Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

Immunochemical analysis of the exposure of high mobility group protein 14 and 17 surfaces in chromatin

Antisera were elicited against synthetic peptides corresponding either to regions common to all members of the high mobility group 14 and 17 protein family protein or to distinct domains of the HMG-14 or HMG-17 subgroup. The antisera were used to probe the accessibility of various HMG domains in chromatin. Competitive enzyme-linked immunosorbent assays indicate that the central region of the proteins, which contains their DNA binding domain and is positively charged, is exposed to a smaller degree than the C-terminal region of the proteins, which has a net negative charge. The C-terminal regions of the HMG-14 and HMG-17 proteins are exposed and available to interact with other proteins.     

Dissection of reverse gyrase activities: insight into the evolution of a thermostable molecular machine

Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.     

Isolation and characterization of the C-terminal nuclease domain from the RecB protein of Escherichia coli.

The RecB subunit of the Escherichia coli RecBCD enzyme has been shown in previous work to have two domains: an N-terminal 100 kDa domain with ATP-dependent helicase activity, and a C-terminal 30 kDa domain. The 30 kDa domain had nuclease activity when linked to a heterologous DNA binding protein, but by itself it appeared unable to bind DNA and lacked detectable nuclease activity. We have expressed and isolated this 30 kDa domain, called RecB(N), and show that it does have nuclease activity detectable at high protein concentration in the presence of polyethylene glycol, added as a molecular crowding agent. The activity is undetectable in a mutant RecB(N)protein in which an aspartate residue has been changed to alanine. Structural analysis of the wild-type and mutant RecB(N)proteins by second derivative absorbance and circular dichroism spectroscopy indicates that both are folded proteins with very similar secondary and tertiary structures. The results show that the Asp-->Ala mutation has not caused a significant structural change in the isolated domain and they support the conclusion that the C-terminal domain of RecB has the sole nuclease active site of RecBCD.     

Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA

RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.     

Partial proteolysis of simian virus 40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly

Simian virus 40 large tumor antigen (Tag) is a multi-functional viral protein that binds specifically to SV40 origin DNA, serves as the replicative DNA helicase, and orchestrates the assembly and operation of the viral replisome. Tag associated with Mg-ATP forms hexamers and, in the presence of SV40 origin DNA, double hexamers. Limited tryptic digestion of monomeric Tag revealed three major stable structural domains. The N-terminal domain spans amino acids 1-130, the central domain comprises amino acids 131-476, and the C-terminal domain extends from amino acid 513 to amino acid 698. Co-immunoprecipitation of digestion products of monomeric Tag suggests that the N-terminal domain associates stably with sequences located in the central region of the same Tag molecule. Hexamer formation protected the tryptic cleavage sites in the exposed region between the central and C-terminal domains. Upon hexamerization, this exposed region also became less accessible to a monoclonal antibody whose epitope maps in that region. The tryptic digestion products of the soluble hexamer and the DNA-bound double hexamer were indistinguishable. A low-resolution model of the intramolecular and intermolecular interactions among Tag domains in the double hexamer is proposed.     

Quaternary structure and function in phage lambda repressor: 1H-NMR studies of genetically altered proteins

The quaternary structure and dynamics of phage lambda repressor are investigated in solution by 1H-NMR methods. lambda repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. 1H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the "linker" polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84----Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

The N-terminal domain of PILB from Neisseria meningitidis is a disulfide reductase that can recycle methionine sulfoxide reductases

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.     

Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2

Monoclonal antibodies were prepared against the high mobility group (HMG) proteins 1, 2a, and 2b from hen erythrocyte chromatin. One antibody that recognized multiple sites along HMG-1, -2a, and -2b reacted strongly with HMG proteins from all vertebrates tested. In contrast, five antibodies that detected unique epitopes on chicken HMG-1 and -2a recognized antigenic sites that exhibited restricted phylogenic distributions. The differential reactivity of these antibodies on vertebrate proteins was in agreement with traditional taxonomy in that the avian HMGs were most closely related to those from reptiles and less related to those from mammals, amphibians, bonyfish, and especially the jawless fish. Mononucleosomes generated by mild digestion of erythrocyte chromatin with micrococcal nuclease were highly enriched in HMG-2a. One antigenic determinant located within the N-terminal domain of HMG-2a was freely accessible to its antibody when the protein was bound to these mononucleosomes. In contrast, two antibodies that recognized determinants in the central region of HMG-2a exhibited little chromatin binding activity. The masking of the central domain by DNA binding was presumably not responsible for these results because all three determinants were available for antibody binding when HMG-2a was bound to DNA in vitro. Therefore, the central region of HMG-2a may be masked from antibody binding by protein-protein interactions in chromatin.     

Production of HMG-3 by limited trypsin digestion of purified high-mobility-group nonhistone chromatin proteins

Three isolated nonhistone proteins (HMG-1, HMG-2 and HMG-E) have been purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions, and subjected to limited proteolysis. When treated with trypsin, the three proteins exhibited similar patterns of degradation, as judged by SDS and acid/urea gel electrophoresis. In particular, the first product, P1 (a relatively stable intermediate in each digestion), was a protein analogous to HMG-3, a principal degradation product in preparations of calf thymus high-mobility-group proteins. At least in the case of HMG-E, the products formed by tryptic attack on P1 are the two individual DNA binding domains of HMG-E. P1 derived from HMG-E and one of the individual DNA binding domains of HMG-E were purified by chromatography on columns containing DNA-cellulose or phosphocellulose. The properties of these two portions of HMG-E are consistent with our recently postulated three-domain structure for HMG-1 and its homologs (Reeck, G.R., Isackson, P.J. and Teller, D.C. (1982) Nature 300, 76-78). Thus, P1 consists of two DNA-binding domains of approximately equal molecular weight covalently linked together. From chromatography on DNA-cellulose columns, it is clear that P1 binds to DNA more tightly than does HMG-E. The highly acidic C-terminal domain of HMG-E (which is removed by trypsin in generating P1) thus counteracts the DNA binding of the two other domains of HMG-E (at least in the protein's interaction with purified DNA).     

Comparative analysis of polyphenol oxidase from plant and fungal species

Polyphenol oxidase from plants and fungi is a metalloenzyme containing a type-3 copper center and is homologous to oxygen-carrying hemocyanin of molluscs. Molluscan hemocyanin consists of two domains, an N-terminal domain containing the copper center and a smaller C-terminal domain, connected by an alpha-helical linker. It is presumed that the same is true of polyphenol oxidase from plants and fungi although the structure of a polyphenol oxidase containing the C-terminal domain has not been determined. We show that a number of important structural features are conserved in the N-terminal domains of polyphenol oxidases from various plants and fungi, including a tyrosine motif which can be considered a landmark indicating the beginning of the linker region connecting the N- and C-terminal domains. Our sequence alignments and secondary structure predictions indicate that the C-terminal domains of polyphenol oxidases are likely to be similar in tertiary structure to that of hemocyanin. Detailed bioinformatics analyses of the linker regions predict that this section of the polypeptide chain is intrinsically disordered (lacking fixed tertiary structure) and contains a site of proteolytic processing as well as a potential phosphorylation site.     

The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.     

Homology model building of the HMG-1 box structural domain.

Nucleoproteins belonging to the HMG-1/2 family possess homologous domains approximately 75 amino acids in length. These domains, termed HMG-1 boxes, are highly structured, compact, and mediate the interaction between HMG-1 box-containing proteins and DNA in a variety of biological contexts. Homology model building experiments on HMG-1 box sequences 'threaded' through the 1H-NMR structure of an HMG-1 box from rat indicate that the domain does not have rigid sequence requirements for its formation. Energy calculations indicate that the structure of all HMG-1 box domains is stabilized primarily through hydrophobic interactions. We have found structural relationships in the absence of statistically significant sequence similarity, identifying several candidate proteins which could possibly assume the same three-dimensional conformation as the rat HMG-1 box motif. The threading technique provides a method by which significant structural similarities in a diverse protein family can be efficiently detected, and the 'structural alignment' derived by this method provides a rational basis through which phylogenetic relationships and the precise sites of interaction between HMG-1 box proteins and DNA can be deduced.     

