Mammalian High Mobility Group Protein Research Articles

Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Exploring the Conformations and Binding Location of HMGA2·DNA Complexes Using Ion Mobility Spectrometry and 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.

AT-hook peptides bind the major and minor groove of AT-rich DNA duplexes.

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as ‘AT-hooks’, are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three ‘AT-hook’ peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, ‘key-locked’ conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the “AT-hook” DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple “AT-hook” DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the “AT-hook” DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin’s cellular targets.

The Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2): Biochemical and Biophysical Properties, and Its Association with Adipogenesis.

The mammalian high-mobility-group protein AT-hook 2 (HMGA2) is a small DNA-binding protein and consists of three “AT-hook” DNA-binding motifs and a negatively charged C-terminal motif. It is a multifunctional nuclear protein directly linked to obesity, human height, stem cell youth, human intelligence, and tumorigenesis. Biochemical and biophysical studies showed that HMGA2 is an intrinsically disordered protein (IDP) and could form homodimers in aqueous buffer solution. The “AT-hook” DNA-binding motifs specifically bind to the minor groove of AT-rich DNA sequences and induce DNA-bending. HMGA2 plays an important role in adipogenesis most likely through stimulating the proliferative expansion of preadipocytes and also through regulating the expression of transcriptional factor Peroxisome proliferator-activated receptor γ (PPARγ) at the clonal expansion step from preadipocytes to adipocytes. Current evidence suggests that a main function of HMGA2 is to maintain stemness and renewal capacity of stem cells by which HMGA2 binds to chromosome and lock chromosome into a specific state, to allow the human embryonic stem cells to maintain their stem cell potency. Due to the importance of HMGA2 in adipogenesis and tumorigenesis, HMGA2 is considered a potential therapeutic target for anticancer and anti-obesity drugs. Efforts are taken to identify inhibitors targeting HMGA2.

Tight Binding of Natural Polyphenols to the Intrinsically Disordered Mammalian High Mobility Group Protein At-Hook 2

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered protein (IDP) that specifically recognizes the minor-groove of AT-rich DNA sequence. It works as a multifunctional DNA-binding protein and plays important roles in tumorigenesis and adipogenesis. Previous studies showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. In this study, we found that several natural polyphenols, such as aurintricarboxylic acid, gallic acid, ellagic acid, and (−)-epigallocatechin gallate (EGCG) tightly bind to HMGA2 and, as a result, strongly inhibit HMGA2 binding to AT-rich DNA sequences. Since these polyphenols can inhibit adipogenesis in vitro and in animal models, our results suggest that their anti-obesity activities may stem from their inhibition of HMGA2-DNA interactions. Because polyphenols are naturally occurring chemical compounds enriched in vegetables and fruits, our discovery strongly support that vegetables and fruits are natural remedies to treat obesity.

Identification of Suramin as a Potent and Specific Inhibitor of the Mammalian High Mobility Group Protein at-Hook 2 (HMGA2)-DNA Interactions

Identification of Suramin as a Potent and Specific Inhibitor of the Mammalian High Mobility Group Protein at-Hook 2 (HMGA2)-DNA Interactions

The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2)

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged “AT-hooks” and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus greatly contribute to the homodimer formation.

Isomerization kinetics of AT hook decapeptide solution structures.

