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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) Amino acid sequence of mouse HMGA2.The positively charged “AT hooks” and the negatively charged C-terminus are highlighted by underlines and a box, respectively. (B) The experimental (open dots with dotted line) and calculated (solid line) CD spectra of HMGA2 in BPES buffer. The CD spectra were calculated by the program CONTIN as described in the text. The solid dots are residuals. (C) Gel filtration chromatography profile of HMGA2 in solution. Recombinant HMGA2 was resolved by gel filtration in the Sephacryl S-100 column as described under “Materials and Methods.” V0 is the void volume; A, C, and R represent the elution volume of albumin, chymotrypsinogen A, and ribonuclease A, respectively.
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pone.0130478.g001: (A) Amino acid sequence of mouse HMGA2.The positively charged “AT hooks” and the negatively charged C-terminus are highlighted by underlines and a box, respectively. (B) The experimental (open dots with dotted line) and calculated (solid line) CD spectra of HMGA2 in BPES buffer. The CD spectra were calculated by the program CONTIN as described in the text. The solid dots are residuals. (C) Gel filtration chromatography profile of HMGA2 in solution. Recombinant HMGA2 was resolved by gel filtration in the Sephacryl S-100 column as described under “Materials and Methods.” V0 is the void volume; A, C, and R represent the elution volume of albumin, chymotrypsinogen A, and ribonuclease A, respectively.

Mentions: Mouse HMGA2 is a 108 amino acid residue protein. Inspection of its primary structure gives some unique features. It has 25 basic amino acid residues (either Lys or Arg), 12 acidic amino acid residues (either Glu or Asp), 15 Pro, and 10 Gly. The high contents of the basic amino acid residues make HMGA2 a high isoelectric point protein (the isoelectric point was estimated to be about 11; the estimated net charge at pH 7.0 is about +13). The charge distribution of HMGA2 is asymmetrical, with the positively charged residues mainly concentrated in the center of the sequence and the negatively charged residues at the C-terminus (Fig 1A). Several programs, such as PONDR [39], DISOPRED [40], and GlobPlot [41], were used to predict its secondary structure. All predicted that HMGA2 in the native state does not adopt a defined structure. Indeed, our CD studies showed that HMGA2 is an unstructured protein. Fig 1B shows the CD spectrum of HMGA2 in which the strong peak near 200 nm indicates an unordered conformation. The CD results were further analyzed using three CD analysis programs CONTIN, CDSSTR, and SELCON3 that are included in a downloadable software package CDPro [42]. These analyses showed that about 80% of HMGA2 is unstructured (Fig 1B; S1 Table, Supplementary data). Interestingly, all programs suggest that HMGA2 has about 20% β-sheet component. It is possible that part of HMGA2 has an extended structure similar to β strands. A comparison of the experimental and calculated CD spectra is shown in Fig 1B. Similar results were obtained in our NMR analyses and DSC experiments [29].


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) Amino acid sequence of mouse HMGA2.The positively charged “AT hooks” and the negatively charged C-terminus are highlighted by underlines and a box, respectively. (B) The experimental (open dots with dotted line) and calculated (solid line) CD spectra of HMGA2 in BPES buffer. The CD spectra were calculated by the program CONTIN as described in the text. The solid dots are residuals. (C) Gel filtration chromatography profile of HMGA2 in solution. Recombinant HMGA2 was resolved by gel filtration in the Sephacryl S-100 column as described under “Materials and Methods.” V0 is the void volume; A, C, and R represent the elution volume of albumin, chymotrypsinogen A, and ribonuclease A, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130478.g001: (A) Amino acid sequence of mouse HMGA2.The positively charged “AT hooks” and the negatively charged C-terminus are highlighted by underlines and a box, respectively. (B) The experimental (open dots with dotted line) and calculated (solid line) CD spectra of HMGA2 in BPES buffer. The CD spectra were calculated by the program CONTIN as described in the text. The solid dots are residuals. (C) Gel filtration chromatography profile of HMGA2 in solution. Recombinant HMGA2 was resolved by gel filtration in the Sephacryl S-100 column as described under “Materials and Methods.” V0 is the void volume; A, C, and R represent the elution volume of albumin, chymotrypsinogen A, and ribonuclease A, respectively.
Mentions: Mouse HMGA2 is a 108 amino acid residue protein. Inspection of its primary structure gives some unique features. It has 25 basic amino acid residues (either Lys or Arg), 12 acidic amino acid residues (either Glu or Asp), 15 Pro, and 10 Gly. The high contents of the basic amino acid residues make HMGA2 a high isoelectric point protein (the isoelectric point was estimated to be about 11; the estimated net charge at pH 7.0 is about +13). The charge distribution of HMGA2 is asymmetrical, with the positively charged residues mainly concentrated in the center of the sequence and the negatively charged residues at the C-terminus (Fig 1A). Several programs, such as PONDR [39], DISOPRED [40], and GlobPlot [41], were used to predict its secondary structure. All predicted that HMGA2 in the native state does not adopt a defined structure. Indeed, our CD studies showed that HMGA2 is an unstructured protein. Fig 1B shows the CD spectrum of HMGA2 in which the strong peak near 200 nm indicates an unordered conformation. The CD results were further analyzed using three CD analysis programs CONTIN, CDSSTR, and SELCON3 that are included in a downloadable software package CDPro [42]. These analyses showed that about 80% of HMGA2 is unstructured (Fig 1B; S1 Table, Supplementary data). Interestingly, all programs suggest that HMGA2 has about 20% β-sheet component. It is possible that part of HMGA2 has an extended structure similar to β strands. A comparison of the experimental and calculated CD spectra is shown in Fig 1B. Similar results were obtained in our NMR analyses and DSC experiments [29].

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