Crystal structure of the BMP-2-BRIA ectodomain complex

Bone morphogenetic proteins (BMPs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP-2 can induce ectopic bone and cartilage formation in adult vertebrates and is involved in central steps in early embryonal development in animals. Signaling by these cytokines requires binding of two types of transmembrane serine/threonine receptor kinase chains classified as type I and type II. Here we report the crystal structure of human dimeric BMP-2 in complex with two high affinity BMP receptor IA extracellular domains (BRIAec). The receptor chains bind to the 'wrist' epitopes of the BMP-2 dimer and contact both BMP-2 monomers. No contacts exist between the receptor domains. The model reveals the structural basis for discrimination between type I and type II receptors and the variability of receptor-ligand interactions that is seen in BMP-TGF-beta systems.     

Crystal structure of the human TbetaR2 ectodomain--TGF-beta3 complex

Transforming growth factor-beta (TGF-beta) is the prototype of a large family of structurally related cytokines that play key roles in maintaining cellular homeostasis by signaling through two classes of functionally distinct Ser/Thr kinase receptors, designated as type I and type II. TGF-beta initiates receptor assembly by binding with high affinity to the type II receptor. Here, we present the 2.15 A crystal structure of the extracellular ligand-binding domain of the human TGF-beta type II receptor (ecTbetaR2) in complex with human TGF-beta3. ecTbetaR2 interacts with homodimeric TGF-beta3 by binding identical finger segments at opposite ends of the growth factor. Relative to the canonical 'closed' conformation previously observed in ligand structures across the superfamily, ecTbetaR2-bound TGF-beta3 shows an altered arrangement of its monomeric subunits, designated the 'open' conformation. The mode of TGF-beta3 binding shown by ecTbetaR2 is compatible with both ligand conformations. This, in addition to the predicted mode for TGF-beta binding to the type I receptor ectodomain (ecTbetaR1), suggests an assembly mechanism in which ecTbetaR1 and ecTbetaR2 bind at adjacent positions on the ligand surface and directly contact each other via protein--protein interactions.     

Crystal structure of the human MUS81-EME2 complex

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Crystal structure of the koh-amylose complex

The crystal structure of potassium hydroxide complexed amylose, obtained by heterogeneous deacetylation of amylose triacetate, has been determined through a combined stereochemical structure-refinement and X-ray diffraction-analysis. The structure crystallizes in an orthorhombic unit-cell with parameters a  8.84, b  12.31, and c (fiber repeat)  22.41 Å, and with P2₁2₁2₁ symmetry. The conformation of the amylose chain is a distorted, left-handed helix with 6 d-glucose residues per turn. Each three-residue asymmetric unit is complexed with one molecule of potassium hydroxide and three molecules of water. The K⁺ ion coordinates with four oxygen atoms of the amylose chain and with two other oxygen atoms, and this coordination is probably the cause for the more-extended amylose chain-conformation than would be predicted from a φ, ψ map. The distortions in the chain are primarily manifested by different O-6 rotations and by slightly different bridge and φ, ψ angles for the individual residues. The structure is extensively hydrogen bonded, although largely through water molecules, which accounts for its ready water solubility. The left-handed conformation of the chain in this structure is consistent with the conformations of amylose triacetate and V-amylose, both of which are left-handed.     

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.     

Crystal structure of the ascorbate peroxidase-ascorbate complex

Heme peroxidases catalyze the H2O2-dependent oxidation of a variety of substrates, most of which are organic. Mechanistically, these enzymes are well characterized: they share a common catalytic cycle that involves formation of a two-electron, oxidized Compound I intermediate followed by two single-electron reduction steps by substrate. The substrate specificity is more diverse--most peroxidases oxidize small organic substrates, but there are prominent exceptions--and there is a notable absence of structural information for a representative peroxidase-substrate complex. Thus, the features that control substrate specificity remain undefined. We present the structure of the complex of ascorbate peroxidase-ascorbate. The structure defines the ascorbate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase catalysis for more than 20 years. A new mechanism for electron transfer is proposed that challenges existing views of substrate oxidation in other peroxidases.     

Crystal structure of quinone reductase 2 in complex with resveratrol

Resveratrol has been shown to have chemopreventive, cardioprotective, and antiaging properties. Here, we report that resveratrol is a potent inhibitor of quinone reductase 2 (QR2) activity in vitro with a dissociation constant of 35 nM and show that it specifically binds to the deep active-site cleft of QR2 using high-resolution structural analysis. All three resveratrol hydroxyl groups form hydrogen bonds with amino acids from QR2, anchoring a flat resveratrol molecule in parallel with the isoalloxazine ring of FAD. The unique active-site pocket in QR2 could potentially bind other natural polyphenols such as flavonoids, as proven by the high affinity exhibited by quercetin toward QR2. K562 cells with QR2 expression suppressed by RNAi showed similar properties as resveratrol-treated cells in their resistance to quinone toxicity. Furthermore, the QR2 knockdown K562 cells exhibit increased antioxidant and detoxification enzyme expression and reduced proliferation rates. These observations could imply that the chemopreventive and cardioprotective properties of resveratrol are possibly the results of QR2 activity inhibition, which in turn, up-regulates the expression of cellular antioxidant enzymes and cellular resistance to oxidative stress.     

Crystal structure of beta-cyclodextrin-dimethylsulfoxide inclusion complex

beta-Cyclodextrin (beta-CD) crystallizes from 27% DMSO-water as beta-CD.0.5DMSO.7.35H(2)O in the monoclinic space group P2(1) with unit cell constants: a=15.155(1), b=10.285(1), c=20.906(1) A, beta=109.86(1) degrees. Anisotropic refinement of 888 atomic parameters against 9,127 X-ray diffraction data converged at an R-factor of 0.055. The beta-CD macrocycle adopts a 'round' conformation stabilized by intramolecular, interglucose O-3(n) triplebond O-2(n+1) hydrogen bonds. In the beta-CD cavity, DMSO, water sites W-1, W-3 (occupancies 0.5, 0.25, 0.75) are not located concurrently with the water site W-2 because the interatomic distances to W-2 are too short (1.56-1.75 A). DMSO is placed in the beta-CD cavity such that its S-atom is shifted from the O-4 plane center to the beta-CD O-6-side ca. 0.9 A and the C-S bond which is inclined 13.6 degrees to the beta-CD molecular axis. It is maintained in position by hydrogen bonding to water site W-3 and the O-31-H group. 7.35 water molecules are extensively disordered in 13 positions both inside (W-1-W-4) and outside (W-5-W-13) the beta-CD cavity. They act as hydrogen bonding mediators contributing significantly to the stability of the crystal structure.     

Crystal structure of the S15-rRNA complex

In bacterial ribosomes, the small (30S) ribosomal subunit is composed of 16S rRNA and 21 distinct proteins. Ribosomal protein S15 is of particular interest because it binds primarily to 16S rRNA and is required for assembly of the small subunit and for intersubunit association, thus representing a key element in the assembly of a whole ribosome. Here we report the 2.8 ? resolution crystal structure of the highly conserved S15-rRNA complex. Protein S15 interacts in the minor groove with a G-U/G-C motif and a three-way junction. The latter is constrained by a conserved base triple and stacking interactions, and locked into place by magnesium ions and protein side chains, mainly through interactions with the unique three-dimensional geometry of the backbone. The present structure gives insights into the dual role of S15 in ribosome assembly and translational regulation.     

Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071

In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5′-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis.     

Crystal structure of beta-cyclodextrin-benzoic acid inclusion complex

The inclusion complex of beta-cyclodextrin (beta-CD) with benzoic acid (BA) has been characterized crystallographically. Two beta-CDs cocrystallize with two BAs, 0.7 ethanol and 20.65 water molecules [2(C(6)H(10)O(5))(7).2(C(7)H(6)O(2)).0.7(C(2)H(6)O).20.65H(2)O] in the triclinic space group P1 with unit cell constants: a=15.210(1), b=15.678(1), c=15.687(1) A, alpha=89.13(1), beta=74.64(1), gamma=76.40(1) degrees. The anisotropic refinement of 1840 atomic parameters against 16,201 X-ray diffraction data converged at R=0.078. In the crystal lattice, beta-CD forms dimers stabilized by direct O-2(m)_1/O-3(m)_1...O-2(n)_2/O-3(n)_2 hydrogen bonds (intradimer) and by indirect O-6(m)_1...,O-6(n)_2 hydrogen bonds with one or two bridging water molecules joined in between (interdimer). These dimers are stacked like coins in a roll constructing endless channels where the guest molecules are included. The BA molecules protrude with their COOH groups at the beta-CD O-6-sides and are maintained in positions by hydrogen bonding to the surrounding O-6-H groups and water molecules. Water molecules (20.65) are distributed over 30 positions in the interstices between beta-CD molecules, except the water sites W-1, W-2 that are located in the channel of the beta-CD dimer. Water site W-2 is hydrogen bonded to the disordered ethanol molecule (occupancy 0.7).     

Crystal structure of a lysozyme-tetrasaccharide lactone complex

The binding of a proposed transition-state analogue, the δ-lactone derived from tetra-N-acetylchitotetraose, to lysozyme in the crystal at pH 2.6 has been studied by X-ray diffraction techniques to a resolution of 2.5 Å. The tetrasaccharide lactone is bound in sites A, B, C, D with sugar residues located in sites A, B and C in similar positions to those observed previously in the complex with tri-N-acetylchitotriose. Analysis of the electron density map for site D, by direct model-building and with a computer model-building programme, indicates that the δ-lactone ring is in a conformation close to a sofa or a boat which brings the hydroxymethyl group C₍₆₎O₍₆₎ axial. These studies provide support for the role of strain in the proposed mechanism of lysozyme catalysis. The orientation of the lactone group in site D is slightly different from that originally derived by hypothetical model-building.     

