Probing the outer mitochondrial membrane in cardiac mitochondria with nanoparticles.

The outer mitochondrial membrane (OMM) is the last barrier between the mitochondrion and the cytoplasm. Breaches of OMM integrity result in the release of cytochrome c oxidase, triggering apoptosis. In this study, we used calibrated gold nanoparticles to probe the OMM in rat permeabilized ventricular cells and in isolated cardiac mitochondria under quasi-physiological ionic conditions and during permeability transition. Our experiments showed that under control conditions, the OMM is not permeable to 6-nm particles. However, 3-nm particles could enter the mitochondrial intermembrane space in mitochondria of permeabilized cells and isolated cardiac mitochondria. Known inhibitors of the voltage-dependent anion channel (VDAC), König polyanion, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited this entrance. Thus, 3-nm particles must have entered the mitochondrial intermembrane space through the VDAC. The permeation of the isolated cardiac mitochondria OMM for 3-nm particles was approximately 20 times that in permeabilized cells, suggesting low availability of VDAC pores within the cell. Experiments with expressed green fluorescent protein showed the existence of intracellular barriers restricting the VDAC pore availability in vivo. Thus, our data showed that 1), the physical diameter of VDAC pores in cardiac mitochondria is >or=3 nm but

Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.

To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.

The crystal structure of the fab fragment of the monoclonal antibody MAK33. Implications for folding and interaction with the chaperone bip.

The Fab fragment of the murine monoclonal antibody, MAK33, directed against human creatine kinase of the muscle-type, was crystallized and the three-dimensional structure was determined to 2.9 A. The antigen-binding surface of MAK33 shows a convex overall shape typical for immunoglobulins binding large antigens. The structure allows us to analyze the environment of cis-prolyl-peptide bonds whose isomerization is of key importance in the folding process. These residues seem to be involved with not only domain stability but also seem to play a role in the association of heavy and light chains, reinforcing the importance of beta-strand recognition in antibody assembly. The structure also allows the localization of segments of primary sequence postulated to represent binding sites for the ER-specific chaperone BiP within the context of the entire Fab fragment. These sequences are found primarily in beta-strands that are necessary for interactions between the individual domains.

Crystal structure of a brominated RNA helix with four mismatched base pairs: An investigation into RNA conformational variability.

The X-ray crystal structure of a brominated RNA helix with four mismatched base pairs and sequence r(UG(Br)C(Br)CAGUUCGCUGGC)(2) was determined to 2.1 A using the methods of multiwavelength anomalous diffraction (MAD) applied to the bromine K-absorption edge. There are three molecules in the asymmetric unit with unique crystal-packing environments, revealing true conformational variability at high resolution for this sequence. The structure shows that the sequence itself does not define a consistent pattern of solvent molecules, with the exception of the mismatched base pairs, implying that specific RNA-protein interactions would occur only with the nucleotides. There are a number of significant tertiary interactions, some of which are a result of the brominated base pairs and others that are directly mediated by the RNA 2' hydroxyl groups. The mismatched base pairs exhibit a solvent network as well as a stacking pattern with their nearest neighbors that validate previous thermodynamic analysis.

Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.

The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein. Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.

Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

BACKGROUND: The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation of methane to methanol. The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through the sMMO reductase. Although the X-ray structure of the hydroxylase is known, little structural information is available regarding protein B. RESULTS: Wild-type protein B from Methylococcus capsulatus (Bath) is very susceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein. We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydrophobic contacts. CONCLUSIONS: A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography. The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.

Crystal structure of the tandem phosphatase domains of RPTP LAR.

Most receptor-like protein tyrosine phosphatases (RPTPs) contain two conserved phosphatase domains (D1 and D2) in their intracellular region. The carboxy-terminal D2 domain has little or no catalytic activity. The crystal structure of the tandem D1 and D2 domains of the human RPTP LAR revealed that the tertiary structures of the LAR D1 and D2 domains are very similar to each other, with the exception of conformational differences at two amino acid positions in the D2 domain. Site-directed mutational changes at these positions (Leu-1644-to-Tyr and Glu-1779-to-Asp) conferred a robust PTPase activity to the D2 domain. The catalytic sites of both domains are accessible, in contrast to the dimeric blocked orientation model previously suggested. The relative orientation of the LAR D1 and D2 domains, constrained by a short linker, is stabilized by extensive interdomain interactions, suggesting that this orientation might be favored in solution.

Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions.

The crystal structure of the nonheme iron-containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 A resolution. The enzyme is composed of two copies each of three subunits (alpha 2 beta 2 gamma 2), and all three subunits are almost completely alpha-helical, with the exception of two beta hairpin structures in the alpha subunit. The active site of each alpha subunit contains one dinuclear iron center, housed in a four-helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4 degrees C, a bridging acetate ion, which is replaced at -160 degrees C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural differences identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position.

Intramolecular interactions of the regulatory domains of the Bcr-Abl kinase reveal a novel control mechanism.

BACKGROUND: The Abl nonreceptor tyrosine kinase is implicated in a range of cellular processes and its transforming variants are involved in human leukemias. The N-terminal regulatory region of the Abl protein contains Src homology domains SH2 and SH3 which have been shown to be important for the regulation of its activity in vivo. These domains are often found together in the same protein and biochemical data suggest that the functions of one domain can be influenced by the other. RESULTS: We have determined the crystal structure of the Abl regulatory region containing the SH3 and SH2 domains. In general, the individual domains are very similar to those of previously solved structures, although the Abl SH2 domain contains a loop which is extended so that one side of the resulting phosphotyrosine-binding pocket is open. In our structure the protein exists as a monomer with no intermolecular contacts to which a biological function may be attributed. However, there is a significant intramolecular contact between a loop of the SH3 domain and the extended loop of the SH2 domain. This contact surface includes the SH2 loop segment that is responsible for binding the phosphate moiety of phosphotyrosine-containing proteins and is therefore critical for orienting peptide interactions. CONCLUSIONS: The crystal structure of the composite Abl SH3-SH2 domain provides the first indication of how SH2 and SH3 domains communicate with each other within the same molecule and why the presence of one directly influences the activity of the other. This is the first clear evidence that these two domains are in contact with each other. The results suggest that this direct interaction between the two domains may affect the ligand binding properties of the SH2 domain, thus providing an explanation for biochemical and functional data concerning the Bcr-Abl kinase.

Sequence variation as a strategy for crystallizing RNA motifs.

The determination of RNA structures by X-ray crystallography is an exciting and developing field. At present, the crystallographic characterization of RNA is limited by the difficulty in obtaining large, high quality crystals. This paper outlines several techniques for improving the likelihood of obtaining RNA crystals, for improving the size of those crystals, and for extending the limit of the diffraction maxima. Sequence variations have proven to be more effective in changing the quality of the crystals than variations in crystallization conditions, often making the difference between obtaining true single crystals and multiply twinned crystalline material.

HPLC purification of RNA for crystallography and NMR.

Homogeneous preparations of milligram quantities of RNA are a prerequisite for their characterization by biophysical methods such as crystallography or NMR spectroscopy. Methods for obtaining milligram quantities of pure synthetic RNA are described in this paper. These methods employ anion exchange HPLC for purifying full-length sequence from failure sequences and incompletely deprotected material. RNA molecules with little or extensive amounts of secondary structure could be purified. In cases where the RNA molecule was tightly folded, the cation in the eluent buffer influenced both the distinction of the peaks during chromatography and the final folded conformation. Finally, two RNA sequences were chemically synthesized, deprotected, purified, and crystallized using this methodology.

Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin.

