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Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD 3 ), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD 3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2 , which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43 , with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD 3 to identify mechanistically and structurally novel molecules for the development of new medicines.

The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered "undruggable" due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all-l- and all-d-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-d peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target.

Conformationally stabilized α-helical peptides are capable of inhibiting disease-relevant intracellular or extracellular protein-protein interactions in vivo. We have previously reported that the employment of ring-closing metathesis to introduce a single all-hydrocarbon staple along one face of an α-helical peptide greatly increases α-helical content, binding affinity to a target protein, cell penetration through active transport, and resistance to proteolytic degradation. In an effort to improve upon this technology for stabilizing a peptide in a bioactive α-helical conformation, we report the discovery of an efficient and selective bis ring-closing metathesis reaction leading to peptides bearing multiple contiguous staples connected by a central spiro ring junction. Circular dichroism spectroscopy, NMR, and computational analyses have been used to investigate the conformation of these "stitched" peptides, which are shown to exhibit remarkable thermal stabilities. Likewise, trypsin proteolysis assays confirm the achievement of a structural rigidity unmatched by peptides bearing a single staple. Furthermore, fluorescence-activated cell sorting (FACS) and confocal microscopy assays demonstrate that stitched peptides display superior cell penetrating ability compared to their stapled counterparts, suggesting that this technology may be useful not only in the context of enhancing the drug-like properties of α-helical peptides but also in producing potent agents for the intracellular delivery of proteins and oligonucleotides.

Mdm2 is a major negative regulator of the tumor suppressor p53 protein, a protein that plays a crucial role in maintaining genome integrity. Inactivation of p53 is the most prevalent defect in human cancers. Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. We present here a 2.0 Å resolution structure of the Mdm2 protein with a bound stapled p53 peptide. Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. The structure represents the first crystal structure of an i, i + 7 stapled peptide bound to its target and reveals that rather than acting solely as a passive conformational brace, a staple can intimately interact with the surface of a protein and augment the binding interface., (© 2011 American Chemical Society)

The introduction of all-hydrocarbon i,i+3 staples into alpha-helical peptide scaffolds via ring-closing olefin metathesis (RCM) between two alpha-methyl,alpha-pentenylglycine residues incorporated at i and i+3 positions, which lie on the same face of the helix, has been investigated. The reactions were found to be highly dependent upon the side-chain stereochemistry of the amino acids undergoing RCM. The i,i+3 stapling system established here provides a potentially useful alternative to the well-established i,i+4 stapling system now in widespread use.

To determine the factors involved in the specific recognition function of a bacterial 8-oxoguanine (oxoG) DNA glycosylase MutM, a series of potentials of mean force and thermodynamic integration simulations were performed with the wild type and a single-point E3Q mutant of MutM bound to oxoG and G-containing DNA, respectively. Interestingly, the mutation of the catalytically important Glu3 (E3) residue to Gln (Q) significantly changes the free-energy surface so that oxoG can bind stably in the active site of the enzyme. Free-energy simulations with the protonated and deprotonated E3 residue further showed that the protonation of the catalytically important E3 residue plays a key role in distinguishing oxoG versus G in the active site by lowering the free energy of oxoG preferentially in the active site. The results suggest that MutM utilizes the thermodynamic recognition mechanism for stable binding of the lesion base in the active site of the enzyme in addition to kinetic discrimination at the early stage of the base extrusion for facilitated extrusion of oxoG.

Recent work has shown that the incorporation of an all-hydrocarbon "staple" into peptides can greatly increase their alpha-helix propensity, leading to an improvement in pharmaceutical properties such as proteolytic stability, receptor affinity, and cell permeability. Stapled peptides thus show promise as a new class of drugs capable of accessing intractable targets such as those that engage in intracellular protein-protein interactions. The extent of alpha-helix stabilization provided by stapling has proven to be substantially context dependent, requiring cumbersome screening to identify the optimal site for staple incorporation. In certain cases, a staple encompassing one turn of the helix (attached at residues i and i+4) furnishes greater helix stabilization than one encompassing two turns (i,i+7 staple), which runs counter to expectation based on polymer theory. These findings highlight the need for a more thorough understanding of the forces that underlie helix stabilization by hydrocarbon staples. Here we report all-atom Monte Carlo folding simulations comparing unmodified peptides derived from RNase A and BID BH3 with various i,i+4 and i,i+7 stapled versions thereof. The results of these simulations were found to be in quantitative agreement with experimentally determined helix propensities. We also discovered that staples can stabilize quasi-stable decoy conformations, and that the removal of these states plays a major role in determining the helix stability of stapled peptides. Finally, we critically investigate why our method works, exposing the underlying physical forces that stabilize stapled peptides.