Quarternary Structure and Function in Phage λ Repressor: 1H-NMR Studies of Genetically Altered Proteins

Abstract

The quaternary structure and dynamics of phage λ repressor are investigated in solution by ¹H-NMR methods. λ repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. ¹H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the “linker” polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84→Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit

Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding protein that is required for multiple processes in cellular DNA metabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates in multiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multiple domains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X2-C-X13-C-X2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate the roles of these domains in RPA function. The central DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part of the N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residues in RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these residues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.     

LAST motifs and SMART domains in gene 32 protein: an unfolding story of autoregulation?

Bacteriophage T4 gene 32 protein is a classical single strand-specific DNA binding protein. It is a single polypeptide chain of 301 amino acid residues that consists of three structural domains, each of which has a binding function. The N-terminal domain is involved in homotypic protein-protein interaction (the basis of binding cooperativity), the core domain binds single strands directly, and the C-terminal domain has a role in heterotypic protein-protein association. The three domains have traditionally been thought to be independent of each other. However, the observation of a striking repetition of a basic, polar sequence (the "LAST" Motif), seen in both the N-terminal and core domains, suggests a linkage between these domains. Moreover, the C-domain and adjoining portion (flap) of the core are highly acidic, and are potential mimics of single-stranded DNA. With these observations, I construct a model in which this flap is associated with the ssDNA binding site in the absence of DNA, and upon cooperative protein binding to DNA, the flap now associates with the N-terminal domain of the adjacent DNA-bound protein. The flap thus acts as a gate, which might slow the binding of the protein to DNA. This could lead to the regulation of the protein's various interactions with other proteins, as well as affect its ability to lower DNA melting temperature.     

Crystal structure of the N-terminal domain of MukB: a protein involved in chromosome partitioning.

BACKGROUND: The 170 kDa protein MukB has been implicated in ATP-dependent chromosome partitioning during cell division in Escherichia coli. MukB shares its dimeric structure and domain architecture with the ubiquitous family of SMC (structural maintenance of chromosomes) proteins that facilitate similar functions. The N-terminal domain of MukB carries a putative Walker A nucleotide-binding region and the C-terminal domain has been shown to bind to DNA. Mutant phenotypes and a domain arrangement similar to motor proteins that move on microtubules led to the suggestion that MukB might be a motor protein acting on DNA. RESULTS: We have cloned, overexpressed and crystallized a 26 kDa protein consisting of 227 N-terminal residues of MukB from E. coli. The structure has been solved using multiple anomalous dispersion and has been refined to 2.2 A resolution. The N-terminal domain of MukB has a mixed alpha/beta fold with a central six-stranded antiparallel beta sheet. The putative nucleotide-binding loop, which is part of an unexpected helix-loop-helix motif, is exposed on the surface and no nucleotide-binding pocket could be detected. CONCLUSIONS: The N-terminal domain of MukB has no similarity to the kinesin family of motor proteins or to any other nucleotide-binding protein. Together with the finding of the exposed Walker A motif this observation supports a model in which the N- and C-terminal domains come together in the dimer of MukB to form the active site. Conserved residues on one side of the molecule delineate a region of the N-terminal domain that is likely to interact with the C-terminal domain.     

Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation

Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.     

Solution structure and interdomain interactions of the Saccharomyces cerevisiae "TATA binding protein" (TBP) probed by radiolytic protein footprinting

Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains. Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex. Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation. Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA. Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA. In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding. These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein. The functional implications of these results are discussed.