The success of cisplatin in cancer chemotherapy derives from its ability to crosslink DNA and alter the structure. Most cisplatin-DNA adducts are intrastrand d(GpG) and d(ApG) crosslinks, which unwind and bend the duplex to facilitate the binding of proteins that contain one or more high-mobility group (HMG) domains. When HMG-domain proteins such as HMG1, IXR (intrastrand-crosslink recognition) protein from yeast, or human upstream-binding factor (hUBF) bind cisplatin intrastrand crosslinks, they can be diverted from their natural binding sites on the genome and shield the adducts from excision repair. These activities sensitize cells to cisplatin and contribute to its cytotoxic properties. Crystallographic information about the structure of cisplatin-DNA adducts has been limited to short single-stranded deoxyoligonucleotides such as cis-[Pt(NH3)2(d(pGpG))]. Here we describe the X-ray structure at 2.6 A resolution of a double-stranded DNA dodecamer containing this adduct. Our information provides, to our knowledge, the first crystallographic look at a platinated DNA duplex and should help the design of new platinum and other metal crosslinking antitumour drug candidates. Moreover, the structure reveals a unique fusion of A- and B-type DNA segments that could be of more general importance.

Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states.

BACKGROUND: The hydroxylase component of soluble methane monooxygenase (sMMO) contains a dinuclear iron center responsible for the oxidation of methane to methanol. As isolated, the center is in the oxidized, diiron(III) state. The 2.2 A resolution X-ray structure of the oxidized hydroxylase, Hox, from Methylococcus capsulatus (Bath) was previously determined at 4 degrees C. In this structure the two iron atoms are bridged by a glutamate, a hydroxide ion, and an acetate ion, and additionally coordinated to two His residues, three Glu residues, and a water molecule. RESULTS: The 1.7 A resolution crystal structures of the sMMO hydroxylase from Methylococcus capsulatus (Bath) in both its oxidized diiron(III), Hox, and dithionite-treated, reduced diiron(II), Hred, oxidation states were determined at -160 degrees C. The structure of the diiron center in Hox differs from that previously reported at 2.2 A resolution and 4 degrees C. Although the hydroxide bridge is retained, the bidentate, bridging ligand assigned as acetate is replaced by a weakly coordinating monoatomic water bridge. In the resulting four-membered Fe(OH)Fe(OH2) ring, the Fe ... Fe distance is shortened from 3.4 A to 3.1 A. In protomer A of Hred, the hydroxide bridge is displaced by an oxygen atom of Glu243, which undergoes a carboxylate shift from its terminal monodentate binding mode in Hox to a mode in which the carboxylate is both monoatomic bridging and bidentate chelating. We therefore conclude that the center has been reduced to the diiron(II) oxidation state. Both iron atoms are coordinated to five ligands and weakly to a sixth water molecule in the resulting structure. The diiron center in protomer B of Hred has the same composition as those in Hox. In both the oxidized and reduced structures, the diiron core is connected through hydrogen bonds involving exogenous species to Thr213 in the active site cavity. CONCLUSIONS: The diiron center in Hox can change its exogenous ligand coordination and geometry, a property that could be important in the catalytic cycle of sMMO. In Hred, a carboxylate shift occurs, extruding hydroxide ion and opening coordination sites for reaction with O2 to form the diiron(III) peroxo intermediate, Hperoxo. Residue Thr213 may function in catalysis.

Geometry of the soluble methane monooxygenase catalytic diiron center in two oxidation states.

