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

Frost L, Baez MA, Harrilal C, Garabedian A, Fernandez-Lima F, Leng F - PLoS ONE (2015)

Bottom Line: 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.Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Sciences Institute, Florida International University, Miami, Florida, United States of America; Department of Chemistry and Biochemistry, Florida International University, Miami, Florida, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

A possible model for the HMGA2 homodimerization.Blue lines represent the protein backbone. Electrostatic interactions between the positively charged “AT hooks” (red rectangle with two red circles) and the negatively charged C-terminus (yellow oval) coordinate the dimer formation. (A) represents HMGA2 monomers. (B) and (C) represent different interchangeable conformations of HMGA2 homodimers. (C) is more consistent with our EDC cross-linking and sedimentation velocity results. The HMGA2 homodimers may be an ensemble of different conformers.
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pone.0130478.g007: A possible model for the HMGA2 homodimerization.Blue lines represent the protein backbone. Electrostatic interactions between the positively charged “AT hooks” (red rectangle with two red circles) and the negatively charged C-terminus (yellow oval) coordinate the dimer formation. (A) represents HMGA2 monomers. (B) and (C) represent different interchangeable conformations of HMGA2 homodimers. (C) is more consistent with our EDC cross-linking and sedimentation velocity results. The HMGA2 homodimers may be an ensemble of different conformers.

Mentions: Our preliminary results showed that the quaternary structure stems from electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus. As described above, the charge distribution of HMGA2 is asymmetrical (Fig 1A). This unique property provides an opportunity for the protein to self-associate. As demonstrated in Fig 5, HMGA2Δ94–108, a mutant protein without the negatively charged C-terminus, cannot be cross-linked into dimers by EDC. The CTP containing the negatively charged C-terminus tightly binds to HMGA2Δ94–108 (Fig 6). Moreover, we showed that the negatively charged C-terminus binds to the second “AT-hook” (data not shown) therefore promoting the dimer formation. A possible mechanism would be that the dimer association process is initiated by the charge neutralization and subsequently enforced by the hydrophobic interaction and hydrogen bonding from the peptide backbone. Fig 7 is a schematic graph explaining this mechanism. The “unstructured” monomers (Fig 7A) interact with each other to form a homodimer (Fig 7B and 7C) through electrostatic interactions between the highly charged “AT-hooks” (red rectangle with two red circles) and the highly charged C-terminus (yellow oval). The homodimers do not have regular secondary structure and are composed of an ensemble of different conformers with distinct and dynamic and angles [39,47,50]. They may have local and limited residual structure that is critical for self-association. This dimerization process is reminiscent of the initial step of the amyloid aggregation of a large number of neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. In these cases the proteins change their random coil to amyloidenenic β-sheet conformation therefore leading to self-association [51–53]. As demonstrated previously, any generic proteins including intrinsically unstructured proteins have the potential to form amyloid aggregates under suitable conditions [54,55]. This property results from the inherent physicochemical properties of the polypeptide backbone (hydrophobicity and hydrogen bonding) rather than the specific interaction between the side chains [54,56,57]. Although charge was considered to be the key parameter to prevent protein association or aggregation [58], HMGA2 has an asymmetrical charge distribution that would promote self-association. Indeed, recent evidence showed that the electrostatic interaction can promote protein or polypeptide’s self-association. Tjernberg et al.[59] showed that peptides as few as 4 residues can form well defined amyloid fibril. Both charge attraction and hydrophobic interaction are required. Goers el al. showed that several unstructured polycations, such as spermine, polylysine, polyarginine, and polyethyleneimine tightly bind to -synuclein, an IDP, and catalyze its oligomerization [60]. This process was mediated by the electrostatic interaction between the negatively charged C-terminus and the polycations, and may be enforced by the hydrophobic interaction and hydrogen bonding of the peptide backbone [48]. These studies suggest that the intrinsically “unstructured” proteins or polypeptides can interact with each other to form higher structures.


