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.     

13C NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate

Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L. R., Sherry, A. D., Newkirk, M. M., & Gray D. M. (1988) J. Biol. Chem. 263, 18864-18872]. Thus, these lysines are probably directly involved in the nucleic acid binding function of the protein. Negatively charged chelates of lanthanide ions were used to perturb the 13C NMR resonances of labeled lysyl and amino-terminal residues of the gene 5 protein. The terbium chelate was found to bind tightly (Ka approximately 10(5) M-1) to the protein with a stoichiometry of 1 chelate molecule per protein dimer. 13C resonances of Lys-24, Lys-46, and Lys-69 were maximally shifted by the terbium chelate and were maximally relaxed by the gadolinium chelate. Also, the terbium chelate was excluded by the oligomer d(pA)7. Computer fits of the induced chemical shifts of 13C resonances with those expected for various positions of the terbium chelate failed to yield a possible chelate binding site unless the chemical shift for Lys-24 was excluded from the fitting process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein).

The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.     

Effect of charge reversal mutations on the ligand- and membrane-binding properties of liver fatty acid-binding protein

Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength. This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA), with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932-16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those within the alpha-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein undergoes a conformational change, resulting in DAUDA release.     

Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2

We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B' motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B' as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.     

Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2-286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action.     

Analysis of site-specific N-homocysteinylation of human serum albumin in vitro and in vivo using MALDI-ToF and LC-MS/MS mass spectrometry

Elevated levels of homocysteine (Hcy) are associated with cardiovascular and neurodegenerative diseases in humans. Hcy becomes a component of human proteins as a result of N-homocysteinylation of protein lysine residues by Hcy-thiolactone, which affects the protein's structure and function, and contributes to Hcy-related pathology. Albumin is the major target for N-homocysteinylation in human blood in vivo. Previous work has identified Lys-525 as a predominant site of N-homocysteinylation in vitro and in vivo. Here we show that Lys-4, Lys-12, Lys-137, Lys-159, Lys-205, and Lys-212 of human albumin are susceptible to N-homocysteinylation in vitro and provide evidence that two of those residues, Lys-137 and Lys-212, in addition to Lys-525, are N-homocysteinylated in vivo in human plasma.     

A C-terminal segment with properties of alpha-helix is essential for DNA binding and in vivo function of zinc finger protein Rme1p

Rme1p plays important roles in the control of meiosis and in cell cycle progression through binding to upstream regions of IME1 and CLN2 in Saccharomyces cerevisiae. Rme1p has three zinc finger segments, and two of them are atypical. To determine DNA binding domain of Rme1p, a series of Rme1p derivatives fused with maltose-binding protein were purified and characterized by gel mobility shift assay. We show that not only three zinc fingers, but also the neighboring C-terminal region is essential for DNA binding. Mutational analysis of this region revealed that basic residues Arg-287, Lys-290, and Arg-291 and the hydrophobic residues Phe-288, Leu-292, Ile-295, and Leu-296 are critical for DNA binding. In addition, double substitutions by proline at Asn-289 and Lys-293, each of which was not essential for DNA binding, abolished DNA binding. These results suggest that the C-terminal segment forms an amphipathic helical structure. Furthermore, it was shown that the mutations in the important basic residues abolish or impair Rme1p function in vivo for repression and inhibition of spore formation. Thus, the C-terminal segment is essential and acts as a novel accessory domain for DNA binding by zinc fingers.     

The active site of the Escherichia coli MutY DNA adenine glycosylase.

Escherichia coli MutY is an adenine DNA glycosylase active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. Although MutY can form a covalent intermediate with its DNA substrates, its possession of 3' apurinic lyase activity is controversial. To study the reaction mechanism of MutY, the conserved Asp-138 was mutated to Asn and the reactivity of this mutant MutY protein determined. The glycosylase activity was completely abolished in the D138N MutY mutant. The D138N mutant and wild-type MutY protein also possessed different DNA binding activities with various mismatches. Several lysine residues were identified in the proximity of the active site by analyzing the imino-covalent MutY-DNA intermediate. Mutation of Lys-157 and Lys-158 both individually and combined, had no effect on MutY activities but the K142A mutant protein was unable to form Schiff base intermediates with DNA substrates. However, the MutY K142A mutant could still bind DNA substrates and had adenine glycosylase activity. Surprisingly, the K142A mutant MutY, but not the wild-type enzyme, could promote a beta/delta-elimination on apurinic DNA. Our results suggest that Asp-138 acts as a general base to deprotonate either the epsilon-amine group of Lys-142 or to activate a water molecule and the resulting apurinic DNA then reacts with Lys-142 to form the Schiff base intermediate with DNA. With the K142A mutant, Asp-138 activates a water molecule to attack the C1' of the adenosine; the resulting apurinic DNA is cleaved through beta/delta-elimination without Schiff base formation.     

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.     

Effect of single mutations on the specificity of Escherichia coli FPG protein for excision of purine lesions from DNA damaged by free radicals

The formamidopyrimidine N-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that is specific for the removal of purine-derived lesions from DNA damaged by free radicals and other oxidative processes. We investigated the effect of single mutations on the specificity of this enzyme for three purine-derived lesions in DNA damaged by free radicals. These damaging agents generate a multiplicity of base products in DNA, with the yields depending on the damaging agent. Wild type Fpg protein (wt-Fpg) removes 8-hydroxyguanine (8-OH-Gua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from damaged DNA with similar specificities. We generated five mutant forms of this enzyme with mutations involving Lys-57-->Gly (FpgK57G), Lys-57-->Arg (FpgK57R), Lys-155-->Ala (FpgK155A), Pro-2-->Gly (FpgP2G), and Pro-2-->Glu (FpgP2E), and purified them to homogeneity. FpgK57G and FpgK57R were functional for removal of FapyAde and FapyGua with a reduced activity when compared with wt-Fpg. The removal of 8-OH-Gua was different in that the specificity of FpgK57G was significantly lower for its removal from irradiated DNA, whereas wt-Fpg, FpgK57G, and FpgK57R excised 8-OH-Gua from H2O2/Fe(III)-EDTA/ascorbic acid-treated DNA with almost the same specificity. FpgK155A and FpgP2G had very low activity and FpgP2E exhibited no activity at all. Michaelis-Menten kinetics of excision was measured and kinetic constants were obtained. The results indicate an important role of Lys-57 residue in the activity of Fpg protein for 8-OH-Gua, but a lesser significant role for formamidopyrimidines. Mutations involving Lys-155 and Pro-2 had a dramatic effect with Pro-2-->Glu leading to complete loss of activity, indicating a significant role of these residues. The results show that point mutations significantly change the specificity of Fpg protein and suggest that point mutations are also expected to change specificities of other DNA repair enzymes.     

Structure of nucleosomes. Localization of the H2A and H2B histone segments interacting with DNA using DNA-protein crosslinking]

Histones' H2A and H2B peptidic points which interact with nucleosomal DNA have been identified by using the methods of DNA--protein covalent cross-linking. H2B can be linked to DNA via its N-terminal tail and via several lysines contained within residues 24-34. The most prominent site of histone H2A covalent linking to DNA is His-123, the less prominent being Lys-119 and Lys-124.     

Intramolecular allosteric interaction in the phospholipase C-δ1 pleckstrin homology domain

Protein activities are generally regulated by intramolecular allosteric interactions, by which spatially separated sites in a protein molecule communicate. Intramolecular allosteric interactions in the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain were investigated by solution NMR spectroscopy for selectively [α-¹⁵N]Lys-labeled proteins. The results of NMR analyses indicated that the binding of inositol 1,4,5-trisphosphate (IP₃) to the protein induces local environmental changes at all lysine residues, including residues such as Lys-43 spatially separated from the specific IP₃ binding site consisting of Lys-30, Lys-32, and Lys-57. IP₃ binding also induces conformational stabilization of a characteristic short α-helix (α2) from residues 82 to 87. Mutational analyses indicated that an interaction network mainly consisting of the side chains of Lys-30, Lys-32, and Lys-43 exists in the ligand-free protein, and it was therefore predicted that binding of IP₃ to the specific site modifies the interaction network, resulting in formation of a new interaction network, in which the side chains of Lys-57 and Phe-87 contribute to stable IP₃ binding. These results provide evidence for intramolecular interactions in the PLC-δ1 PH domain, the function of which could be allosterically regulated by modifications at sites spatially separated from the ligand-binding site through the intramolecular interaction network.     

