Acidic C-terminal Tail Research Articles

A critical role in structure-specific DNA binding for the acetylatable lysine residues in HMGB1

The structure-specific DNA-binding protein HMGB1 (high-mobility group protein B1) which comprises two tandem HMG boxes (A and B) and an acidic C-terminal tail, is acetylated in vivo at Lys(2) and Lys(11) in the A box. Mutation to alanine of both residues in the isolated A domain, which has a strong preference for pre-bent DNA, abolishes binding to four-way junctions and 88 bp DNA minicircles. The same mutations in full-length HMGB1 also abolish its binding to four-way junctions, and binding to minicircles is substantially impaired. In contrast, when the acidic tail is absent (AB di-domain) there is little effect of the double mutation on four-way junction binding, although binding to minicircles is reduced approximately 15-fold. Therefore it appears that in AB the B domain is able to substitute for the non-functional A domain, whereas in full-length HMGB1 the B domain is masked by the acidic tail. In no case does single substitution of Lys(2) or Lys(11) abolish DNA binding. The double mutation does not significantly perturb the structure of the A domain. We conclude that Lys(2) and Lys(11) are critical for binding of the isolated A domain and HMGB1 to distorted DNA substrates.

Acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 and ssDNA compete for the same binding surface

ssDNA-binding proteins are key components of the machinery that mediates replication, recombination, and repair. Prokaryotic ssDNA-binding proteins share a conserved DNA-binding fold and an acidic C-terminal tail. It has been proposed that in the absence of ssDNA, the C-terminal tail contacts the ssDNA-binding cleft, therefore predicting that the binding of ssDNA and the C-terminal tail is mutually exclusive. Using chemical cross-linking, competition studies, and NMR chemical-shift mapping, we demonstrate that: (i) the C-terminal peptide of the gene 2.5 protein cross-links to the core of the protein only in the absence of ssDNA, (ii) the cross-linked species fails to bind to ssDNA, and (iii) a C-terminal peptide and ssDNA bind to the same overall surface of the protein. We propose that the protection of the DNA-binding cleft by the electrostatic shield of the C-terminal tail observed in prokaryotic ssDNA-binding proteins, ribosomal proteins, and high-mobility group proteins is an evolutionarily conserved mechanism. This mechanism prevents random binding of charged molecules to the nucleic acid-binding pocket and coordinates nucleic acid-protein and protein-protein interactions.

HMGB1 protein inhibits DNA replication in vitro: A role of the acetylation and the acidic tail

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.

Mapping Intramolecular Interactions between Domains in HMGB1 using a Tail-truncation Approach

The mechanism underlying negative regulation of HMGB1-DNA interaction by the acidic C-terminal tail is ill defined. To address this issue, we have devised a novel NMR chemical-shift perturbation mapping strategy to elucidate interactions between the tail, which consists solely of aspartic acid and glutamic acid residues, and the two well characterized HMG-box DNA-binding domains. A series of HMGB1 tail-truncation mutants differing from each other by five residues was generated. Chemical-shift perturbation mapping using these mutants shows that tails of different lengths bind with different affinities. Nevertheless, the truncated tails bind along the same path on the HMG boxes as the full-length tail, differences in length being manifested in differences in the “reach”. The tail makes extensive contacts with the DNA-binding surfaces of both HMG boxes, thus explaining the basis of negative regulation of HMGB1–DNA interaction by the tail.

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

Dynamic DNA Helicase-DNA Polymerase Interactions Assure Processive Replication Fork Movement

A single copy of bacteriophage T7 DNA polymerase and DNA helicase advance the replication fork with a processivity greater than 17,000 nucleotides. Nonetheless, the polymerase transiently dissociates from the DNA without leaving the replisome. Ensemble and single-molecule techniques demonstrate that this dynamic processivity is made possible by two modes of DNA polymerase-helicase interaction. During DNA synthesis the polymerase and the helicase interact at a high-affinity site. In this polymerizing mode, the polymerase dissociates from the DNA approximately every 5000 bases. The polymerase, however, remains bound to the helicase via an electrostatic binding mode that involves the acidic C-terminal tail of the helicase and a basic region in the polymerase to which the processivity factor also binds. The polymerase transfers via the electrostatic interaction around the hexameric helicase in search of the primer-template.

Polymerase Switching in DNA Replication

Our view of DNA replication has been of two coupled DNA polymerases anchored to the replication fork helicase in a "replisome" complex, synthesizing leading and lagging strands simultaneously. New evidence suggests that three DNA polymerases can be accommodated into the replisome and that polymerases and repair factors are dynamically recruited and engaged without dismantling of the replisome.