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

Frost L, Baez MA, Harrilal C, Garabedian A, Fernandez-Lima F, Leng F - PLoS ONE (2015)

A possible model for the HMGA2 homodimerization.Blue lines represent the protein backbone. Electrostatic interactions between the positively charged “AT hooks” (red rectangle with two red circles) and the negatively charged C-terminus (yellow oval) coordinate the dimer formation. (A) represents HMGA2 monomers. (B) and (C) represent different interchangeable conformations of HMGA2 homodimers. (C) is more consistent with our EDC cross-linking and sedimentation velocity results. The HMGA2 homodimers may be an ensemble of different conformers.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482583&req=5

pone.0130478.g007: A possible model for the HMGA2 homodimerization.Blue lines represent the protein backbone. Electrostatic interactions between the positively charged “AT hooks” (red rectangle with two red circles) and the negatively charged C-terminus (yellow oval) coordinate the dimer formation. (A) represents HMGA2 monomers. (B) and (C) represent different interchangeable conformations of HMGA2 homodimers. (C) is more consistent with our EDC cross-linking and sedimentation velocity results. The HMGA2 homodimers may be an ensemble of different conformers.
Mentions: Our preliminary results showed that the quaternary structure stems from electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus. As described above, the charge distribution of HMGA2 is asymmetrical (Fig 1A). This unique property provides an opportunity for the protein to self-associate. As demonstrated in Fig 5, HMGA2Δ94–108, a mutant protein without the negatively charged C-terminus, cannot be cross-linked into dimers by EDC. The CTP containing the negatively charged C-terminus tightly binds to HMGA2Δ94–108 (Fig 6). Moreover, we showed that the negatively charged C-terminus binds to the second “AT-hook” (data not shown) therefore promoting the dimer formation. A possible mechanism would be that the dimer association process is initiated by the charge neutralization and subsequently enforced by the hydrophobic interaction and hydrogen bonding from the peptide backbone. Fig 7 is a schematic graph explaining this mechanism. The “unstructured” monomers (Fig 7A) interact with each other to form a homodimer (Fig 7B and 7C) through electrostatic interactions between the highly charged “AT-hooks” (red rectangle with two red circles) and the highly charged C-terminus (yellow oval). The homodimers do not have regular secondary structure and are composed of an ensemble of different conformers with distinct and dynamic and angles [39,47,50]. They may have local and limited residual structure that is critical for self-association. This dimerization process is reminiscent of the initial step of the amyloid aggregation of a large number of neurodegenerative diseases including Alzheimer’s disease and Parkinson’s disease. In these cases the proteins change their random coil to amyloidenenic β-sheet conformation therefore leading to self-association [51–53]. As demonstrated previously, any generic proteins including intrinsically unstructured proteins have the potential to form amyloid aggregates under suitable conditions [54,55]. This property results from the inherent physicochemical properties of the polypeptide backbone (hydrophobicity and hydrogen bonding) rather than the specific interaction between the side chains [54,56,57]. Although charge was considered to be the key parameter to prevent protein association or aggregation [58], HMGA2 has an asymmetrical charge distribution that would promote self-association. Indeed, recent evidence showed that the electrostatic interaction can promote protein or polypeptide’s self-association. Tjernberg et al.[59] showed that peptides as few as 4 residues can form well defined amyloid fibril. Both charge attraction and hydrophobic interaction are required. Goers el al. showed that several unstructured polycations, such as spermine, polylysine, polyarginine, and polyethyleneimine tightly bind to -synuclein, an IDP, and catalyze its oligomerization [60]. This process was mediated by the electrostatic interaction between the negatively charged C-terminus and the polycations, and may be enforced by the hydrophobic interaction and hydrogen bonding of the peptide backbone [48]. These studies suggest that the intrinsically “unstructured” proteins or polypeptides can interact with each other to form higher structures.

Bottom Line: 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.Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular Sciences Institute, Florida International University, Miami, Florida, United States of America; Department of Chemistry and Biochemistry, Florida International University, Miami, Florida, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus