Biophysical Analysis of Kindlin-3 Reveals an Elongated Conformation and Maps Integrin Binding to the Membrane-distal ��-Subunit NPXY Motif

Kindlin-3, a 75-kDa protein, has been shown to be critical for hemostasis, immunity, and bone metabolism via its role in integrin activation. The Kindlin family is hallmarked by a FERM domain comprised of F1, F2, and F3 subdomains together with an N-terminal F0 domain and a pleckstrin homology domain inserted in the F2 domain. Recombinant Kindlin-3 was cloned, expressed, and purified, and its domain organization was studied by x-ray scattering and other techniques to reveal an extended conformation. This unusual elongated structure is similar to that found in the paralogue Talin head domain. Analytical ultracentrifugation experiments indicated that Kindlin-3 forms a ternary complex with the Talin and β-integrin cytoplasmic tails. NMR showed that Kindlin-3 specifically recognizes the membrane-distal tail NPXY motif in both the β_1A and β_1D isoforms, although the interaction is stronger with β_1A. An upstream Ser/Thr cluster in the tails also plays a critical role. Overall these data support current biological, clinical, and mutational data on Kindlin-3/β-tail binding and provide novel insights into the overall conformation and interactions of Kindlin-3.     

Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C₄-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.     

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T_m = 52 °C, enhanced the protein thermostability by 36 °C (T_m = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.     

Structural insights into the substrate specificity of Streptococcus pneumoniae β(1,3)-galactosidase BgaC

The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5–8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)₈ TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.     

Crystal structure of Archaeoglobus fulgidus CTP:inositol-1-phosphate cytidylyltransferase, a key enzyme for di-myo-inositol-phosphate synthesis in (hyper)thermophiles

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K_m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V_max determined at 90°C was 62.9 μmol · min⁻¹ · mg of protein⁻¹. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X₄-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.     

The power of hard-sphere models: explaining side-chain dihedral angle distributions of Thr and Val

The energy functions used to predict protein structures typically include both molecular-mechanics and knowledge-based terms. In contrast, our approach is to develop robust physics- and geometry-based methods. Here, we investigate to what extent simple hard-sphere models can be used to predict side-chain conformations. The distributions of the side-chain dihedral angle χ₁ of Val and Thr in proteins of known structure show distinctive features: Val side chains predominantly adopt χ₁ = 180°, whereas Thr side chains typically adopt χ₁ = 60° and 300° (i.e., χ₁ = ±60° or g− and g⁺ configurations). Several hypotheses have been proposed to explain these differences, including interresidue steric clashes and hydrogen-bonding interactions. In contrast, we show that the observed side-chain dihedral angle distributions for both Val and Thr can be explained using only local steric interactions in a dipeptide mimetic. Our results emphasize the power of simple physical approaches and their importance for future advances in protein engineering and design.     

Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66

Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln¹⁰⁰ to Ile⁷³² of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 Å resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold; domain A contained the enzyme''s catalytic module, comprising a (β/α)₈-barrel; and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, −4 to −1, in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-d-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp³⁸⁵ side chain. These structures collectively indicated that Asp³⁸⁵ was the catalytic nucleophile and that Glu⁴⁵³ was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme''s catalytic machinery.     

Sensing Domain Dynamics in Protein Kinase A-I�� Complexes by Solution X-ray Scattering