The HMGB1 acidic tail regulates HMGB1 DNA binding specificity by a unique mechanism

HMGB1 is a conserved chromosomal protein composed of two DNA-binding domains and an acidic C-terminal tail. There were evidences suggesting that the C-terminal tail contributed to the DNA binding specificity of the N-terminal DNA-binding domains. However, the mechanism underlining this observation is largely unknown. Our data first confirmed the previous study with NMR that showed a direct interaction between HMGB1’s C-terminal tail and its N-terminal domains. We further demonstrated that this interaction can be competed more efficiently by a DNA with four-way junction structure than by a linear double-stranded DNA. Mutations within the N-terminal region, that disrupt its binding to the C-terminal tail, abolished HMGB1’s ability to distinguish the linear DNA and the four-way junction DNA. Those data suggested a unique mechanism designed by nature that utilizes a protein’s negatively charged C-terminal tail to enhance its DNA-binding domain’s specificity to certain structured DNAs.

Structural and Biophysical Coupling of Heparin and Activin Binding to Follistatin Isoform Functions

Follistatin (FS) regulates transforming growth factor-beta superfamily ligands and is necessary for normal embryonic and ovarian follicle development. Follistatin is expressed as two splice variants (FS288 and FS315). Previous studies indicated differences in heparin binding between FS288 and FS315, potentially influencing the physiological functions and locations of these isoforms. We have determined the structure of the FS315-activin A complex and quantitatively compared heparin binding by the two isoforms. The FS315 complex structure shows that both isoforms inhibit activin similarly, but FS315 exhibits movements within follistatin domain 3 (FSD3) apparently linked to binding of the C-terminal extension. Surprisingly, the binding affinities of FS288 and FS315 for heparin are similar at lower ionic strengths with FS315 binding decreasing more sharply as a function of salt concentration. When bound to activin, FS315 binds heparin similarly to the FS288 isoform, consistent with the structure of the complex, in which the acidic residues of the C-terminal extension cannot interact with the heparin-binding site. Activin-induced binding of heparin is unique to the FS315 isoform and may stimulate clearance of FS315 complexes.

Single Molecule Force Spectroscopy of Salt-dependent Bacteriophage T7 Gene 2.5 Protein Binding to Single-stranded DNA

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.

Conformational Properties of the SDS-Bound State of α-Synuclein Probed by Limited Proteolysis: Unexpected Rigidity of the Acidic C-Terminal Tail

Alpha-synuclein (alpha-syn) is a "natively unfolded" protein constituting the major component of intracellular inclusions in several neurodegenerative disorders. Here, we describe proteolysis experiments conducted on human alpha-syn in the presence of SDS micelles. Our aim was to unravel molecular features of micelle-bound alpha-syn using the limited proteolysis approach. The nonspecific proteases thermolysin and proteinase K, as well as the Glu-specific V8-protease, were used as proteolytic probes. While alpha-syn at neutral pH is easily degraded to a variety of relatively small fragments, in the presence of 10 mM SDS the proteolysis of the protein is rather selective. Complementary fragments 1-111 and 112-140, 1-113 and 114-140, and 1-123 and 124-140 are obtained when thermolysin, proteinase K, and V8 protease, respectively, are used. These results are in line with a conformational model of alpha-syn in which it acquires a folded helical structure in the N-terminal region in its membrane-bound state. At the same time, they indicate that the C-terminal portion of the molecule is rather rigid, as seen in its relative resistance to extensive proteolytic degradation. It is likely that, under the specific experimental conditions of proteolysis in the presence of SDS, the negatively charged C-terminal region can be rigidified by binding a calcium ion, as shown before with intact alpha-syn. In this study, some evidence of calcium binding properties of isolated C-terminal fragments 112-140, 114-140, and 124-140 was obtained by mass spectrometry measurements, since molecular masses for calcium-loaded fragments were obtained. Our results indicate that the C-terminal portion of the membrane-bound alpha-syn is quite rigid and structured, at variance from current models of the membrane-bound protein deduced mostly from NMR. Considering that the aggregation process of alpha-syn is modulated by its C-terminal tail, the results of this study may provide useful insights into the behavior of alpha-syn in a membrane-mimetic environment.

