Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA.     

Nature of full-length HMGB1 binding to cisplatin-modified DNA

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.     

Intercalating residues determine the mode of HMG1 domains A and B binding to cisplatin-modified DNA

Cisplatin exerts its anticancer activity by forming covalent adducts with DNA. High-mobility group (HMG)-domain proteins recognize the major 1,2-intrastrand cisplatin-DNA cross-links and can mediate cisplatin cytotoxicity. The crystal structure of HMG1 domain A bound to cisplatin-modified DNA, further analyzed here, reveals intercalation of a key Phe37 residue. Other published structures of HMG domains bound to DNA, including NHP6A and HMG-D, similarly indicate amino acid side chains intercalating into linear DNA to form a bend. To delineate the importance of such side chain intercalations and further to explore the binding modes of different HMG domains toward prebent DNA structures, site-directed mutagenesis was used to generate HMG1 domain A and domain B mutants. The affinities of these mutant proteins for cisplatin-modified DNA were determined in gel electrophoresis mobility shift assays. The results indicate that intercalating residues at positions 16 or 37 can both contribute to the binding affinity. The data further reveal that the length of the loop between helices I and II is not critical for binding affinity. Footprinting analyses indicate that the position of the intercalating residue dictates the binding mode of the domain toward platinated DNA. Both congruent and offset positioning of the HMG domain with respect to the locus of the cisplatin-induced bend in the DNA were encountered. Packing interactions in the crystal structure suggest how full-length HMG1 might bind to DNA by contacting more than one duplex simultaneously. Taken together, these results demonstrate that cisplatin modification of DNA provides an energetically favorable, prebent target for HMG domains, which bind to these targets through one or more side chain and favorable hydrophobic surface interactions.     

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.     

Recognition of DNA interstrand cross-link of antitumor cisplatin by HMGB1 protein

Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands.     

Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins

The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.     

Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA

High mobility group (HMG) 1 protein and its two homologous DNA-binding domains, A and B ("HMG-boxes"), can bend and supercoil DNA in the presence of topoisomerase I, as well as recognize differently bent and distorted DNA structures, including four-way DNA junctions, supercoiled DNA and DNA modified with anticancer drug cisplatin. Here we show that the lysine-rich part of the linker region between A and B domains of HMG-1, the (85)TKKKFKD(91) sequence that is attached to the N-terminus of the B domain within HMG-1, is a prerequisite for a preferential binding of the B domain to supercoiled DNA. The above sequence is also essential for a high-affinity binding of the B domain to DNA containing a site-specific major 1,2-d(GpG) intrastrand DNA adduct of cisplatin. Mutation of Arg(97), but not Lys(90) [Lys(90) forms a specific cross-link with platinum(II) in major groove of cisplatin-modified DNA; Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180--2188], to alanine significantly (>40-fold) reduces affinity of the B domain to cisplatin-modified DNA, inhibits the ability of the B domain to bend (ligase-mediated circularization) or supercoil DNA, and results in a loss of the preferential binding of the B domain to supercoiled DNA without affecting the structural-specificity of the HMG-box for four-way DNA junctions. Some of the reported activities of the B domain are enhanced when the B domain is covalently linked to the A domain. We propose that binding of the A/B linker region within the major DNA groove helps the two HMG-1 domains to anchor to the minor DNA groove to facilitate their DNA binding and other activities.     

Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important

Human calbindin D(28k) is a Ca(2+) binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D(28k) appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D(28k) binds Ca(2+) with lower affinity than does the reduced protein.     

Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1)

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.     

The DNA binding and bending activities of truncated tail-less HMGB1 protein are differentially affected by Lys-2 and Lys-81 residues and their acetylation

The role of lysines 2 and 81 as target sites for acetylation in full-length HMGB1 and truncated tail-less protein, respectively, has been studied by mutation analysis for the abilities of these proteins to bind and bend DNA. The DNA bending ability of truncated tail-less HMGB1 containing Lys-2 mutated to alanine does not differ from that of the wild-type protein, while the same mutation of Lys-81 reduced the bending capacity of the mutant protein. These data demonstrate that Lys-81 is critical for the DNA bending ability of truncated HMGB1. Such a conclusion is further confirmed by the experiments carried out with CBP-acetylated proteins: acetylation of Lys-2 in mutant protein K81/A81 alleviated DNA bending and induced DNA end-joining. On the contrary, the acetylation of Lys-81 in the mutant K2/A2 enhanced the bending potential of HMGB1∆C. Regarding the ability of HMGB1 to specifically bind bent DNA, the individual mutations of either K2 or K81 as well as the double mutation of both residues to alanine were found to completely abolish binding of truncated tail-less HMGB1 to cisplatin-modified DNA. We conclude that unlike the case with the bending ability of truncated HMGB1, where Lys-81 has a primary function, Lys-2 and Lys-81 are both critical for the protein''s binding to cisplatin-modified DNA. The mutation K2/A2 in full-length HMGB1 and acidic tail removal induce the same conformational changes. Any further substitutions at the acetylable lysines in the truncated form of HMGB1 do not have an additional effect.     

MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.     

Structure-expression relationship of tumor necrosis factor receptor mutants that increase expression

The extracellular domain of the p55 TNF receptor (TNFrED) is an important therapeutic protein for targeting tumor necrosis factor-alpha (TNF-alpha). The expression level of the TNFrED is low for bioproduction, which is presumably associated with the complication of pairing 24 cysteine residues to form correct disulfide bonds. Here we report the application of the yeast display method to study expression of TNFrED, a multimeric receptor. Randomly mutated libraries of TNFrED were screened, and two mutants were identified that express several-fold higher protein levels compared with the wild type while still retaining normal binding affinity for TNF-alpha. The substituted residues responsible for the higher protein expression in both mutants were identified as proline, and both proline residues are adjacent to cysteine residues involved in disulfide bonds. Analysis of the mutant residues revealed that the improved level of expression is due to conformational restriction of the substituted residues to that of the folded state seen in the crystal structures of TNFrED thereby forcing the neighboring cysteine residues into the correct orientation for proper disulfide bond formation.     

The MntC crystal structure suggests that import of Mn2+ in cyanobacteria is redox controlled

The MntC protein is the periplasmic solute-binding protein component of the high-affinity manganese ATP-binding cassette-type transport system in the cyanobacterium Synechocytis PCC sp. 6803. We have determined the structure of recombinant MntC at 2.9 A resolution by X-ray crystallography using a combination of multi-wavelength anomalous diffraction and molecular replacement. The presence of Mn2+ in the metal ion-binding site was ascertained by use of anomalous difference electron density maps using diffraction data collected at the Mn absorption edge. The MntC protein is similar to previously determined metal ion-binding, solute-binding proteins with two globular domains connected by an extended alpha-helix. However, the metal ion-binding site is asymmetric, with two of the four ligating residues (Glu220 and Asp295) situated closer to the ion than the two histidine residues (His89 and His154). A unique characteristic of the MntC is the existence of a disulfide bond between Cys219 and Cys268. Analysis of amino acid sequences of homologous proteins shows that conservation of the cysteine residues forming the disulfide bond occurs only in cyanobacterial manganese solute-binding proteins. One of the monomers in the MntC asymmetric unit trimer is disordered significantly in the globular domain containing the disulfide bond. The electron density on the manganese ion and on the disulfide bond in this monomer indicates that reduction of this bond changes the relative position of the lower domain and of the Glu220 ligand, potentially lowering the affinity towards Mn2+. This is confirmed by reduction of the disulfide bond in vitro, showing the release of bound Mn2+. We propose that the reduction or oxidation state of the disulfide bond can alter the binding affinity of the protein towards Mn2+ and thus determine whether these ions will be transported into the cytoplasm, or be available for photosystem II biogenesis in the periplasm.     

Disulfide bond formation between RNA binding domains is used to regulate mRNA binding activity of the chloroplast poly(A)-binding protein

Binding of the chloroplast poly(A)-binding protein, RB47, to the psbA mRNA is regulated in response to light and is required for translation of this mRNA in chloroplasts. The RNA binding activity of RB47 can be modulated in vitro by oxidation and reduction. Site-directed mutations to individual cysteine residues in each of the four RNA binding domains of RB47 showed that changing single cysteines to serines in domains 2 or 3 reduced, but did not eliminate, the ability of RB47 to be redox-regulated. Simultaneously changing cysteines to serines in both domains 2 and 3 resulted in the production of RB47 protein that was insensitive to redox regulation but retained the ability to bind the psbA mRNA at high affinity. The poly(A)-binding protein from Saccharomyces cerevisiae lacks cysteine residues in RNA binding domains 2 and 3, and this poly(A)-binding protein lacks the ability to be regulated by oxidation or reduction. These data show that disulfide bond formation between RNA binding domains in a poly(A)-binding protein can be used to regulate the ability of this protein to bind mRNA and suggest that redox regulation of RNA binding activity may be used to regulate translation in organisms whose poly(A)-binding proteins contain these critical cysteine residues.