Crystal structure of a papain-E-64 complex

E-64 [1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl] amino]-4-guanidinobutane] is an irreversible inhibitor of many cysteine proteases. A papain-E-64 complex was crystallized at pH 6.3 by using the hanging drop method. Three different crystal forms grew in 3-7 days; the form chosen for structure analysis has space group P212121, with a = 42.91(4) A, b = 102.02(6) A, c = 49.73(2) A, and Z = 4. Diffraction data were measured to 2.4-A resolution, giving 9367 unique reflections. The papain structure was solved by use of the molecular replacement method, and then the inhibitor was located from a difference electron density map and fitted with the aid of a PS330 computer graphics system. The structure of the complex was refined to R = 23.3%. Our analysis shows that a covalent link is formed between the sulfur of the active-site cysteine 25 and the C-2 atom of the inhibitor. Contrary to earlier predictions, the E-64 inhibitor clearly interacts with the S subsites on the enzyme rather than the S' subsites, and papain's histidine 159 imidazole group plays a binding rather than a catalytic role in the inactivation process.     

Crystal structure of a gamma-herpesvirus cyclin-cdk complex

Several gamma-herpesviruses encode proteins related to the mammalian cyclins, regulatory subunits of cyclin-dependent kinases (cdks) essential for cell cycle progression. We report a 2.5 A crystal structure of a full-length oncogenic viral cyclin from gamma-herpesvirus 68 complexed with cdk2. The viral cyclin binds cdk2 with an orientation different from cyclin A and makes several novel interactions at the interface, yet it activates cdk2 by triggering conformational changes similar to cyclin A. Sequences within the viral cyclin N-terminus lock part of the cdk2 T-loop within the core of the complex. These sequences and others are conserved amongst the viral and cellular D-type cyclins, suggesting that this structure has wider implications for other cyclin-cdk complexes. The observed resistance of this viral cyclin-cdk complex to inhibition by the p27(KIP:) cdk inhibitor is explained by sequence and conformational variation in the cyclin rendering the p27(KIP:)-binding site on the cyclin subunit non-functional.     

Crystal structure of the excisionase-DNA complex from bacteriophage lambda

The excisionase (Xis) protein from bacteriophage lambda is the best characterized member of a large family of recombination directionality factors that control integrase-mediated DNA rearrangements. It triggers phage excision by cooperatively binding to sites X1 and X2 within the phage, bending DNA significantly and recruiting the phage-encoded integrase (Int) protein to site P2. We have determined the co-crystal structure of Xis with its X2 DNA-binding site at 1.7A resolution. Xis forms a unique winged-helix motif that interacts with the major and minor grooves of its binding site using an alpha-helix and an ordered beta-hairpin (wing), respectively. Recognition is achieved through an elaborate water-mediated hydrogen-bonding network at the major groove interface, while the preformed hairpin forms largely non-specific interactions with the minor groove. The structure of the complex provides insights into how Xis recruits Int cooperatively, and suggests a plausible mechanism by which it may distort longer DNA fragments significantly. It reveals a surface on the protein that is likely to mediate Xis-Xis interactions required for its cooperative binding to DNA.     

Crystal structure of the Rna14-Rna15 complex

A large protein machinery is required for 3'-end processing of mRNA precursors in eukaryotes. Cleavage factor IA (CF IA), a complex in the 3'-end processing machinery in yeast, contains four subunits, Rna14, Rna15, Clp1, and Pcf11. Rna14 has a HAT (half a TPR) domain at the N terminus and a region at the C terminus that mediates interactions with Rna15. Rna15 contains a RNA recognition module (RRM) at the N terminus, followed by a hinge region. These two proteins are homologs of CstF-77 and CstF-64 in the cleavage stimulation factor (CstF) of the mammalian 3'-end processing machinery. We report the first crystal structure of Rna14 in complex with the hinge region of Rna15, and the structures of the HAT domain of Rna14 alone in two different crystal forms. The complex of the C-terminal region of Rna14 with the hinge region of Rna15 does not have strong interactions with the HAT domain of Rna14, and this complex is likely to function independently of the HAT domain. Like CstF-77, the HAT domain of Rna14 is also a tightly associated dimer with a highly elongated shape. However, there are large variations in the organization of this dimer among the Rna14 structures, and there are also significant structural differences to CstF-77. These observations suggest that the HAT domain and especially its dimer may have some inherent conformational variability.     

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.     

Crystal structure of EMS16 in complex with the integrin alpha2-I domain

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.