BACKGROUND: The hydroxylase component of soluble methane monooxygenase (sMMO) contains a dinuclear iron center responsible for the oxidation of methane to methanol. As isolated, the center is in the oxidized, diiron(III) state. The 2.2 A resolution X-ray structure of the oxidized hydroxylase, Hox, from Methylococcus capsulatus (Bath) was previously determined at 4 degrees C. In this structure the two iron atoms are bridged by a glutamate, a hydroxide ion, and an acetate ion, and additionally coordinated to two His residues, three Glu residues, and a water molecule. RESULTS: The 1.7 A resolution crystal structures of the sMMO hydroxylase from Methylococcus capsulatus (Bath) in both its oxidized diiron(III), Hox, and dithionite-treated, reduced diiron(II), Hred, oxidation states were determined at -160 degrees C. The structure of the diiron center in Hox differs from that previously reported at 2.2 A resolution and 4 degrees C. Although the hydroxide bridge is retained, the bidentate, bridging ligand assigned as acetate is replaced by a weakly coordinating monoatomic water bridge. In the resulting four-membered Fe(OH)Fe(OH2) ring, the Fe ... Fe distance is shortened from 3.4 A to 3.1 A. In protomer A of Hred, the hydroxide bridge is displaced by an oxygen atom of Glu243, which undergoes a carboxylate shift from its terminal monodentate binding mode in Hox to a mode in which the carboxylate is both monoatomic bridging and bidentate chelating. We therefore conclude that the center has been reduced to the diiron(II) oxidation state. Both iron atoms are coordinated to five ligands and weakly to a sixth water molecule in the resulting structure. The diiron center in protomer B of Hred has the same composition as those in Hox. In both the oxidized and reduced structures, the diiron core is connected through hydrogen bonds involving exogenous species to Thr213 in the active site cavity. CONCLUSIONS: The diiron center in Hox can change its exogenous ligand coordination and geometry, a property that could be important in the catalytic cycle of sMMO. In Hred, a carboxylate shift occurs, extruding hydroxide ion and opening coordination sites for reaction with O2 to form the diiron(III) peroxo intermediate, Hperoxo. Residue Thr213 may function in catalysis.

Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane.

The 2.2 A crystal structure of the 251K alpha 2 beta 2 gamma 2 dimeric hydroxylase protein of methane monooxygenase from Methylococcus capsulatus (Bath) reveals the geometry of the catalytic di-iron core. The two iron atoms are bridged by exogenous hydroxide and acetate ligands and further coordinated by four glutamate residues, two histidine residues and a water molecule. The dinuclear iron centre lies in a hydrophobic active-site cavity for binding methane. An extended canyon runs between alpha beta pairs, which have many long alpha-helices, for possible docking of the reductase and coupling proteins required for catalysis.

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.

Crystallization and preliminary X-ray analysis of the methane monooxygenase hydroxylase protein from Methylococcus capsulatus (Bath).

Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition alpha 2 beta 2 gamma 2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2(1)2(1)2(1), with one dimer of relative molecular mass M(r) = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 A, b = 110.1 A, c = 333.5 A. The crystals diffract uniformly beyond 2.5 A resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.

DNA-nogalamycin interactions.

The anthracycline antibiotic nogalamycin differs from the more common daunomycin-type anthracyclines by substitution on both ends of the intercalating chromophore, giving nogalamycin the approximate shape of a dumbbell. The chromophore of daunomycin is substituted on only one end. In nogalamycin, the positively charged amino sugar substituent of daunomycin is replaced by an uncharged nogalose sugar and a methyl ester group. The other end of nogalamycin, where daunomycin is unsubstituted, is fused to a bicyclo amino sugar with a positively charged dimethylamino group. Much larger DNA fluctuations are required for intercalative entry of nogalamycin than for entry of daunomycin. This report describes the X-ray crystal structure of the complex between nogalamycin and the self-complementary DNA hexamer d(me5CGTsAme5CG). The DNA contains cytosines methylated at the 5-positions and a phosphorothioate linkage at the TpA step. Nogalamycin intercalates at the terminal CpG steps and interacts with both strands in both grooves of the DNA. Large conformational adjustments in both nogalamycin and the DNA are necessary to form a stable, intercalative complex. The interactions of the bases with the nogalamycin substituents lead to sliding of bases relative to each other along the normal to Watson-Crick hydrogen bonds. The planarities of base pairs surrounding the intercalation site are distorted. The backbones of the two strands are distorted asymmetrically by nogalamycin with large deviations from standard B-DNA geometry. The complex between nogalamycin and DNA illustrates the conformational flexibility of DNA. The hydrogen-bonding interactions between nogalamycin and DNA do not suggest a sequence-specific binding of the drug, although additional secondary effects might lead to differences between various intercalation sites.

Ternary interactions of spermine with DNA: 4'-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.

Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate.

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.