Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations

Eph receptors and their ephrin ligands are important mediators of cell-cell communication. They are divided in two subclasses based on their affinities for each other and on sequence conservation. Receptor-ligand binding within each subclass is fairly promiscuous, while binding cross the subclasses happens rarely. EphA4 is an exception to this general rule, since it has long been known to bind both A- and B-class ephrin ligands but the reason for this exceptional behavior has not been worked out at molecular level. Recent structural and biochemical studies on EphA4 ligand-binding domain alone and in complex with its ligands have addressed this question. However, the published structures of EphA4/ephrin complexes differ considerably from each other and strikingly different explanations for the exceptional promiscuity of EphA4 were proposed. To address these contradictory findings, we have determined a crystal structure of the EphA4 ligand-binding domain at 2.3 Å resolution and show that the receptor has an unprecedented ability to exist in two very different, well-ordered conformations even in the unbound state. Our results suggest that the ligand promiscuity of the Ephs is directly correlated with the structural flexibility of the ligand-binding surface of the receptor.     

Crystal structure of human synbindin reveals two conformations of longin domain

Transport protein particle (TRAPP) is a large multiprotein complex that involves in ER-to-Golgi and intra-Golgi traffic. Synbindin, the human ortholog of yeast Trs23, is one component of the TRAPP complexes. In the hippocampal neurons the synbindin/syndecan complex is involved in synaptic membrane trafficking and thereby regulates the formation of dendritic spines. Here we present the three-dimensional structure of human synbindin, which contains a longin domain (LD) and an atypical PDZ domain (APD). In the crystal, synbindin forms a hexamer, in which the LD forms two different conformations and the APD is quite disordered. These conformational changes of synbindin suggest a possible interaction mode of the LD.     

Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers

BACKGROUND: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rosetta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profile), then the existence of a fusion protein in invertebrates and the coordinated expression of separate mRNAs in mouse suggest that Nit and Fhit function in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions. RESULTS: To satisfy the phylogenetic profile criterion for functional significance of protein fusion events, we cloned additional Nit homologs from organisms with Fhit homologs. We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivative phased by two-wavelength anomalous diffraction. CONCLUSIONS: Nit monomers possess a new alpha-beta-beta-alpha sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit. Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit.     

Molecular dynamics simulation of water permeation through the alpha-hemolysin channel

The alpha-hemolysin (AHL) nanochannel is a non-selective channel that allows for uncontrolled transport of small molecules across membranes leading to cell death. Although it is a bacterial toxin, it has promising applications, ranging from drug delivery systems to nano-sensing devices. This study focuses on the transport of water molecules through an AHL nanochannel using molecular dynamics (MD) simulations. Our results show that AHL can quickly transport water across membranes. The first-passage time approach was used to estimate the diffusion coefficient and the mean exit time. To study the energetics of transport, the potential of mean force (PMF) of a water molecule along the AHL nanochannel was calculated. The results show that the energy barriers of water permeation across a nanopore are always positive along the channel and the values are close to thermal energy (k_BT). These findings suggest that the observed quick permeation of water is due to small energy barriers and a hydrophobic inner channel surface resulting in smaller friction. We speculate that these physical mechanisms are important in how AHL causes cell death.     

Dynamics and energetics of water permeation through the aquaporin channel

Structural properties of water inside bovine aquaporin-1 are investigated by molecular simulation. The calculations, which are based on the recently determined X-ray structure at 2.2 A resolution (Sui et al., Nature 2001;414:872-878), are carried out on one monomeric subunit immersed in a water-n-octane-water bilayer. Molecular dynamics (MD) simulations suggest that His182, a fully conserved residue in the channel pore, is protonated in the delta position. Furthermore, they reveal a highly ordered water structure in the channel, induced by the electrostatic properties of the protein. Multiple-steering MD simulations are used to calculate the free-energy of water diffusion. To the best of our knowledge, this represents the first free-energy calculation based on the new, high-resolution structure of the pore. The calculated barrier is 2.5 kcal/mol, and it is associated to water permeation through the Asn-Pro-Ala (NPA) region of the pore, where water molecules are only hydrogen-bonded with themselves. These findings are fully consistent with those based on the previous MD studies on the human protein (de Groot and Grubmüller, Science 2001;294:2353-2357).     

Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin

The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.     

Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual alpha-helical extensions

Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.     

