational analyses of the Ede1 UBA domain-ubiquitin interaction reveal several key affinity determinants and, unexpectedly, a negative affinity determinant in the wild-type Ede1 protein, implying that high-affinity interactions may not be the sole criterion for optimal function of monoubiquitin-binding endocytic proteins.