The predominant product of aberrant DNA methylation is the genotoxic lesion N7-methyl-2'-deoxyguanosine (m7dG). M7dG is recognized and excised by lesion-specific DNA glycosylases, namely AlkA in E. coli and Aag in humans. Structural studies of m7dG recognition and catalysis by these enzymes have been hampered due to a lack of efficient means by which to incorporate the chemically labile m7dG moiety site-specifically into DNA on a preparative scale. Here we report a solution to this problem. We stabilized the lesion toward acid-catalyzed and glycosylase-catalyzed depurination by 2'-fluorination and toward base-catalyzed degradation using mild, nonaqueous conditions in the DNA deprotection reaction. Duplex DNA containing 2'-fluoro-m7dG (Fm7dG) cocrystallized with AlkA as a host-guest complex in which the lesion-containing segment of DNA was nearly devoid of protein contacts, thus enabling the first direct visualization of the N7-methylguanine lesion nucleobase in DNA. The structure reveals that the base-pairing mode of Fm7dG:C is nearly identical to that of G:C, and Fm7dG does not induce any apparent structural disturbance of the duplex structure. These observations suggest that AlkA and Aag must perform a structurally invasive interrogation of DNA in order to detect the presence of intrahelical m7dG lesions.

Here we present the first structure of a very advanced intermediate in the lesion-extrusion pathway of a DNA glycosylase, human 8-oxoguanine DNA glycosylase (hOGG1), and a substrate DNA containing a mutagenic lesion, 8-oxoguanine (oxoG). The structure was obtained by irradiation and flash-freezing of a disulfide-cross-linked (DXLed) complex of hOgg1 bound to DNA containing a novel photocaged derivative of oxoG. The X-ray structure reveals that, upon irradiation, the oxoG lesion has transited from the exosite to the active site pocket, but has not undergone cleavage by the enzyme. Furthermore, all but one of the specificity-determining interactions between the lesion and the enzyme are unformed in the flashed complex (FC), because active site functionality and elements of the DNA backbone are mispositioned. This structure thus provides a first glimpse into the structure of a very late-stage intermediate in the lesion-extrusion pathway--the latest observed to date for any glycosylase--in which the oxoG has undergone insertion into the enzyme active site following photodeprotection, but the enzyme and DNA have not yet completed the slower process of adjusting to the presence of the lesion in the active site.

Various 4'-F nucleosides have been prepared in only two to three steps via sequential bromination and fluorination of ribofuranoses or nucleosides.

Human cytidine deaminase (CDA) is an enzyme prominent for its role in catalyzing metabolic processing of nucleoside-type anticancer and antiviral agents. It is thus a promising target for the development of small molecule therapeutic adjuvants. We report the first crystal structure of human CDA as a complex with a tight-binding inhibitor, diazepinone riboside 1. The structure reveals that inhibitor 1 is able to establish a canonical pi/pi-interaction with a key active site residue, Phe 137.

The syntheses of stereodiverse libraries of 12 and 19 are reported, where each asterisk represents an independently varied stereocenter. These scaffolds provide additional templates for investigations of geometric diversity in library syntheses. Libraries of these N-Fmoc-amino acids were further functionalized by incorporation into a peptide sequence, demonstrating the utility of 12 and 19 as building blocks for diversity oriented synthesis.

Using olefin cross-metathesis, we synthesized a novel stereodiversified library of compounds 3 containing a trans-1,4-enediol. Screening this library for mu opioid receptor (MOR) affinity identified multiple high-affinity ligands and revealed that the stereochemical configuration varied widely among those ligands having the highest affinity. It was not possible to predict the configurations of the most active compounds 3 on the basis of the configuration of endomorphin-2, a known MOR peptide ligand, validating the diversity-based approach to ligand discovery.

