Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1 at 2.0A resolution

Sphingomonas sp. A1 possesses a high molecular mass (average 25,700 Da) alginate uptake system mediated by a novel pit-dependent ABC transporter. The X-ray crystallographic structure of AlgQ2 (57,200 Da), an alginate-binding protein in the system, was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 18.3% for 15 to 2.0 A resolution data. The refined structure of AlgQ2 was comprised of 492 amino acid residues, 172 water molecules, and one calcium ion. AlgQ2 was composed of two globular domains with a deep cleft between them, which is expected to be the alginate-binding site. The overall structure is basically similar to that of maltose/maltodextrin-binding protein, except for the presence of an N2-subdomain. The entire calcium ion-binding site is similar to the site in the EF-hand motif, but comprises a ten residue loop. This calcium ion-binding site is about 40 A away from the alginate-binding site.     

Crystal structure of human pepsin and its complex with pepstatin.

The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position.     

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.     

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by ¹H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27–36 is essential to bind purine- or pyrimidine-based nucleosides.Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.     

Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG

Abstract

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H⁺ Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H⁺ Symporter (OHS) family. Sequence alignment of these distantly related proteins (～ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.     

Crystal structure of the ENT domain of human EMSY

EMSY is a recently discovered gene encoding a BRCA2-associated protein and is amplified in some sporadic breast and ovarian cancers. The EMSY sequence contains no known domain except for a conserved approximately 100 residue segment at the N terminus. This so-called ENT domain is unique in the human genome, although multiple copies are found in Arabidopsis proteins containing members of the Royal family of chromatin remodelling domains. Here, we report the crystal structure of the ENT domain of EMSY, consisting of a unique arrangement of five alpha-helices that fold into a helical bundle arrangement. The fold shares regions of structural homology with the DNA-binding domain of homeodomain proteins. The ENT domain forms a homodimer via the anti-parallel packing of the extended N-terminal alpha-helix of each molecule. It is stabilized mainly by hydrophobic residues at the dimer interface and has a dissociation constant in the low micromolar range. The dimerisation of EMSY mediated by the ENT domain could provide flexibility for it to bind two or more different substrates simultaneously.     

Crystal structure of beta1,4-galactosyltransferase complex with UDP-Gal reveals an oligosaccharide acceptor binding site

The crystal structure of the catalytic domain of bovine beta1,4-galactosyltransferase (Gal-T1) co-crystallized with UDP-Gal and MnCl(2) has been solved at 2.8 A resolution. The structure not only identifies galactose, the donor sugar binding site in Gal-T1, but also reveals an oligosaccharide acceptor binding site. The galactose moiety of UDP-Gal is found deep inside the catalytic pocket, interacting with Asp252, Gly292, Gly315, Glu317 and Asp318 residues. Compared to the native crystal structure reported earlier, the present UDP-Gal bound structure exhibits a large conformational change in residues 345-365 and a change in the side-chain orientation of Trp314. Thus, the binding of UDP-Gal induces a conformational change in Gal-T1, which not only creates the acceptor binding pocket for N-acetylglucosamine (GlcNAc) but also establishes the binding site for an extended sugar acceptor. The presence of a binding site that accommodates an extended sugar offers an explanation for the observation that an oligosaccharide with GlcNAc at the non-reducing end serves as a better acceptor than the monosaccharide, GlcNAc. Modeling studies using oligosaccharide acceptors indicate that a pentasaccharide, such as N-glycans with GlcNAc at their non-reducing ends, fits the site best. A sequence comparison of the human Gal-T family members indicates that although the binding site for the GlcNAc residue is highly conserved, the site that binds the extended sugar exhibits large variations. This is an indication that different Gal-T family members prefer different types of glycan acceptors with GlcNAc at their non-reducing ends.     

Crystal structure of Thermobifida fusca Cse1 reveals target DNA binding site

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐CRISPR‐associated (Cas) defense system is the only adaptive and inheritable immunity found in prokaryotes. The immunity is achieved through a multistep process of adaptation, expression, and interference. In the Type I‐E system, interference is mediated by the CRISPR‐associated complex for antiviral defense (Cascade), which recognizes invading double‐stranded DNA (dsDNA) through the protospacer adjacent motif (PAM) by one of the Cascade components, Cse1. Here, we report the crystal structure of Thermobifida fusca Cse1 at 3.3 Å resolution. T. fusca Cse1 reveals the chair‐like two‐domain architecture with a well‐defined flexible loop, L1, located at the larger N‐terminal domain, which was not observed in previous structures of the single Cse1 protein. Structure‐based mutagenesis analysis demonstrates that the well‐defined flexible loop and a partially conserved structural motif ([FW]‐X‐[TH]) are involved in PAM binding and recognition, respectively. Moreover, structural docking of T. fusca Cse1 into Escherichia coli Cascade cryoelectron microscopy maps, coupled with structural comparison, reveals a conserved positive patch that is contiguous with Cse2 in the Cascade complex and adjacent to the Cas3 binding site, suggesting its role in R‐loop formation/stabilization and the recruitment of Cas3 for target cleavage. Consistent with the structural observation, the introduction of alanine mutations at this positive patch abolished DNA binding activity by Cse1. Taken together, these results suggest that Cse1 is a critical Cascade component involved in Cascade assembly, dsDNA target recognition, R‐loop formation, and Cas3 recruitment for target cleavage.     

