Search

Aβ binding alcohol dehydrogenase (ABAD) is an NAD-dependent mitochondrial dehydrogenase. The binding between ABAD and Aβ is likely a direct link between Aβ and mitochondrial toxicity in Alzheimer's disease. In this study, surface plasmon resonance (SPR) was employed to determine the temperature dependence of the affinity of the ABAD-Aβ interaction. A van't Hoff analysis revealed that the ABAD-Aβ association is driven by a favorable entropic change (ΔS = 300 ± 30 J mol-1 K-1) which overcomes an unfavorable enthalpy change (ΔH = 49 ± 7 kJ/mol). Therefore, hydrophobic interactions and changes in protein dynamics are the dominant driving forces of the ABAD-Aβ interaction. This is the first dissection of the entropic and enthalpic contribution to the energetics of a protein-protein interaction involving Aft. SPR confirmed the conformational changes in the ABAD-Aβ complex after Aβ binding, consistent with differences seen in the crystal structures of free ABAD and the ABAD-Aβ complex. Saturation transfer difference (STD) NMR experiments directly and unambiguously demonstrated the inhibitory effect of Aβ on the ABAD-NAD interaction. Conversely, NAD inhibits the Aβ-ABAD interaction. Binding of Aβ and binding of NAD to ABAD are likely mutually exclusive. Thus, Aβ binding induces conformational and subsequently functional changes in ABAD, which may have a role in the mechanism of Aβ toxicity in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Solution ¹H NMR has been used to characterize the active site molecular and electronic structure of the cyanide-inhibited 2,4-dimethyldeuterohemin complex of the heme oxygenase from Neisseria meningitidis (NmHO) with respect to the mode of interaction of the C-terminus with the substrate and the spontaneous ‘aging’ of NmHO that results in the cleavage of the C-terminal Arg208-His209 dipeptide. The structure of the portion involving residues Ala12-Phe192 is found to be essentially identical to that of the protohemin complex in either solution or crystal. However, His207 from the C-terminus is found to interact strongly with the substrate 1CH3, as opposed to the 8CH3 in the protohemin complex. The different mode of interaction of His207 with the alternate substrates is attributed to the 2-vinyl group of protohemin sterically interfering with the optimal orientation of the proximal helix Asp27 carboxylate that serves as acceptor to the strong H-bond by the peptide of His207. The 2,4-dimethyldeuterohemin HO complex ‘ages’ in manner similary to that of protohemin, (Liu, Y., Ma, L.-H., Satterlee, J.D., Zhang, X., Yoshida, T., and La Mar, G. N., (2006) Biochemistry 45, 3875–3886) with mass spectrometry and N-terminal sequencing indicating that the Arg208-His209 dipeptide is cleaved. The 2,4-dimethyldeutero-hemin complex of WT HO populates an equilibrium isomer stabilized in low phosphate concentration for which the axial His imidazole ring is rotated by ∼20° from that in the WT. The His ring reorientation is attributed to Asp24 serving as the H-bond acceptor to the His207 peptide NH, rather than to the His23 ring NδH as in the crystals. The functional implications of the altered C-terminal interaction with substrate modification are discussed. [ABSTRACT FROM AUTHOR]

The substrate and active site residues of the low-spin hydroxide complex of the protohemin complex of Neisseria meningitidis heme oxygenase (NmHO) have been assigned by saturation transfer between the hydroxide and previously characterized aquo complex. The available dipolar shifts allowed the quantitation of both the orientation and anisotropy of the paramagnetic susceptibility tensor. The resulting positive sign, and reduced magnitude of the axial anisotropy relative to the cyanide complex, dictate that the orbital ground state is the conventional "dπ" (dxy²(dxz, dyz)³); and not the unusual "dxy" (dxz²dyz²dxy) orbital ground state reported for the hydroxide complex of the homologous heme oxygenase (HO) from Pseudomonas aeruginosa (Caignan, G.; Deshmukh, R.; Zeng, Y.; Wilks, A.; Bunce, R. A.; Rivera, M. J. Am. Chem. Soc. 2003, 125, 11842-11852) and proposed as a signature of the HO distal cavity. The conservation of slow labile proton exchange with solvent from pH 7.0 to 10.8 confirms the extraordinary dynamic stability of NmHO complexes. Comparison of the diamagnetic contribution to the labile proton chemical shifts in the aquo and hydroxide complexes reveals strongly conserved bond strengths in the distal H-bond network, with the exception of the distal His53 Nϵ1H. The iron-ligated water is linked to His53 primarily by a pair of nonligated, ordered water molecules that transmit the conversion of the ligated H-bond donor (H2O) to a H-bond acceptor (OH-), thereby increasing the H-bond donor strength of the His53 side chain. [ABSTRACT FROM AUTHOR]