The mammalian high mobility group protein HMGA2 contains three DNA binding motifs associated with many physiological functions including oncogenesis, obesity, stem cell youth, human height, and human intelligence. In the present paper, trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) has been utilized to study the conformational dynamics of the third DNA binding motif using the "AT hook" decapeptide unit (Lys(1)-Arg(2)-Prol(3)-Arg(4)-Gly(5)-Arg(6)-Prol(7)-Arg(8)-Lys(9)-Trp(10), ATHP) as a function of the solvent state. Solvent state distributions were preserved during electrospray ion formation, and multiple IMS bands were identified for the [M + 2H](2+) and for the [M + 3H](3+) charge states. Conformational isomer interconversion rates were measured as a function of the trapping time for the [M + 2H](2+) and [M + 3H](3+) charge states. Candidate structures were proposed for all IMS bands observed. Protonation site, proline residue conformation, and side chain orientations were identified as the main motifs governing the conformational interconversion processes. Conformational dynamics from the solvent state distribution to the gas-phase "de-solvated" state distribution demonstrated that ATHP is "structured", and relative abundances are associated with the relative stability between the proposed conformers. The most stable ATHP [M + 2H](2+) conformation at the "de-solvated" state corresponds to the AT hook motif observed in AT-rich DNA regions.

Interaction of Saccharomyces cerevisiae HMO2 Domains with Distorted DNA

The Saccharomyces cerevisiae high mobility group protein HMO2 is a component of the chromatin remodeling complex INO80. In this capacity, it has been shown to direct INO80 to DNA double-strand breaks, thereby contributing to double-strand break repair. Consistent with such function, HMO2 binds DNA ends, protecting them from exonucleolytic degradation. We show here that both domains of HMO2, HMO2-BoxA and HMO2-BoxB, bind preferentially to distorted DNA, with HMO2-BoxA binding preferentially to four-way DNA junctions and DNA with tandem mismatches and HMO2-BoxB binding four-way junctions as well as DNA with stem-loop structures, tandem mismatches, and abasic sites. As previously reported for mammalian high mobility group proteins, the acidic C-terminal extension significantly attenuates DNA binding. Notably, the unique ability of HMO2 to protect DNA ends is conferred by the Box A domain. Considering the reported roles for INO80 in other events such as recovery of stalled replication forks and nucleotide excision repair, we assessed the effect of DNA damaging agents on an hmo2Δ strain; while modest growth inhibition is seen upon exposure to UV light, exposure to hydroxyurea, which causes replication fork arrest, induces severe growth deficiency. These data suggest that HMO2 may also participate in directing the INO80 complex to sites such as stalled replication forks; the preferred binding of HMO2 domains to damaged DNA and intermediates in homologous recombination is consistent with such function.

DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

The mammalian high-mobility group protein AT hook 2 (HMGA2) is a DNA binding protein that specifically recognizes the minor groove of AT-rich DNA sequences. Disruption of its expression pattern is directly linked to oncogenesis and obesity. In this paper, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA segment containing five pairs of restriction enzyme sites, which can be used to produce a set of DNA fragments of identical length to study protein-induced DNA bending. The DNA fragments of identical length can also be generated using PCR amplification. Since pBendAT does not contain more than three consecutive AT base pairs, it is suitable for the assessment of DNA bending induced by proteins recognizing AT-rich DNA sequences. Indeed, using pBendAT, we demonstrated that HMGA2 is a DNA bending protein and bends all three tested DNA binding sequences of HMGA2, SELEX1, SELEX2, and PRDII. The DNA bending angles were estimated to be 34.2 degrees , 33.5 degrees , and 35.4 degrees , respectively.

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)2 is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5′-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3′ (FL-AT-1) and 5′-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3′ (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to ∼200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.

Specific Recognition of AT-Rich DNA Sequences by the Mammalian High Mobility Group Protein AT-hook 2: A SELEX Study

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a transcriptional factor involved in cell differentiation and transformation. Disruption of its normal expression pattern is directly linked to oncogenesis and obesity. HMGA2 contains three "AT-hook" DNA binding domains, which specifically bind to the minor groove of AT-rich sequences. Using a PCR-based systematic evolution of ligands by exponential enrichment (SELEX) procedure, we have identified two consensus sequences for HMGA2, 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These two consensus sequences have a unique and interesting feature: the first five base pairs are AT-rich, the middle four base pairs are GC-rich, and the last six base pairs are AT-rich. Our results showed that all three of these segments are critical for high-affinity binding of HMGA2 to DNA. For example, if one of the AT-rich sequences is mutated to a non-AT-rich sequence, the DNA binding affinity of HMGA2 is reduced at least 100-fold. Intriguingly, if the GC-segment is replaced by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step toward finding its binding sites within the genome.