Crystal structure of the peptidoglycan recognition protein at 1.8 A resolution reveals dual strategy to combat infection through two independent functional homodimers

The mammalian peptidoglycan recognition protein-S (PGRP-S) binds to peptidoglycans (PGNs), which are essential components of the cell wall of bacteria. The protein was isolated from the samples of milk obtained from camels with mastitis and purified to homogeneity and crystallized. The crystals belong to orthorhombic space group I222 with a = 87.0 Å, b = 101.7 Å and c = 162.3 Å having four crystallographically independent molecules in the asymmetric unit. The structure has been determined using X-ray crystallographic data and refined to 1.8 Å resolution. Overall, the structures of all the four crystallographically independent molecules are identical. The folding of PGRP-S consists of a central β-sheet with five β-strands, four parallel and one antiparallel, and three α-helices. This protein fold provides two functional sites. The first of these is the PGN-binding site, located on the groove that opens on the surface in the direction opposite to the location of the N terminus. The second site is implicated to be involved in the binding of non-PGN molecules, it also includes putative N-terminal segment residues (1-31) and helix α2 in the extended binding. The structure reveals a novel arrangement of PGRP-S molecules in which two pairs of molecules associate to form two independent dimers. The first dimer is formed by two molecules with N-terminal segments at the interface in which non-PGN binding sites are buried completely, whereas the PGN-binding sites of two participating molecules are fully exposed at the opposite ends of the dimer. In the second dimer, PGN-binding sites are buried at the interface while non-PGN binding sites are fully exposed at the opposite ends of the dimer. This form of dimeric arrangement is unique and seems to be aimed at enhancing the capability of the protein against specific invading bacteria. This mode of functional dimerization enhances efficiency and specificity, and is observed for the first time in the family of PGRP molecules.     

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 ? resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ～392 residues and two L subunits of ～150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ～100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 ? away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.     

Crystal structure of the kinase and UBA domains of SNRK reveals a distinct UBA binding mode in the AMPK family

Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel member of the AMP-activated protein kinase (AMPK) family and is involved in many metabolic processes. Here we report the crystal structure of an N-terminal SNRK fragment containing kinase and adjacent ubiquitin-associated (UBA) domains. This structure shows that the UBA domain binds between the N- and C-lobes of the kinase domain. The mode of UBA binding in SNRK largely resembles that in AMPK and brain specific kinase (BRSK), however, unique interactions play vital roles in stabilizing the KD-UBA interface of SNRK. We further propose a potential role of the UBA domain in the regulation of SNRK kinase activity. This study provides new insights into the structural diversities of the AMPK kinase family.     

Structural characterisation of the insecticidal toxin XptA1, reveals a 1.15 MDa tetramer with a cage-like structure

A recently identified class of proteins conferring insecticidal activity to several bacteria within the Enterobacteriaceae family have potential for control of commercially important insect pests. Here, we report the first purification, biophysical characterisation and 3-D structural analysis of one of the toxin components, XptA1, from Xenorhabdus nematophila PMFI296 to a resolution of 23 A. Membrane binding studies indicate that the three-component toxin system has a different mode of action from that of proteins from Bacillus thuringiensis (Bt). Biophysical characterisation of XptA1 suggests a mechanism of action of XptA1 whereby it first binds to the cell membrane forming a structure with a central cavity and forms a complex with its partners XptB1 and XptC1 producing the full insecticidal toxin. The structure of XptA1 is shown by a combination of electron microscopy, ultracentrifugation and circular dichroism spectroscopy to be a 1.15 MDa tetramer with a cage-like structure. Each of the four symmetry-related subunits has three well-defined domains and a longitudinal twist with one end narrower than the other. One third of the residues of XptA1 are alpha-helical and it is suggested the subunits associate partly via an alpha-helical coiled-coil interaction. XptA1 itself shows the same secondary structure at neutral pH and in an alkaline environment up to pH10.5. This pH tolerance indicates that the folded XptA1 can pass through the midgut of Lepidopteran insects susceptible to the insecticidal toxin complex. This implies therefore that its folded structure is important for its biological activity.     

The structure of beta-carbonic anhydrase from the carboxysomal shell reveals a distinct subclass with one active site for the price of two

CsoSCA (formerly CsoS3) is a bacterial carbonic anhydrase localized in the shell of a cellular microcompartment called the carboxysome, where it converts HCO(3)(-) to CO(2) for use in carbon fixation by ribulose-bisphosphate carboxylase/oxygenase (RuBisCO). CsoSCA lacks significant sequence similarity to any of the four known classes of carbonic anhydrase (alpha, beta, gamma, or delta), and so it was initially classified as belonging to a new class, epsilon. The crystal structure of CsoSCA from Halothiobacillus neapolitanus reveals that it is actually a representative member of a new subclass of beta-carbonic anhydrases, distinguished by a lack of active site pairing. Whereas a typical beta-carbonic anhydrase maintains a pair of active sites organized within a two-fold symmetric homodimer or pair of fused, homologous domains, the two domains in CsoSCA have diverged to the point that only one domain in the pair retains a viable active site. We suggest that this defunct and somewhat diminished domain has evolved a new function, specific to its carboxysomal environment. Despite the level of sequence divergence that separates CsoSCA from the other two subclasses of beta-carbonic anhydrases, there is a remarkable level of structural similarity among active site regions, which suggests a common catalytic mechanism for the interconversion of HCO(3)(-) and CO(2). Crystal packing analysis suggests that CsoSCA exists within the carboxysome shell either as a homodimer or as extended filaments.     

Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes

Arrestins are multi-functional proteins that regulate signaling and trafficking of the majority of G protein-coupled receptors (GPCRs), as well as sub-cellular localization and activity of many other signaling proteins. We report the first crystal structure of arrestin-3, solved at 3.0 Å resolution. Arrestin-3 is an elongated two-domain molecule with overall fold and key inter-domain interactions that hold the free protein in the basal conformation similar to the other subtypes. Arrestin-3 is the least selective member of the family, binding a wide variety of GPCRs with high affinity and demonstrating lower preference for active phosphorylated forms of the receptors. In contrast to the other three arrestins, part of the receptor-binding surface in the arrestin-3 C-domain does not form a contiguous β-sheet, which is consistent with increased flexibility. By swapping the corresponding elements between arrestin-2 and arrestin-3 we show that the presence of this loose structure is correlated with reduced arrestin selectivity for activated receptors, consistent with a conformational change in this β-sheet upon receptor binding.     

Sequence,structure and function-based classification of the broadly conserved FAH superfamily reveals two distinct fumarylpyruvate hydrolase subfamilies

Fumarylacetoacetate hydrolase (FAH) superfamily proteins are found ubiquitously in microbial pathways involved in the catabolism of aromatic substances. Although extensive bioinformatic data on these proteins have been acquired, confusion caused by problems with the annotation of these proteins hinders research into determining their physiological functions. Here we classify 606 FAH superfamily proteins using a maximum likelihood (ML) phylogenetic tree, comparative gene-neighbourhood patterns and in vitro enzyme assays. The FAH superfamily proteins used for the analyses are divided into five distinct subfamilies, and two of them, FPH-A and FPH-B, contain the majority of the proteins of undefined function. These subfamilies include clusters designated FPH-I and FPH-II, respectively, which include two distinct types of fumarylpyruvate hydrolase (FPH), an enzyme involved in the final step of the gentisate pathway. We determined the crystal structures of these FPH enzymes at 2.0 Å resolutions and investigate the substrate binding mode by which these types of enzymes can accommodate fumarylpyruvate as a substrate. Consequentially, we identify the molecular signatures of the two types of FPH enzymes among the broadly conserved FAH superfamily proteins. Our studies allowed us to predict the relationship of unknown FAH superfamily proteins using their sequence information.     

Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties

Background  

The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.     

Crystal structure of the cysteine-rich secretory protein stecrisp reveals that the cysteine-rich domain has a K+ channel inhibitor-like fold

Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-angstrom resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N-terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family.     

Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs

The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 "nonstructural" proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 A as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular beta-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the "gag-knuckle fold group" of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.     

Crystal structure of the N-terminal ankyrin repeat domain of TRPV3 reveals unique conformation of finger 3 loop critical for channel function

In all six members of TRPV channel subfamily, there is an ankyrin repeat domain (ARD) in their intracellular Ntermini. Ankyrin (ANK) repeat, a common motif with typically 33 residues in each repeat, is primarily involved in protein-protein interactions. Despite the sequence similarity among the ARDs of TRPV channels, the structure of TRPV3-ARD, however, remains unknown. Here, we report the crystal structure of TRPV3-ARD solved at 1.95 ? resolution, which reveals six-ankyrin repeats. While overall structure of TRPV3-ARD is similar to ARDs from other members of TRPV subfamily; it, however, features a noticeable finger 3 loop that bends over and is stabilized by a network of hydrogen bonds and hydrophobic packing, instead of being flexible as seen in known TRPV-ARD structures. Electrophysiological recordings demonstrated that mutating key residues R225, R226, Q255, and F249 of finger 3 loop altered the channel activities and pharmacology. Taken all together, our findings show that TRPV3-ARD with characteristic finger 3 loop likely plays an important role in channel function and pharmacology.