The hydrogen bonding of ligated water in ferric, high-spin, resting-state substrate complexes of heme oxygenase from Neisseria meningitidis has been systematically perturbed by variable electron-withdrawing substituents on the hemin periphery. The pattern of 1H NMR-detected dipolar shifts due to the paramagnetic anisotropy is strongly conserved among the four complexes, with the magnitude of dipolar shifts or anisotropy increasing in the order of substituent formyl < vinyl < methyl. The magnetic anisotropy is axial and oriented by the axial Fe-His23 bond, and while individual anisotropies have uncertainties of ∼5%, the relative values of Δχ (and the zero-field splitting constant, D ∝ Δχax) are defined to 1%. The unique changes in the axial field strength implied by the variable zero-field splitting are in accord with expectations for the axial water serving as a stronger H-bond donor in the order of hemin substituents formyl > vinyl > methyl. These results establish the axial anisotropy (and D) as a sensitive probe of the H-bonding properties of a ligated water in resting-state, substrate complexes of heme oxygenase. Correction of observed labile proton chemical shifts for paramagnetic influences indicates that Gin49 and His53, some ∼ 10 Å from the iron, sense the change in the ligated water H-bonding to the three nonligated ordered water molecules that link the two side chains to the iron ligand. The present results augur well for detecting and characterizing changes in distal water H-bonding upon mutagenesis of residues in the distal network of ordered water molecules and strong H-bonds. [ABSTRACT FROM AUTHOR]

Solution ¹H NMR spectroscopy and mass spectrometry are utilized to characterize the irreversible "aging" of native heme oxygenase from N. meningitidis, NmHO. 2D NMR characterization of the cyanide-inhibited substrate complex shows that the C-terminal interaction between Arg208His209 and the exposed pyrrole of the protohemin substrate in the "native" NmHO complex is lost in the "aging". Mass spectrometry and N-terminal sequencing of wild type and "aged" NmHO reveal that the "aging" process involves cleavage of the Arg208His209 dipeptide. The construction of the double deletion mutant without Arg208His209 and its NMR comparison as both the resting state substrate complex and its cyanide-inhibited complex with the "aged" NmHO reveal that cleavage of the C-terminal dipeptide is the only modification during the aging. Comparison of cyanide ligand binding constants reveal a factor ~1.7 greater CN- affinity in the native than "aged" NmHO. The rate of protohemin degradation and its stereoselectivity are unaffected by the C-terminal truncation. However, the free α-biliverdin yield in the presence of desferrioxamine is significantly increased in the "aged" NmHO and its deletion mutant relative to WT, arguing for a role of the NmHO C-terminus in modulating product release. The facile cleavage of Arg208His209 in the resting state complex, with a half-life of ~24 h at 25 °C, suggests that previous characterization of NmHO may have been carried out on a mixture of native and "aged" NmHO, and may account for the "lost" C-terminal residues in the crystal structures. [ABSTRACT FROM AUTHOR]

Heme oxygenase, HO, from the pathogenic bacterium Neisseria meningitidis catabolizes heme for the iron necessary for infection. The enzyme, labeled HemO, exhibits less sequence homology to mammalian HO than another studied HO from Corynebacterium diphtheriae. Solution 1H NMR has been utilized to define the active site molecular and electronic structure of the cyanide-inhibited, substrate- bound complex for comparison with those provided by several crystal structures. Extensive assignments by solely 1H NMR 2D methods reveal a structure that is very strongly conserved with respect to the crystal structure, although 1H/2H exchange indicates dynamically much more stable distal and proximal helices than those for other HOs. Several residues found with alternate orientations in crystal structures of water- and NO-ligated complexes were shown to occupy positions found solely in the NO complex, confirming that there are structural accommodations in response to ligating the substrate complex with a diatomic, H-bond acceptor ligand. The observed dipolar shifts allow the determination of the magnetic axes that show that the Fe--CN unit is tilted &ap; 10° toward the α-meso position, thereby facilitating the α-stereoselectivity of the enzyme. Numerous labile protons with larger than usual low-field bias are identified and, in common with the other HO complexes, shown to participate in an extended, distal side H-bond network. This H-bond network orders several water molecules, most, but not all, of which have been detected crystallographically. A series of three C-terminal residues, His207-Arg208His209, are not detected in crystal structures. However, 1H NMR finds two residues, His207 and likely Arg208 in contact with pyrrole D, which in crystal structures is exposed to solvent. The nature of the NOEs leads us to propose a H-bond between the proximally oriented His207 ring and the carboxylate of Asp27 and a salt- bridge between the terminus of Arg208 and the reoriented 7-propionyl carboxylate. While numerous ordered water molecules are found near both propionates in the crystal structure, we find much larger water NOEs to the 6- than 7-propionate, suggesting that water molecules near the 7-propionate have been expelled from the cavity by the insertion of Arg208 into the distal pocket. The conversion of the 7-propionate link from the N-terminal region (Lys 16) to the C-terminal region (Arg208) in the ligated substrate complex both closes the heme cavity more tightly and may facilitate product exit, the rate-limiting step in the enzyme activity. [ABSTRACT FROM AUTHOR]

The reactions of the anticancer complex trans-[PtCI[SUB2]{(E)-HN=C(OMe)Me}[SUB2]] (trans-EE) with a series of ribo and deoxyribodinucleotides have been studied by HPLC and 2D [SUP1]H, [SUB15]N] HMQC NMR spectroscopy and compared with those of the inactive trans isomer of cisplatin, trans-[PtCI[SUB2](NH[SUB3])[SUB2]] (transDDP). Reactions of trans-EEwith r(ApG) and d(ApG) take place through solvolysis of the starting substrate and subsequent formation of trans G-N7/monochloro and G-N7/monoaqua adducts. Slowly, the monofunctional adducts evolve to a bifunctional adduct forming an unprecedented and unexpected A-N3/ G-N7 platinum cross-link spanning two trans positions. For stereochemical reasons, trans platinum complexes cannot form N7/N7 cross-links between adjacent purines in di- or polynucleotides. For the reverse sequence r(GpA), no chelate structure was formed even after a two-week reaction. The reaction of transDDP with r(ApG) produces many more products than the analogous reaction with trans-EE. One of these products was identified as the A-N3/G-N7 trans-chelate. [ABSTRACT FROM AUTHOR]

One flexible star-shaped bridging molecule tris-(4-cyanophenyl)amine (TCPA) is synthesized from tris-(4-iodophenyl)amine by treatment with CuCN in hexamethylphosphorotriamide (HMPA) as a catalyst in high yield. Anion-template-controlled reactions of TCPA and silver salts give a series of coordination polymers (CPs) with various topological patterns. The complex [Ag(TCPA)(ClO4)]n·nC6H6(1) forms an undulating 4.82network, which displays unusual net entanglement. Two 4.82nets interweave each other to give rise to 2-fold interpenetrating basic layers, which link together via double μ-Operchloratebridging interactions. The complex [Ag(TCPA)(CF3SO3)]n·2nC6H6(2) forms noninterpenetrating (6,3) nets. The solid state structure of [Ag3(TCPA)(CF3CO2)3]n·nCH2Cl2(3) also is a (6,3) honeycomb network, differently, including a sphere-type Ag4(CF3CO2)62−cluster-anion in the host frameworks, which is a rare example of the host cationic network containing a guest cluster-anion. The complexes [Ag(TCPA)(BF4)·0.5H2O]n·1.5nC6H6(4), [Ag(TCPA)(ClO4)·0.5H2O]n·nC7H8·nH2O (5), and [Ag(TCPA)(CF3SO3)·0.5H2O]n·1.5nC6H6(6) exhibit three similar (10,3) networks which form rare net entanglements. Four (10,3) nets interweave to generate a 4-fold interpenetrating assemble, which are closely united together via specific μ2-Owaterbridging interactions. The complex [Ag4(TCPA)2(CF3CO2)3(CF3SO3)]n·nC6H6·nCH2Cl2(7) is a three-dimensional (3D) structure formed via multiple “zigzag” Ag4chains. In this AgI-TCPA system, the structural diversities and topological differences of these networks result from the flexibilities of the bridging ligand TCPA and the template-effect of anions. The free rotation of three Ph−CN “arms” around the central N moiety results in TCPA adopting different conformation in the self-assembly process by using variational solvent system and/or counterions. [ABSTRACT FROM AUTHOR]

Protein splicing is a precise autocatalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid-base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved "B-block" histidine. In this study, we have combined NMR pKa, determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pKa shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pka of 7.3 ± 0.6 in a precursor and a pKa of <3.5 in a spliced intein. The pKa values and QM/MM data suggest that the B-block histidine has a dual role in the acid-base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond at the N-terminal splicing junction. The proposed pKa shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the near absolute sequence conservation of this residue. [ABSTRACT FROM AUTHOR]

In situ living cell protein analysis would enable the structural identification and functional interrogation of intracellular proteins in native cellular environments. Previously, we have presented an in situ mass spectrometry (MS) strategy to identify protein and protein/metal ion complex with relatively small molecular weight (Anal. Chem. 2016, 88, 10860-10866). However, it is still challenging to directly identify larger proteins and protein/ligand complexes in cell, due to numerous nonspecific bindings of ligands, solvents, and other cellular constituents. Here we present a versatile single-step mass spectrometric strategy, "in-cell" mass spectrometry ("in-cell" MS), for in situ protein identification and dynamic protein-ligand interaction monitoring directly from living cells. "In-cell" MS combined all-ion-fragmentation mode with our previous method; thus, on a high-resolution MS instrument, we can greatly improve the signal/noise ratio of the larger proteins and protein/ligand complexes. Meanwhile, we also achieved a much wider mass range for protein complex and detection of 17 proteins with molecular weight ranging from 4 to 44 kDa. In addition, "in-cell" MS could also monitor dynamic protein interactions in living cells. Calcium-regulated calmodulin-melittin interaction was tested to demonstrate the proof of concept. "In-cell" MS provides an alternative for in situ analysis of living cells, which might contribute to rapid protein analysis and quality control in biochemistry laboratories, protein engineering, and even protein industry. [ABSTRACT FROM AUTHOR]

The identification of endogenous proteins as well as their binding to metal ions in living cells is determined by combining pulsed electrophoretic separations with nanoelectrospray ionization followed by mass spectrometric detection. This approach avoids problems resulting from the complicated cellular environment. In this manner, we demonstrate the rapid identification (300 ms or less) of intact proteins from living E. coli cells including the complexation of calmodulin with calcium ion. The latter showed different binding states from those observed in in vitro studies. These observations also reveal in vitro measurements do not necessarily represent the actual situation in living cells. We conclude that the attempted in situ measurement of intracellular proteins with minimal sampling processes should be preferred. [ABSTRACT FROM AUTHOR]

The copper chaperone Cox17 (cytochrome c oxidase copper chaperone) has been shown to facilitate the delivery of cisplatin to mitochondria, which contributes to the overall cytotoxicity of the drug [Zhao et al. (2014) Chem.Commun. 50, 2667-2669].Kinetic data indicate that Cox17 has reactivity similar to glutathione (GSH), the most abundant thiol-rich molecule in the cytoplasm. In the present study, we found that GSH significantly modulates the reaction of platinum complexes with Cox17. GSH enhances the reactivity of three anti-cancer drugs (cisplatin, carboplatin and oxaliplatin) to Cox17, but suppresses the reaction of transplatin. Surprisingly, the pre-formed cisplatin-GSH adducts are highly reactive to Cox17; over 90% platinum transfers from GSH to Cox17. On the other hand, transplatin-GSH adducts are inert to Cox17. These different effects are consistent with the drug activity of these platinum complexes. In addition, GSH attenuates the protein aggregation of Cox17 induced by platination. These results indicate that the platinum-protein interactions could be substantially influenced by the cellular environment. [ABSTRACT FROM AUTHOR]

Asplatin, a fusion of aspirin and cisplatin, exhibits significant cytotoxicity in tumor cells and almost fully overcomes the drug resistance of cisplatin resistant cells. Asplatin is highly accumulated in cancer cells and is activated upon the reduction by ascorbic acid. [ABSTRACT FROM AUTHOR]

The first crystal trans-structure of a naturally occurring split intein has been determined for the Npu (Nostoc punctiforme PCC73102) DnaE split intein. Guided by this structure, the residues NArg50 and CSer35, well conserved in DnaE split inteins, are identified to be critical in the trans-splicing of Npu DnaE split intein. An in vitro splicing assay demonstrates that NArg50 and CSer35 play synergistic roles in modulating its intein activity. The C-terminal CAsn36 exhibits two orientations of its side chain and interacts with both NArg50 and CSer35 through hydrogen bonding. These interactions likely facilitate the cyclization of asparagine in the course of protein splicing. The mutation of either residue reduces intein activity, and correlates with the low activity of the Ssp (Cyanobacterium synechocystis sp. strain PCC6803) DnaE split intein. On the other hand, NArg50 also forms a hydrogen bond with the highly conserved F-block CAsp17, thus influencing the N-S acyl shift during N-terminal cleavage. Sequence alignments show that residues NArg50 and CSer35 are rather conserved in those split inteins that lack a penultimate histidine residue. The conserved non-catalytic residues of split inteins modulate the efficiency of protein trans-splicing by hydrogen-bond interactions with the catalytic residues at the splice junction. [ABSTRACT FROM AUTHOR]

Although much effort has been made for studies on aptamer-target interactions due to promising applications of aptamers in biomedical and analytical fields, measurement of the aptamer-target binding constant and binding site still remains challenging. Herein, we report a sensitive label-free chemiluminescence (CL) strategy to determine the target concentration and, more importantly, to measure the target-aptamer binding constant and binding site. This approach is suitable for multiple types of targets, including small molecules, peptides, and proteins that can enhance the CL initiated by N-(aminobutyl)-N-ethylisoluminol functionalized gold colloids, making the present method a general platform to investigate aptamer-target interactions. This approach can achieve extremely high sensitivity with nanogram samples for measuring the target-aptamer binding constant. And the measurement could be rapidly performed using a simple and low-cost CL system. It provides an effective tool for studying the binding of biologically important molecules to nucleic acids and the selection of aptamers. Besides, we have also discovered that the 14-mer aptamer fragment itself split from the ATP-binding aptamer could selectively capture ATP. The binding constant, site, and conformation between ATP and the 14-mer aptamer fragment were obtained using such a novel CL strategy and molecular dynamic simulation. [ABSTRACT FROM AUTHOR]

Arsenic is a biologically interesting element with both antitumor and carcinogenic effects. Zinc finger proteins (ZFPs) have been confirmed to be the cellular targets of arsenite; however, arsenite inhibits ZFPs much less efficiently in vitro than in vivo. The molecular mechanism of this difference is unknown. In this work, we found that the reaction of arsenite with ZFPs relies on the presence of small biomolecules such as glutathione (GSH), histidine, and cysteine (Cys). The weak acidity also enhances the reaction. Further study shows that the coordination of zinc is much more susceptible than that of arsenic to these solution conditions, which enhance the competition of arsenic. Notably, different from C3H-type ZFPs, the C2H2-type ZFPs are more significantly influenced by the presence of thiol-containing molecules in the reaction. GSH and Cys can facilitate the reaction by participation of the coordination to As(III) together with C2H2-type ZFPs. Consequently, the reactions are promoted both thermodynamically and kinetically via the formation of ternary complexes GSH-As-ZFP or Cys-As-ZFP. These results indicate that the reactions between arsenite and proteins are considerably modulated by environments such as the small biomolecules and the acidity of the solution. This finding clarifies the discrepancy observed in the reactions of arsenite in vitro versus in cells, and provides an insight into the molecular mechanism of arsenite. [ABSTRACT FROM AUTHOR]

Human copper transporter 1 (hCTR1) facilitates the cellular uptake of cisplatin, and the extracellular N-terminal domain has been proven to coordinate to platinum drugs. It has been reported that the intracellular C-terminal motif is crucial for the function of hCTR1 in cisplatin influx. In this work, we conduct reactions of the intracellular motif with platinum drugs. The octapeptide from the C-terminal domain of hCTR1 is used, and the reactions are investigated using ultraviolet, high-performance liquid chromatography, electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. Results show that the C8 peptide is highly reactive to cisplatin and oxaliplatin, and the -HCH sequence is the most favorable binding site of platinum agents. Cisplatin first binds to the cysteine residues in the reaction with the C8 peptide. The ammine ligand, even trans to a thiol ligand, can remain coordinated in platination adducts for a >12 h reaction. Intramolecular platinum migration was observed in the C8 peptide, and the ammine ligands remain coordinated to platinum during this process. This result indicates that hCTR1 can transfer cisplatin in the active form through a trans chelation process. These findings provide insight into the mechanism of the C-terminus of hCTR1 in the transfer of platinum drugs from the trimeric pore of hCTR1 to the cytoplasm. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that sulfur-containing molecules can play important roles in the activity of platinum anticancer drugs. Although nuclear DNA is retained to be the ultimate target, these platinum compounds can readily react with a variety of other substrates containing a soft donor atom, such as proteins, peptides, and low molecular weight biomolecules, before reaching DNA. In a recent study it was demonstrated that the DNA platination rate of a trans-geometry antitumor drug was dramatically enhanced by methionine binding, thus suggesting that the thioether could serve as a catalyst for DNA platination. In this work we performed detailed studies on the reactions of a widely investigated and very promising trans-platinum complex having two iminoethers and two chlorido ligands, trans-EE, with methionine (Met) and guanosine 5′-monophosphate (GMP). The results show that in the reaction of trans-EE with methionine the bisadduct is the dominant species in the early stage of the reaction. The reaction is also influenced by chloride concentration: at low NaCl the bis-methionine adduct is formed in preference, whereas the monoadduct is favored at high NaCl concentration. Not only the monomethionine complex, trans-PtCl(E-iminoether)2(AcMet), but also the bis-methionine adduct, trans-Pt(E-iminoether)2(AcMet)2, which has already lost both leaving chlorides, can react with GMP to form the ternary platinum complex trans-Pt(E-iminoether)2(AcMet)(GMP). The latter reaction discloses the possibility of direct coordination to DNA of a platinum-protein adduct, in which the two carrier ligands remain intact; this is not the case of cis-oriented platinum complexes, like cisplatin, for which formation of a ternary complex is usually accompanied by loss of at least one carrier ligand. Interestingly, isomerization from S to N coordination of one methionine takes place in the bis-methionine complex at neutral pH, while the monoadduct appears to be stable. The shift from S to N coordination of one methionine in the trans-bis-methionine adduct can easily account for the obtainment of the cis isomer in the bis-chelated Pt(Met-S,N)2 end product. [ABSTRACT FROM AUTHOR]

Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pKa of 6.1, two units above the normal pKa of aspartate. The elevated pKa of D422 is coupled to the depressed pKa of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pKa to normal, while a D422G mutation increases the C1 pKa from 7.5 to 8.5. The pKa coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC50 of ∼2 μm, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ∼40 μm, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria. [ABSTRACT FROM AUTHOR]

One flexible, discrete coordination cage [Cu2(3-BPFA)4(H2O)2](ClO4)4·4CH3OH (1), and two cation-clusters with µ2-Cl bridging [Ni2(µ-Cl)(3-BPFA)4(H2O)2](ClO4)3 (2) and [Co2(µ-Cl)(3-BPFA)4(H2O)2](ClO4)4·4CH3OH (3), containing the ferrocenyl functionality were prepared via coordination-driven self-assembly and Cl-anion template from CuII, NiII and CoII salts and a flexible two-arm molecule 1,1-bis[(3-pyridylamino)carbonyl]ferrocene (3-BPFA). [ABSTRACT FROM AUTHOR]

Cox17 facilitates the platinum accumulation in mitochondria, which contributes to the overall cytotoxicity of cisplatin. [ABSTRACT FROM AUTHOR]

Methionine binding converts monofunctional platinum antitumor agents to transconfiguration adducts in the reaction with DNA, which suggests the correlation of two classes of platinum complexes in the antitumor mechanism. [ABSTRACT FROM AUTHOR]