Large Scale Preparation of the Mammalian High Mobility Group Protein A2 for Biophysical Studies

Due to asymmetrical charge distribution of the mammalian high mobility group protein A2 (HMGA2), which makes HMGA2 bind to both cation- and anion-exchange columns, we developed a rapid procedure for purifying HMGA2 in the milligram range. This purification procedure greatly facilitated biophysical studies, which require large amounts of the protein.

Energetics of Binding the Mammalian High Mobility Group Protein HMGA2 to poly(dA-dT) 2 and poly(dA)-poly(dT)

The mammalian high mobility group protein A2 (HMGA2) is a chromosomal architectural transcription factor involved in oncogenesis and cell transformation. It has three "AT-hook" DNA binding domains, which specifically bind to the minor groove of AT DNAs. The interaction of HMGA2 with poly(dA-dT)2 and poly(dA)poly(dT) has been investigated using the ethidium displacement assay, isothermal titration calorimetry, and UV melting studies. Each AT hook DNA binding domain was found to bind to 5 bp and each HMGA2 molecule binds to 15 bp. Although an individual AT hook DNA binding domain binds to AT DNAs with moderate affinity, HMGA2 binds with very high affinity to both DNAs in solutions containing 20 mM Na+ at 25 degrees C. The K(a) and binding enthalpy for poly(dA-dT)2 were determined to be, respectively, 1.9x10(14)M(-1) and -29.1(+/-0.5)kcal/mol. The binding reaction is enthalpy-driven with a favorable free energy of -19.5 kcal/mol and unfavorable entropy of -32.5 cal/mol K (-TDeltaS= +9. 7kcal/mol) at a 1M reference state. Interestingly, although HMGA2 binds to poly(dA)poly(dT) with a binding constant of 9.6x10(12) M(-1), the binding reaction is entropy-driven with an unfavorable enthalpy of +0.6 kcal/mol, a free energy of -17.7 kcal/mol and an entropy of +61.4 cal/mol K (-TDeltaS=-18.3 kcal/mol) at the 1 M state. The enthalpy-entropy compensation is similar to that of several minor groove-binding drugs such as netropesin, distamycin A and Hoechst33258 and may be a reflection of dehydration difference of different ligand-DNA complexes. The salt-dependence of the binding constant of HMGA2 with both DNAs showed that electrostatic interaction is a dominant force for the binding reactions. The temperature dependence of binding enthalpy for poly(dA-dT)2 indicates a large heat capacity of binding of -705(+/-113) cal/molK, consistent with an important role of solvent displacement in the linked folding/binding processes in this system.

A mini review of the high mobility group proteins of insects.

High mobility group (HMG) proteins are an abundant class of chromosomal proteins facilitate assembly of higher order structures. The mammalian HMG proteins have been grouped into three distinct families on the basis of their characteristic functional sequence: the HMGB, the HMGN, and the HMGA family. The HMG proteins of Drosophila melanogaster and Chironomus tentans are the best characterized dipteran insect HMG proteins. Three abundant members of this group of nonhistone proteins were detected in those insects. Two of them belong to the HMGB family and one to the HMGA family. The possible relatedness of these proteins to the formation of higher order nucleoprotein structures and their possible role in the regulation of transcription is discussed.