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.     

The type 1 equilibrative nucleoside transporter regulates anxiety-like behavior in mice

Activation of adenosine receptors in the brain reduces anxiety-like behavior in animals and humans. Because nucleoside transporters regulate adenosine levels, we used mice lacking the type 1 equilibrative nucleoside transporter (ENT1) to investigate whether ENT1 contributes to anxiety-like behavior. The ENT1 null mice spent more time in the center of an open field compared with wild-type littermates. In the elevated plus maze, ENT1 null mice entered more frequently into and spent more time exploring the open arms. The ENT1 null mice also spent more time exploring the light side of a light-dark box compared with wild-type mice. Microinjection of an ENT1-specific antagonist, nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside), into the amygdala of C57BL/6J mice reduced anxiety-like behavior in the open field and elevated plus maze. These findings show that amygdala ENT1 modulates anxiety-like behavior. The ENT1 may be a drug target for the treatment of anxiety disorders.     

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

Abstract

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.     

Crystal structure of yeast mitochondrial peripheral membrane protein Tim44p C-terminal domain

The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of TIM23 translocon. Tim44p can tightly associate with the inner mitochondrial membrane. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, we have determined the crystal structure of the yeast Tim44p C-terminal domain to 3.2A resolution using the MAD method. The Tim44p C-terminal domain forms a monomer in the crystal structure and contains six alpha-helices and four antiparallel beta-strands. A large hydrophobic pocket was identified on the Tim44p structure surface. The N-terminal helix A1 is positively charged and the helix A1 protrudes out from the Tim44p main body.     

Crystal structure of a DNA binding protein from the hyperthermophilic euryarchaeon Methanococcus jannaschii

The Sac10b family consists of a group of highly conserved DNA binding proteins from both the euryarchaeotal and the crenarchaeotal branches of Archaea. The proteins have been suggested to play an architectural role in the chromosomal organization in these organisms. Previous studies have mainly focused on the Sac10b proteins from the crenarchaeota. Here, we report the 2.0 A resolution crystal structure of Mja10b from the euryarchaeon Methanococcus jannaschii. The model of Mja10b has been refined to an R-factor of 20.9%. The crystal structure of an Mja10b monomer reveals an alpha/beta structure of four beta-strands and two alpha-helices, and Mja10b assembles into a dimer via an extensive hydrophobic interface. Mja10b has a similar topology to that of its crenarchaeota counterpart Sso10b (also known as Alba). Structural comparison between the two proteins suggests that structural features such as hydrophobic inner core, acetylation sites, dimer interface, and DNA binding surface are conserved among Sac10b proteins. Structural differences between the two proteins were found in the loops. To understand the structural basis for the thermostability of Mja10b, the Mja10b structure was compared to other proteins with similar topology. Our data suggest that extensive ion-pair networks, optimized accessible surface area and the dimerization via hydrophobic interactions may contribute to the enhanced thermostability of Mja10b.     

Crystal structure of human coactosin-like protein at 1.9 A resolution

Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 A resolution. The structure features a central beta-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed.     

Crystal structure of Bordetella pertussis BugD solute receptor unveils the basis of ligand binding in a new family of periplasmic binding proteins

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

Crystal structure of calf spleen purine nucleoside phosphorylase complexed to a novel purine analogue

The combined use of a rapid virtual screen of a small fragment library together with a single point enzyme assay has been used for the discovery of novel PNP inhibitors. The availability of readily soakable crystals of bovine PNP has allowed the approach to be experimentally validated by determining the crystal structure of one of the inhibitor-PNP complexes. Comparison of the experimentally determined binding mode with that predicted by the virtual screening shows them to be similar. This represents a starting point for the growth of the ligand into a higher affinity inhibitor.     

Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays anessential role in autophagy. However, the molecular mechanism by which Beclin 1functions remains largely unknown. Here we report the crystal structure of theevolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Åresolution. Beclin 1 ECD exhibits a previously unreported fold, with threestructural repeats arranged symmetrically around a central axis. Beclin 1 ECDdefines a novel class of membrane-binding domain, with a strong preference forlipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1ECD, comprising three aromatic amino acids, acts as a hydrophobic finger toassociate with lipid membrane, consequently resulting in the deformation ofmembrane and liposomes. Mutation of these aromatic residues rendered Beclin 1unable to stably associate with lipid membrane in vitro and unable tofully rescue autophagy in Beclin 1-knockdown cells in vivo. Theseobservations form an important framework for deciphering the biologicalfunctions of Beclin 1.