The catalytic (C) and regulatory (R) subunits of protein kinase A are exceptionally dynamic proteins. Interactions between the R- and C-subunits are regulated by cAMP binding to the two cyclic nucleotide-binding domains in the R-subunit. Mammalian cells express four different isoforms of the R-subunit (RIα, RIβ, RIIα, and RIIβ) that all interact with the C-subunit in different ways. Here, we investigate the dynamic behavior of protein complexes between RIα and C-subunits using small angle x-ray scattering. We show that a single point mutation in RIα, R333K (which alters the cAMP-binding properties of Domain B) results in a compact shape compared with the extended shape of the wild-type R·C complex. A double mutant complex that disrupts the interaction site between the C-subunit and Domain B in RIα, RIα_ABR333K·C(K285P), results in a broader P(r) curve that more closely resembles the P(r) profiles of wild-type complexes. These results together suggest that interactions between RIα Domain B and the C-subunit in the RIα·C complex involve large scale dynamics that can be disrupted by single point mutations in both proteins. In contrast to RIα·C complexes. Domain B in the RIIβ·C heterodimer is not dynamic and is critical for both inhibition and complex formation. Our study highlights the functional differences of domain dynamics between protein kinase A isoforms, providing a framework for elucidating the global organization of each holoenzyme and the cross-talk between the R- and C-subunits.     

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)₈-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite −8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.     

Structural and biochemical characterization of the salicylyl-acyltranferase SsfX3 from a tetracycline biosynthetic pathway

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal β-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical α/β-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone α/β-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.     

M. tuberculosis sliding β-clamp does not interact directly with the NAD+-dependent DNA ligase

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C222₁ with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent K_d of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD⁺ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria.     

Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)₇-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.     

Human DND1‐RRM2 forms a non‐canonical domain swapped dimer

RNA recognition motif (RRM) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Several variations in the canonical βαββαβ topology have been observed. We have determined the 2.3 Å crystal structure of the human DND1‐RRM2 domain. The structure revealed an interesting non‐canonical RRM fold, which is maintained by the formation of a 3D domain swapped dimer between β₁ and β₄ strands across protomers. We have delineated the structural basis of the stable domain swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our structural and dynamics studies substantiate major determinants and molecular basis for domain swapped dimerization observed in the RRM domain.     

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of β-pore-forming toxins such as aerolysin, C. perfringens ϵ-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of β-strands, two of which span its entire length. Domain II and domain III have three short β-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the β-strands. The sequence of amino acids composing the α-helix and preceding β-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming β-barrel-made pores. These structural features imply that CPE is a β-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long β-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.     

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A

We have analyzed the structure and function of the integrin α₁I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α₁I C139S/E317A was a higher avidity collagen binder than α₁I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α₁I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α₂I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α₂I structure, has not changed its position in the activated α₁I variant. During the integrin activation, Glu³³⁵ on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β₁ subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu³³⁵. This indicates that the stabilization of helix 7 into its downward position is not required if the α₁ MIDAS is already open. To conclude, the activated α₁I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg²⁸⁷-Glu³¹⁷ ion pair has just broken during the integrin activation.     

Mode of Binding of the Tuberculosis Prodrug Isoniazid to Heme Peroxidases: BINDING STUDIES AND CRYSTAL STRUCTURE OF BOVINE LACTOPEROXIDASE WITH ISONIAZID AT 2.7 ? RESOLUTION*

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe⁴²² O, Gln⁴²³ O^ϵ1, and Phe²⁵⁴ O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe³⁺, His¹⁰⁹ N^ϵ2, and Gln¹⁰⁵ N^ϵ2. The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C^β and C^γ atoms of Glu²⁵⁸, and C^γ and C^δ atoms of Arg²⁵⁵. The binding studies indicate that INH binds to LPO with a value of 0.9 × 10⁻⁶ m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H₂O₂ with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.     

Structural Basis for the ABO Blood-Group Dependence of Plasmodium falciparum Rosetting

The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1α₁ domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1α₁ and NTS-DBL1α₁-CIDR1γ reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A₁, weaker binding to groups A₂ and B, and least binding to groups A_x and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α₁-CIDR1γ, reveals extensive contacts between the DBL1α₁ and CIDR1γ and shows that the NTS-DBL1α₁ hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα₁. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.     

Structure and Functional Analysis of LptC, a Conserved Membrane Protein Involved in the Lipopolysaccharide Export Pathway in Escherichia coli

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.     

Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.