A DNA unwinding factor involved in DNA replication in cell-free extracts of Xenopus eggs

Background: Alteration of chromatin structure is a key step in various aspects of DNA metabolism. DNA unwinding factors such as the high mobility group (HMG) proteins are thought to play a general role in controlling chromatin structure and a specific role in controlling DNA replication. For instance, in the in vitro simian virus 40 replication system, minichromosomes containing HMG-17 replicate more efficiently than those without it, suggesting that HMG-17 enhances the rate of replication of a chromatin template by unfolding the higher-order chromatin structure. At present, however, only limited data suggest an involvement of DNA unwinding factors in DNA replication.Results: We purified from Xenopus eggs a novel heterodimeric factor, termed DNA unwinding factor (DUF), that consists of 87 kDa and 140 kDa polypeptides. DUF unwinds closed-circular duplex DNA in the presence of topoisomerase I, but it does not possess a DNA gyrase activity: it does not introduce negative supercoils into DNA at the expense of ATP hydrolysis. Cloning and sequencing of the cDNAs encoding the two polypeptides revealed that the 87 kDa polypeptide is homologous to a mammalian HMG protein, T160/structure-specific recognition protein. The 140 kDa polypeptide is homologous to yeast Cdc68, a protein that controls the expression of several genes during the G1 phase of the cell cycle by modulating chromatin structure. Immunodepletion of DUF from Xenopus egg extracts drastically reduced the ability of the extract to replicate exogenously added sperm chromatin or plasmid DNA.Conclusions: We propose that DUF plays a role in DNA replication in Xenopus egg extracts.

Cdc2 and Mitogen-activated Protein Kinases Modulate DNA Binding Properties of the Putative Transcriptional RegulatorChironomus High Mobility Group Protein I

Cells of the dipteran insect Chironomus contain a high mobility group protein that is homologous to the mammalian high mobility group proteins I/Y (HMGI/Y). These proteins facilitate the assembly of higher order nucleoprotein complexes. In proliferating cells, >30% of Chironomus HMGI was found to be phosphorylated. The phosphorylation sites were mapped to Ser3, Ser22, and Ser72 and were found to be substrates for the kinases Cdc2 (and mitogen-activated protein (MAP)), MAP, and Ca2+/phospholipid-dependent protein kinase, respectively. In mitotically arrested cells, the extent of phosphorylation at Ser3 increased, whereas phosphorylation at Ser22 remained unchanged. In nondividing cells, phosphorylation at Ser3 and Ser22 was strongly reduced. The DNA binding affinity of Chironomus HMGI was not influenced by single phosphorylation at Ser3 or Ser22. In contrast, phosphorylation at both of these sites resulted in a 10-fold weakening of the binding activity and altered the mode of protein-DNA interaction. Since both human and murine HMGI/Y proteins, similarly to the insect HMGI protein, possess phosphorylation sites for Cdc2 and MAP kinases that intersperse the AT-hook DNA-binding motifs, our results may reflect a general mechanism that regulates the properties and function of this class of putative transcriptional regulators.

High mobility group proteins cHMG1a, cHMG1b, and cHMGI are distinctly distributed in chromosomes and differentially expressed during ecdysone dependent cell differentiation.

The mammalian high mobility group proteins HMGI/Y and HMG1/2 are thought to play an architectural role in assembly of nucleoprotein structures. Counterparts to these proteins have recently been found in the cells of the Dipteran insect Chironomus. In this report we investigate the distribution of three abundant HMG proteins in interphase giant chromosomes of the midge, Chironomus. By means of the indirect immunofluorescence technique the cHMG1b and cHMGI proteins were localized in chromosomal puffs, suggesting their involvement in the organization of transcriptionally active chromatin. In contrast, the highly abundant protein cHMG1a was rather uniformly distributed in the chromosomes. The cHMGI protein, but not cHMG1a or cHMG1b, was detected in nucleoli, which may indicate a role in the transcription of ribosomal genes. The regions of the interphase chromosomes containing AT-rich DNA did not contain higher levels of the cHMGI and cHMG1b proteins. A correlation between the specific location of these proteins in chromatin and their synthesis and turnover rates was observed.

Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro.

Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2. These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes. Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II). Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain. No significant change in basal expression was observed for any of the 10 genes examined. Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity. In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone. Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.