We recently reported the use of an exhaustively stereodiversified library based on endomorphin-2 (1) to discover mu opioid receptor (MOR) ligands of type 2-4. Here, we report the synthesis and evaluation of 2,6-dimethyltyrosine analogues 5-10. These analogues showed improved affinity for MOR relative to 2-4. In the cases of 5 and 6, we synthesized and evaluated five stereoisomers of each, thereby discovering stereoisomers with unexpected potency, selectivity, and efficacy. These results illustrate the utility of acyclic, stereodiverse libraries.

Members of the HhH-GPD superfamily of DNA glycosylases are responsible for the recognition and removal of damaged nucleobases from DNA. The hallmark of these proteins is a motif comprising a helix-hairpin-helix followed by a Gly/Pro-rich loop and terminating in an invariant, catalytically essential aspartic acid residue. In this study, we have probed the role of this Asp in human 8-oxoguanine DNA glycosylase (hOgg1) by mutating it to Asn (D268N), Glu (D268E), and Gln (D268Q). We show that this aspartate plays a dual role, acting both as an N-terminal alpha-helix cap and as a critical residue for catalysis of both base excision and DNA strand cleavage by hOgg1. Mutation of this residue to asparagine, another helix-capping residue, preserves stability of the protein while drastically reducing enzymatic activity. A crystal structure of this mutant is the first to reveal the active site nucleophile Lys249 in the presence of lesion-containing DNA; this structure offers a tantalizing suggestion that base excision may occur by cleavage of the glycosidic bond and then attachment of Lys249. Mutation of the aspartic acid to glutamine and glutamic acid destabilizes the protein fold to a significant extent but, surprisingly, preserves catalytic activity. Crystal structures of these mutants complexed with an unreactive abasic site in DNA reveal these residues to adopt a sterically disfavored helix-capping conformation.

Mutation of the active-site residue Cys38 of N-Ada converts it from a sacrificial DNA repair protein to an enzyme that uses methanethiol as an external sacrificial reagent to repair DNA methyl phosphotriesters catalytically.

In this communication, we report the synthesis of an exhaustively stereodiversified library of 16 1,5-enediols (2) and the screening of these compounds for mu opioid receptor (MOR) binding. The stereochemical configuration of 2 strongly impacted the binding affinity, and (S,S,S,R)-2 exhibited a Ki of 8.8 nM for MOR, comparable to that of endomorphin-2 (Ki = 1.2 nM). Moreover, compounds 2 exhibited 5-86-fold selectivity for MOR over delta opioid receptor (DOR) and 16-150-fold selectivity for MOR over kappa opioid receptor (KOR). Additionally, analogues of 2 were synthesized which showed the importance of the trans olefin for receptor binding but that modifications of the C-terminal amino acid were well tolerated. Ligand 11 is noteworthy because it retains only one of the amide bonds present in 1, but binds MOR with an affinity of 10 nM and 110- and 600-fold selectivity for MOR over DOR and KOR. These results demonstrate the utility of stereochemical diversity in the discovery of bioactive small molecules.

[formula: see text] 5-Amino-2'-deoxyuridine 5'-triphosphate, an analogue of deoxythymidine triphosphate, was synthesized and found to be a substrate of Taq DNA polymerase. The DNA-borne analogue underwent selective chemical reaction with permanganate. The use of 5-amino-dU as an interference probe was validated using the Ada protein/ada promoter complex. The performance of 5-amino-dU in interference footprints is similar to that of the previously described analogue 5-hydroxy-dU, but the former is incorporated more readily into DNA during enzymatic polymerization.

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA through direct, irreversible transfer to a cysteine residue on the protein, Cys 69. Methylation of Cys 69 increases the sequence-specific DNA-binding activity of Ada by 10(3)-fold, enabling the methylated protein to activate transcription of a methylation-resistance regulon. The thiolate sulfur atom of Cys 69 is coordinated to a tightly bound zinc ion in the Ada N-terminal domain, and this metal-ligand interaction plays a direct role in promoting the DNA repair chemistry. Ada is thus the founding member of a mechanistic class of proteins that employ metalloactivated thiolates as nucleophiles, other examples of which include protein prenyltransferases and cobalamin-independent methionine synthase. Here we have probed the role of the three other Cys residues in Ada that together with Cys 69 coordinate the zinc through mutation to the alternative ligand residues Asp and His. All of the mutant proteins folded properly and bound zinc, but none of them exhibited measurable levels of DNA repair activity. Significantly, the Cys-to-His mutant proteins retained nearly wild-type sequence-specific DNA-binding activity in the unmethylated state. These findings demonstrate that the three "spectator" Cys ligands communicate chemically with Cys 69 through the bound metal ion.

[reaction: see text] This report describes the parallel synthesis of all 16 stereoisomers of the cis-1,5 enediol module 1. Compounds 1 derive from 2 by silicon-tethered ring-closing metathesis. Such libraries of stereodiversified ligands provide a unique approach to ligand discovery that employs exhaustive searching of conformational space.

The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center. The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes. Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage. Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer. To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated. Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada. The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA. This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.

Small molecules that induce or stabilize the association of macromolecules have proven to be useful effectors of a wide variety of biological processes. To date, all examples of such chemical inducers of dimerization have involved known ligands to well-characterized proteins. The generality of this approach could be broadened by enabling the discovery of heterodimerizers that target known macromolecules having no established ligand, or heterodimerizers that produce a novel biologic response in screens having no predetermined macromolecular target. Toward this end, we report the construction of a diversified library of synthetic heterodimerizers consisting of an invariant ligand that targets the FK506-binding protein (AP1867) attached to 320 substituted tetrahydrooxazepines (THOXs). The THOX components were generated by a combination of liquid- and solid-phase procedures employing sequential Mitsonobu displacements to join two structurally diversified olefin-containing monomers, followed by ruthenium-mediated olefin metathesis to effect closure of the seven-membered ring. The 320 resin-bound THOX ligands were coupled in parallel to AP1867, and the products were released from the resin to yield candidate heterodimerizers in sufficient yield and purity to be used directly in biologic testing. A representative panel of 25 candidate heterodimerizers were tested for their ability to pass through the membrane of human fibrosarcoma cells, and all were found to possess activity in this tissue culture system. These studies pave the way for further studies aimed at using small-molecule inducers of heterodimerization to effect novel biological responses in intact cells.

[figure: see text] We have developed a method for interference footprinting of contacted phosphates in protein-DNA complexes. Template-directed enzymatic polymerization using a synthetic triphosphate analogue (alpha Me-dTTP) generates a product having a modified Internucleotide linkage, which perturbs protein-phosphate contacts. We found that treatment of the methylphosphonodiester-substituted extension product under nonaqueous conditions (MeO-/MeOH) led to the formation of a single cleavage product at each T residue but to two cleavage products when treated under the standard aqueous piperidine cleavage protocol.

[structure] This report describes a modular approach to the synthesis of stereodiversified natural product-like libraries. Monomers 2 and 3 were coupled in parallel by silyl-tethered olefin metathesis to generate all 16 stereoisomers of cis-enediols 1. All 16 stereoisomers were incorporated into chimerae having flanking peptidic segments. These chimerae exhibited a broad range of hydrophobicities, raising the possibility that stereochemical variation might be used to tune the pharmacologic properties of small molecules.

HMG-D is an abundant high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. It is a non-sequence-specific member of a protein family that uses the HMG domain for binding to DNA in the minor groove. The highly charged C-terminal tail of HMG-D contains AK motifs that contribute to high-affinity non-sequence-specific DNA binding. To understand the interactions of the HMG domain and C-terminal tail of HMG-D with DNA in solution, a complex between a high-affinity truncated form of the protein and a disulfide cross-linked DNA fragment was studied using heteronuclear NMR techniques. Despite its relatively high affinity for the single "prebent" site on the DNA, K(d) = 1.4 nM, HMG-D forms a non-sequence-specific complex with the DNA as indicated by exchange broadening of the protein resonances at the DNA interface in solution. The secondary structural elements of the protein are preserved when the protein is complexed with the DNA, and the DNA-binding interface maps to the regions of the protein where the largest chemical shift differences occur. The C-terminal tail of HMG-D confers high-affinity DNA binding, has an undefined structure, and appears to make direct contacts in the major groove of DNA via residues that are potentially regulated by phosphorylation. We conclude that while the HMG domain of HMG-D recognizes DNA with a mode of binding similar to that used by the sequence-specific HMG domain transcription factors, there are noteworthy differences in the structure and interactions of the C-terminal end of the DNA-binding domain and the C-terminal tail.