A Rich1/Amot Complex Regulates the Cdc42 GTPase and Apical-Polarity Proteins in Epithelial Cells

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Secondary structure and dynamics of micelle bound β‐ and γ‐synuclein

We have used solution state NMR spectroscopy to characterize the secondary structure and backbone dynamics of the proteins beta- and gamma-synuclein in their detergent micelle-bound conformations. Comparison of the results with those previously obtained for the Parkinson's disease-linked protein alpha-synuclein shows that structural differences between the three homologous synuclein family members are directly related to variations in their primary amino acid sequences. An 11-residue deletion in the lipid-binding domain of beta-synuclein leads to the destabilization of an entire segment of the micelle-bound helical structure containing the deletion site. The acidic C-terminal tail region of gamma-synuclein, which displays extensive sequence divergence, is more highly disordered than the corresponding regions in the other two family members. The observed structural differences are likely to mediate functional variations between the three proteins, with differences between alpha- and beta-synuclein expected to revolve around their lipid interactions, while differences in gamma-synuclein function are expected to result from different protein-protein interactions mediated by its unique C-terminal tail.

Cloning the genes and DNA binding properties of High Mobility Group B1 (HMGB1) proteins from the human blood flukes Schistosoma mansoni and Schistosoma japonicum

The parasitic helminth Schistosoma mansoni contains three HMGB proteins, HMGB1, HMGB2 and HMGB3, of primary amino acid sequences highly similar to vertebrate proteins. In this report we describe the characterization of the HMGB1 proteins and their genes from S. mansoni and Schistosoma japonicum. The deduced amino acid sequences of HMGB1 proteins from both schistosome species are identical, and comprise 176 residues. The proteins contain the two evolutionarily highly conserved HMG-box domains, A and B, exhibiting 60% similarity to mammalian HMGB1. Unlike the human HMGB1 which contains an unbroken run of 30 glutamic or aspartic residues, the SmHMGB1 or SjHMGB1 proteins possess unusually short acidic C-terminal tails (5 acidic residues interrupted by 2 serines). Southern hybridization and DNA sequencing revealed a single copy HMGB1 gene, composed of 3 exons and two introns, in S. mansoni. The exon/intron boundaries are identical to those of the human HMGB1 gene, with the exception that the second exon of the SmHMGB1 gene which is not split into two exons as in the human HMGB1 gene. RNA blot analysis revealed that the SmHMGB1 gene is constitutively expressed in similar levels both in male and female worms. The single-sized mRNA for SmHMGB1 is consistent with the size derived from the cDNA. Although DNA binding properties of SmHMGB1 (or SjHMGB1) protein seem to be similar to those previously reported with human HMGB1, i.e., preferential binding to supercoiled DNA over linear DNA, specific recognition of DNA four-way junctions, DNA-induced supercoiling in the presence of topoisomerase I, and DNA bending, we have observed two important differences relative to those observed with the human HMGB1: (i) the inability of the isolated SmHMGB1 domain A to bend DNA (as revealed by T4 ligase-mediated circularization assay), and (ii) higher DNA supercoiling and bending potential of the SmHMGB1 protein as compared to its human counterpart. The latter finding may indicate that the long acidic C-tail of human HMGB1 has much stronger repressive role on DNA bending or DNA supercoiling by topoisomerase I at physiological ionic strength than the short C-tail of the SmHMGB1 protein. Considering the important role of HMGB1 in DNA replication, transcription, recombination, and in particularly, the mediation of inflammation responses in mammalian cells, further studies on schistosome HMGB proteins may provide valuable information related to schistosomiasis, where inflammation plays a critical role in this disease.

The Cytotoxic Lymphocyte Protease, Granzyme B, Targets the Cytoskeleton and Perturbs Microtubule Polymerization Dynamics

Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell granules, plays an important role in coordinating apoptosis of CTL and NK target cells. Here, we report that granzyme B targets the cytoskeleton by cleaving and removing the acidic C-terminal tail of alpha-tubulin. Consistent with this, Granzyme B markedly enhanced rates of microtubule polymerization in vitro, most likely by removal of an autoinhibitory domain within the tubulin C terminus. Moreover, delivery of Granzyme B into HeLa target cells promoted dramatic reorganization of the microtubule network in a caspase-independent manner. These data reveal that granzyme B directly attacks a major component of the cell cytoskeleton, which may contribute to the incapacitation of target cells during CTL/NK-mediated killing.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

The Crystal Structure of Yeast Protein Disulfide Isomerase Suggests Cooperativity between Its Active Sites

Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted "U" with the active sites facing each other across the long sides of the "U." The inside surface of the "U" is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.

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.

Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Cross-species Sequence Analysis Reveals Multiple Charged Residue-rich Domains That Regulate Nuclear/Cytoplasmic Partitioning and Membrane Localization of A Kinase Anchoring Protein 12 (SSeCKS/Gravin)

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.