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.     

Regulation of poly(ADP-ribose) polymerase-1 by DNA structure-specific binding

Poly(ADP-ribose) polymerase-1 (PARP-1) is an intracellular sensor of DNA strand breaks and plays a critical role in cellular responses to DNA damage. In normally functioning cells, PARP-1 enzymatic activity has been linked to the alterations in chromatin structure associated with gene expression. However, the molecular determinants for PARP-1 recruitment to specific sites in chromatin in the absence of DNA strand breaks remain obscure. Using gel shift and enzymatic footprinting assays and atomic force microscopy, we show that PARP-1 recognizes distortions in the DNA helical backbone and that it binds to three- and four-way junctions as well as to stably unpaired regions in double-stranded DNA. PARP-1 interactions with non-B DNA structures are functional and lead to its catalytic activation. DNA hairpins, cruciforms, and stably unpaired regions are all effective co-activators of PARP-1 auto-modification and poly(ADP-ribosyl)ation of histone H1 in the absence of free DNA ends. Enzyme kinetic analyses revealed that the structural features of non-B form DNA co-factors are important for PARP-1 catalysis activated by undamaged DNA. K0.5 constants for DNA co-factors, which are structurally different in the degree of base pairing and spatial DNA organization, follow the order: cruciform相似文献   

Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex.

In V(D)J recombination, the RAG1 and RAG2 proteins are the essential components of the complex that catalyzes DNA cleavage. RAG1 has been shown to play a central role in DNA binding and catalysis. In contrast, the molecular roles of RAG2 in V(D)J recombination are unknown. To address this, we individually mutated 36 evolutionarily conserved basic and hydroxy group containing residues within RAG2. Biochemical analysis of the recombinant RAG2 proteins led to the identification of a number of basic residue mutants defective in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target sequence while interacting normally with RAG1. Our findings provide support for the direct involvement of RAG2 in DNA binding during all steps of the cleavage reaction.     

Identification of critical residues in bovine IFNAR-1 responsible for interferon binding

Interferons have antiviral, antigrowth and immunomodulatory effects. The human type I interferons, IFN-alpha, IFN-beta, and IFN-omega, induce somewhat different cellular effects but act through a common receptor complex, IFNAR, composed of subunits IFNAR-1 and IFNAR-2. Human IFNAR-2 binds all type I IFNs but with lower affinity and different specificity than the IFNAR complex. Human IFNAR-1 has low intrinsic binding of human IFNs but strongly affects the affinity and differential ligand specificity of the IFNAR complex. Understanding IFNAR-1 interactions with the interferons is critical to elucidating the differential ligand specificity and activation by type I IFNs. However, studies of ligand interactions with human IFNAR-1 are compromised by its low affinity. The homologous bovine IFNAR-1 serendipitously binds human IFN-alphas with nanomolar affinity. Exploiting its strong binding of human IFN-alpha2, we have identified residues important for ligand binding. Mutagenesis of any of five aromatic residues of bovine IFNAR-1 caused strong decreases in ligand binding, whereas mutagenesis of proximal neutral or charged residues had smaller effects. These residues were mapped onto a homology model of IFNAR-1 to identify the ligand-binding face of IFNAR-1, which is consistent with previous structure/function studies of human IFNAR-1. The topology of IFNAR-1/IFN interactions appears novel when compared with previously studied cytokine receptors.     

A critical role of lysine residues in the stimulation of tissue plasminogen activator by denatured proteins and fibrin clots

Two-chain 70 000-dalton plasminogen activator of tissue origin displays only weak activity toward plasminogen in a two-component system. The rate of activation is enhanced a minimum of 50-fold by the presence of fibrin clots or denatured proteins. The stimulation must depend on both chemical determinants and spatial configuration, since native proteins, including fibrinogen, lack significant stimulatory activity. These studies employed chemical modifications of four stimulatory proteins (fibrin, denatured fibrinogen, denatured IgG and denatured ovalbumin) to identify a critical role for lysine residues. Arginine, aspartic acid, cysteine, cystine, glutamic acid, histidine, methionine, tyrosine and tryptophan were found not to be essential. The critical spatial determinant(s) remain(s) unknown.     

Understanding the role of internal lysine residues of serum albumins in conformational stability and bilirubin binding

The role of internal lysine residues of different serum albumins, viz. from human, rabbit, goat, sheep and buffalo (HSA, RbSA, GSA, SSA and BuSA), in conformational stability and bilirubin binding was investigated after blocking them using acetylation, succinylation and guanidination reactions. No significant change in the secondary structure was noticed whereas the tertiary structure of these proteins was slightly altered upon acetylation or succinylation as revealed by circular dichroism (CD), fluorescence and gel filtration results. Guanidination did not affect the native protein conformation to a measurable extent. Scatchard analysis, CD and absorption spectroscopic results showed marked reductions (5-21-fold decrease in K(a) and approximately 50% decrease in the CD Cotton effect intensity) in the affinity of albumins for bilirubin upon acetylation or succinylation whereas guanidination produced a small change. Interestingly, monosignate CD spectra of bilirubin complexed with GSA, SSA and BuSA were transformed to bisignate CD spectra upon acetylation or succinylation of internal lysine residues whereas spectra remained bisignate in the case of bilirubin bound to acetylated or succinylated derivatives of HSA and RbSA. When probed by CD spectroscopy, bilirubin bound to acetylated or succinylated derivatives of GSA and SSA rapidly switched over to native albumins and not vice versa. These results suggested that salt linkage(s) contributed by internal lysine residue(s) play an important role in the high-affinity binding of bilirubin to albumin and provide stability to the native three-dimensional conformation of the bound pigment. Chloroform severely decreased the intensity of both positive and negative CD Cotton effects of bilirubin complexed with acetylated or succinylated derivatives of all albumins which otherwise increased significantly in the case of bilirubin complexed with native and guanidinated albumin derivatives, except the bilirubin-RbSA complex which showed a small decrease in intensity. These results suggest that the presence of salt linkage(s) in bilirubin-albumin complexation is(are) crucial to bring about effective and efficient stereochemical changes in the bound pigment by co-binding of chloroform which seems to have at least one conserved binding site on these albumins that is shared with bilirubin.     

Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein.

The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.     

NMR assignments for the amino-terminal residues of trp repressor and their role in DNA binding

The trp repressor of Escherichia coli specifically binds to operator DNAs in three operons involved in tryptophan metabolism. The NMR spectra of repressor and a chymotryptic fragment lacking the six amino-terminal residues are compared. Two-dimensional J-correlated spectra of the two forms of the protein are superimposable except for cross-peaks that are associated with the N-terminal region. The chemical shifts and relaxation behavior of the N-terminal resonances suggest mobile "arms". Spin-echo experiments on a ternary complex of repressor with L-tryptophan and operator DNA indicate that the termini are also disordered in the complex, although removal of the arms reduces the DNA binding energy. Relaxation measurements on the armless protein show increased mobility for several residues, probably due to helix fraying in the newly exposed N-terminal region. DNA binding by the armless protein does not reduce the mobility of these residues. Thus, it appears that the arms serve to stabilize the N-terminal helix but that this structural role does not explain their contribution to the DNA binding energy. These results suggest that the promiscuous DNA binding by the arms seen in the X-ray crystal structure is found in solution as well.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (V_H) of a human beta₂ glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (V_L) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4V_H and paired V_L in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of V_H and V_L sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived V_L sequences were expressed with IS4V_H and the V_H of an anti-dsDNA antibody, B3. Six variants of IS4V_H, containing different patterns of arginine residues in CDR3, were paired with B3V_L and IS4V_L. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4V_H CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, V_L containing B3V_L CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in V_L CDR2 or V_L CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Identification of lysine residues in the binding domain of ribonuclease A for the RNase inhibitor from human placenta

Amidination of the available lysine residues of the complex between RNase A and human placental RNase inhibitor has been performed with methyl acetimidate; the conditions of the derivatization preserve the complex functionally intact. Resistance of epsilon-acetimidyllysine residues to hydrolysis by trypsin allowed, after peptide mapping, the identification of lysine residues 7, 31, 41, 61, and 91 as those which were fully protected by the inhibitor from amidination. Lysine residue 37 was partially protected from amidination. In the presence of poly(A), lysine residues 41 and 61 of RNase A were fully protected from amidination, while lysine residues 7, 31, 37, 91, and 104 were only partially protected; the enzyme retained full activity. The results permit identification of lysine residues located in the binding domain of RNase A for the inhibitor. This region is not identical with, but does overlap, the binding domain for poly(A).     

Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.     

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes

Background

Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration.

Methodology/Principal Findings

In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum.

Conclusion

The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.     

A 13C NMR characterization of lysine residues in apolipoprotein B and their role in binding to the low density lipoprotein receptor

NMR spectroscopy of 13C-labeled human low density lipoprotein (LDL) has been employed to characterize the lysine (Lys) residues in apo B-100. Reductive methylation with [13C]formaldehyde converts up to two-thirds of the Lys to the dimethylamino derivative; this pool of Lys is exposed at the surface of the LDL particle. The [13C]dimethyl-Lys which are visualized exhibit resonances at chemical shifts of 42.8 and 43.2 ppm (pH 7.6) indicating that they exist in two different microenvironments; this is a reflection of the native conformation of apo B associated with lipid, because the labeled, reduced, and alkylated protein gives a single resonance when dissolved in 7 M guanidine hydrochloride. The pH dependences of the Lys chemical shifts indicate that the two types of Lys titrate with different pK values; "active" Lys have a pK of 8.9, while "normal" Lys have a pK of 10.5. About 53 active Lys and 172 normal Lys are exposed on the surface of LDL with the remaining 132 Lys which are present in the human apo B-100 molecule being buried and unavailable for methylation. Addition of paramagnetic ions indicates that the active and normal Lys have different exposures to the aqueous phase; apparently this is a reflection of folding of the apo B molecule. The relative involvement of active and normal Lys in binding of apo B-100 to the LDL receptor on fibroblasts was explored by measuring the decrease in receptor binding as a function of the degree of methylation of the two types of Lys. Upper limits of 21 active and 31 normal Lys in the entire apo B-100 molecule are involved in the binding of LDL to the receptor. It is likely that these Lys are located in domains of apo B which contain clusters of basic amino acid residues and also bind heparin. If the sequence corresponding to apo B-48 (residues 1-2151) which does not bind to the receptor is excluded, then the above limits are halved; an upper limit of 10 active Lys may be particularly involved in receptor binding.     

A role of basic residues and the putative intercalating phenylalanine of the HMG-1 box B in DNA supercoiling and binding to four-way DNA junctions

HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of our gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) and helix I (Arg(110)/Lys(114)) of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive.     

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma-1 ligands, [3H](+)pentazocine and [3H]NE-100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di-Leu105,106di-Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti-type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.     

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.     

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.     

Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein

beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.     

A critical role for TORC1 in cellular senescence

Comment on: Kolesnichenko M, et al. Cell Cycle 2012; 11:2391-401 and Pospelova TV, et al. Cell Cycle 2012; 11:2402-407.