Mutational analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).     

Identification of the amino acid residues of proteins S5 and S8 adjacent to each other in the 30 S ribosomal subunit of Escherichia coli.

When Escherichia coli 30 S ribosomal subunits are reacted with protein-protein bifunctional reagents, a number of protein pairs as well as aggregates containing three or more ribosomal proteins are formed. In the present study we have purified one of the protein pairs obtained by reaction of 30 S ribosomal subunits with either radioactive or nonradioactive dimethylsuberimidate. Following molecular weight determination and ammonolysis, the pair was shown to consist of ribosomal proteins S5 and S8. The "native" structure of the complex was surmised from its capacity to be reconstituted into a biologically active 30 S ribosomal subunit. From peptide maps and primary structure determination of various peptides it was demonstrated that the cross-linking bond between ribosomal proteins S5 and S8 involves primarily the residues Lys-93 of protein S8 and the COOH-terminal lysine (Lys-166) of ribosomal protein S5. This result is substantiated by the finding that a mutant carrying an altered S5 lacking the COOH-terminal lysine yields a greatly reduced amount of S5-S8 cross-link. In addition to the points of cross-linking it was found that Lys-30, Lys-68, and Lys-86 of S8 and Lys-5 of S5 react with dimethylsuberimidate, indicating that these residues are available for reaction and suggesting their topographical localization on the ribosomal surface.     

Role of N-linked oligosaccharide flexibility in mannose phosphorylation of lysosomal enzyme cathepsin L

Mannose phosphorylation of N-linked oligosaccharides by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is a key step in the targeting of lysosomal enzymes in mammalian cells and tissues. The selectivity of this process is determined by lysine-based phosphorylation signals shared by lysosomal enzymes of diverse structure and function. By introducing new glycosylation sites at several locations on the surface of mouse procathepsin L and modeling oligosaccharide conformations for sites that are phosphorylated, it was shown that the inherent flexibility of N-linked oligosaccharides can account for the specificity of the transferase for oligosaccharides at different locations on the protein. By using this approach, the physical relationship between the lysine-based signal and the site of phosphorylation of mannose residues was determined. The analysis also revealed the existence of additional independent lysine-based phosphorylation signals on procathepsin L, which account for the low level of phosphorylation observed when the primary Lys-54/Lys-99 signal is ablated. Mutagenesis of residues that surround Lys-54 and Lys-99 and demonstration of mannose phosphorylation of a glycosylated derivative of green fluorescent protein provide strong evidence that the cathepsin L phosphorylation signal is a simple structure composed of as few as two well placed lysine residues.     

Molecular mechanism for functional interaction between DnaA protein and acidic phospholipids: identification of important amino acids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be reactivated from the ADP-bound form to its ATP-bound form through stimulation of ADP release by acidic phospholipids such as cardiolipin. We previously reported that two potential amphipathic helices (Lys-327 to Ile-344 and Asp-357 to Val-374) of DnaA protein are involved in the functional interaction between DnaA and cardiolipin. In relation to one of these helices (Asp-357 to Val-374), we demonstrated that basic amino acids in the helix, especially Lys-372, are vital for this interaction. In this study, we have identified an amino acid in the second potential amphipathic helix (Lys-327 to Ile-344), which would also appear to be involved in the interaction. We constructed three mutant dnaA genes with a single mutation (dnaAR328E, dnaAR334E, and dnaAR342E) and examined the function of the mutant proteins. DnaAR328E, but not DnaAR334E and DnaAR342E, was found to be more resistant to inhibition of its ATP binding activity by cardiolipin than the wild-type protein. The stimulation of ADP release from DnaAR328E by cardiolipin was also weaker than that observed with the other mutants and the wild-type protein. These results suggest that Arg-328 of DnaA protein is involved in the functional interaction of this protein with acidic phospholipids. We propose that acidic phospholipids bind to two basic amino acid residues (Arg-328 and Lys-372) of DnaA protein and change the higher order structure of its ATP-binding pocket, which in turn stimulates the release of ADP from the protein.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.