The 10kDa amino-terminal fragment of Escherichia coli Ada protein (N-Ada10) repairs methyl phosphotriesters in DNA and possesses a tightly bound zinc ion. The complete resonance assignments of this protein domain have been obtained using multidimensional homonuclear and heteronuclear NMR experiments. The assignments served to study the internal mobility of this protein domain via 15N relaxation experiments. This involved the measurement of longitudinal and transverse 15N relaxation rates, as well as the amide proton solvent exchange rates. Relaxation rates in the rotating frame, R1 rho, of 15N nuclei were measured at different spin-lock field strengths, leading to the detection of two slow conformational exchange processes at Gly-25 and Gln-73. For the latter, which is next to the active site of this protein domain, the characteristic time of this process was found to be around 60 microseconds. The other relaxation experiments unveiled some regions of fast internal motions, faster than the overall correlation time. These motions were found in the N- and C- terminal tails, in segment 33-35 which forms the turn between beta-strands S1 and S2, and residues 47-52 located in a long loop preceding strand S3. The latter loop belongs to the potential DNA binding surface of N-Ada10. While the structure from residue 18 to residue 26 appears not well defined in the calculated structure, the relaxation experiments do not indicate higher mobility for this region. Residues at the N-terminal portion, including the first helix, the sequentially adjacent loop, and part of the second helix, exhibit internal motions close to the time scale of the overall rotational correlation time. This appears to be related to the fact that the first helix has no hydrogen bonds or salt bridges to the rest of the protein and is stabilized only by the involvement of some of its side chains in a hydrophobic core consisting of the side chains of two phenylalanines, a tryptophan, a leucine, and a valine. The four cysteines which bind the zinc show motions on different time scales ranging from microseconds to picoseconds. Thus the motions in the immediate region around the bound zinc of the DNA methyl phosphotriester repair domain are of relatively small amplitude but take place over a wide time range. On the other hand, high mobility is found in the turn connecting S1 and S2 and in the loop preceding S3, regions of the potential DNA binding surface.

The Escherichia coli Ada protein repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl-transfer process appears to be autocatalyzed by coordination of the acceptor residue, Cys-69, to a tightly bound zinc ion. Upon methyl transfer, Ada acquires the ability to bind DNA sequence-specifically and thereby to induce genes that confer resistance to methylating agents. The solution structure of an N-terminal 10-kDa fragment of Ada, which retains zinc binding and DNA methyl phosphotriester repair activities, was determined using multidimensional heteronuclear nuclear magnetic resonance techniques. The structure reveals a zinc-binding motif unlike any observed thus far in transcription factors or zinc-containing enzymes and provides insight into the mechanism of metalloactivated DNA repair.

In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.

The overproduction, purification, and determination of the active-site catalytic nucleophile of the DNA (cytosine-5)-methyltransferase (DCMtase) enzyme M.HaeIII are reported. Incubation of purified M.HaeIII with an oligodeoxynucleotide specifically modified with the mechanism-based inhibitor 5-fluoro-2'-deoxycytidine [Osterman, D. G., et al. (1988) Biochemistry 27, 5204-5210], in the presence of the cofactor S-adenosyl-L-methionine (AdoMet), resulted in the formation of a covalent DNA-M.HaeIII complex, which was purified to homogeneity. Characterization of the intact complex showed it to consist of one molecule of the FdC-containing duplex oligonucleotide, one molecule of M.HaeIII, and one methyl group derived from AdoMet. Exhaustive proteolysis, reduction, and alkylation of the DNA-M.HaeIII complex led to the isolation of two DNA-bound peptides--one each from treatment with Pronase or trypsin--which were subjected to peptide sequencing in order to identify the DNA attachment site. Both peptides were derived from the region of M.HaeIII containing a Pro-Cys sequence that is conserved in all known DCMtases. At the position of this conserved Cys residue (Cys71), in the sequence of each peptide, was found an unidentified amino acid residue; all other amino acid residues were in accord with the known sequence. It is thus concluded that Cys71 of M.HaeIII forms a covalent bond to DNA during catalytic methyl transfer. This finding represents a direct experimental verification for the hypothesis that the conserved Cys residue of DCMtases is the catalytic nucleophile [Wu, J. C., & Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786].(ABSTRACT TRUNCATED AT 250 WORDS)

Mitomycin C (1) is a clinically used antitumor antibiotic that binds covalently to deoxyribonucleic acid under reductive or acidic catalysis. We have determined the structures of the adducts resulting from attack of reductively activated 1 on the dinucleoside phosphate d(GpC) to be N2-(2'' beta, 7''-diaminomitosen-1''alpha-yl)-2'-deoxyguanosine (2) and its 1'' beta-isomer (3). This represents a revision of the previously reported structures for these adducts in that the mitomycin residue is linked to the N2- rather than O6-position of 2'-deoxyguanosine. This revision is the result of applying to the mitomycin case a newly developed general method that leads to unambiguous assignment of the linkage position in complex alkylated guanosines. The method as described here takes advantage of the resolution enhancement gained by calculation of the second derivatives of absorbance Fourier transform infrared spectra. In addition, we present 1H NMR data that corroborate the assigned structures of 2 and 3 and that should serve as a useful reference for future investigations into the binding of mitomycin C to DNA. The convenient synthesis of adducts 2 and 3 from deoxyguanosine and mitomycin C reported here should facilitate such investigations as well. Furthermore, we demonstrate a useful acetylation procedure for adducts and metabolites of mitomycin C that furnishes spectroscopically superior chemical derivatives (e.g., triacetates 4 and 5, derived from acetylation of adducts 2 and 3).

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2'' beta, 7''-diaminomitosen-1''-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2'' beta, 7''-diaminomitosen-1'' alpha-yl)guanine (5) and its 1'' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2'' beta, 7''-diaminomitosen-1'' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.

The MerR protein from the Tn501 mercury resistance operon is a metalloregulatory transcriptional switch, converting from repressor to activator on binding of Hg(II). We have determined via binding studies with 203Hg(II) that a single Hg(II) atom binds to the MerR dimer (32 kDa) with a half-saturation concentration of 10(-7) M in the presence of up to 10(-3) M exogenous thiols. This 10(4) selective binding is specific for the binding of Hg(II) and corresponds to concentrations of metal that induce mercury(II) resistance in vivo. Kd values for MerR binding, in the absence and presence of Hg(II), to a 305 bp DNA fragment containing the 18 bp dyad symmetry element, DS1, located at -35 to -10 upstream of the mer structural genes, were determined by a gel shift assay. A Kd of 10(-10) M for free MerR and 10(-11) M for Hg(II)-MerR complexes was revealed. Measurements of koff values, by this assay, show equally long-lived complexes of MerR-DNA (51-min half-life) and Hg(II)-MerR-DNA (49-min half-life), suggesting that Hg(II) accelerates MerR binding to DNA rather than influencing the dissociation rate of the protein-DNA complex. In contrast, 203Hg(II) studies reveal that mercuric ions rapidly dissociate and associate with MerR-DNA complexes. Extensive footprinting studies by DNase I, methylation protection, and hydroxyl radicals indicate MerR stays bound to DS1 even on addition of Hg(II) and shares no interaction in vitro with a second dyad symmetry element, DS2, centered at -79/-80.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial resistance to mercury(II) compounds is controlled by the metalloregulatory MerR protein, a transcriptional repressor and a mercuric ion dependent activator of the mer operon. Site-directed mutagenesis of all four cysteine residues in the Tn501 MerR protein has led to the specific replacement of C82, C115, and C117 with alanine and of C126 with serine. Mutation of C82 and C126 abolishes transcriptional activation in vivo while mutation of C115 and C117 leads to a slight increase and dramatic decrease in transcriptional activation, respectively. All four mutants are competent, to varying degrees, to repress mer transcription. Characterization of the four purified mutant proteins in vitro demonstrates that only the C126S MerR mutant is most notably deficient in stoichiometric Hg(II) binding. All four mutant proteins possess similar DNA binding properties, and the C82 mutant is most affected in the ability to form stable dimers. Given an observed stoichiometry of one Hg(II) per MerR dimer, it is likely that the transcriptionally activating MerR species is a metal-bridged dimer. It is most likely that one C126 per subunit provides high-avidity bidentate ligation to Hg(II), but it remains possible that C82 may be a secondary Hg(II) ligand (e.g., in a tetracoordinate thiol ligation array).