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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

The CTP-TMR and HMGA2Δ95–108 co-eluting in gel-filtration chromatography.The CTP-TMR was prepared as described under “Materials and Methods” and incubated with HMGA2Δ95–108 at 24°C for 30 min in BPES buffer. The CTP-TMR and HMGA2Δ95–108 mixture was then subjected to a Sephacryl S-100 HR filtration column (1×50 cm) equilibrated with BPES buffer. Gel filtration profile of the CTP-TMR binding to HMGA2Δ95–108 was monitored by a graph of OD556 versus elution volume (A) and a 15% SDS PAGE gel (B). Lanes 1 to 8 of the SDS-PAGE gel (B) correspond to the fractions 1 to 8 labeled in panel a. Free HMGA2Δ95–108 and the CTP-TMR were eluted at 22 and 30 ml respectively in the column.
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pone.0130478.g006: The CTP-TMR and HMGA2Δ95–108 co-eluting in gel-filtration chromatography.The CTP-TMR was prepared as described under “Materials and Methods” and incubated with HMGA2Δ95–108 at 24°C for 30 min in BPES buffer. The CTP-TMR and HMGA2Δ95–108 mixture was then subjected to a Sephacryl S-100 HR filtration column (1×50 cm) equilibrated with BPES buffer. Gel filtration profile of the CTP-TMR binding to HMGA2Δ95–108 was monitored by a graph of OD556 versus elution volume (A) and a 15% SDS PAGE gel (B). Lanes 1 to 8 of the SDS-PAGE gel (B) correspond to the fractions 1 to 8 labeled in panel a. Free HMGA2Δ95–108 and the CTP-TMR were eluted at 22 and 30 ml respectively in the column.

Mentions: We next investigated what factors are critical to HMGA2 homodimer formation. One possible factor is electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus. We therefore made a mutant HMGA2Δ95–108 that lacks the negatively charged C-terminus, and reasoned that it should not form homodimers. Indeed, our EDC chemical cross-linking experiments showed that HMGA2Δ95–108 could not form homodimers (compare lanes 2 & 3 with lanes 5 & 6 of Fig 5). These results suggest that the negatively charged C-terminus is required for the dimer formation. We then, used tetramethylrhodamine-5-maleimide (TMR) to label a 14 amino acid residue C-terminal peptide (the CTP) of HMGA2 to produce the CTP-TMR. The CTP-TMR was incubated with HMGA2Δ95–108 and subjected to a pre-equilibrated gel filtration column. Fig 6 shows the elution profile of the gel filtration experiment. Our results demonstrated that the CTP-TMR was co-eluted with HMGA2Δ95–108. Interestingly, there are two co-elution peaks (Fig 6). Possibly, the first peak represents two CTP-TMR molecules binding to one HMGA2Δ95–108 and the second peak represents one CTP-TMR molecule binding to one HMGA2Δ95–108. An alternative possibility would be that the first peak contains one molecule of the CTP-TMR binding to two molecules of HMGA2Δ95–108 and the second peak corresponds to one molecule of the CTP-TMR binding to one molecule of HMGA2∆95–108. Further studies are required to determine the binding stoichiometry.


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)

The CTP-TMR and HMGA2Δ95–108 co-eluting in gel-filtration chromatography.The CTP-TMR was prepared as described under “Materials and Methods” and incubated with HMGA2Δ95–108 at 24°C for 30 min in BPES buffer. The CTP-TMR and HMGA2Δ95–108 mixture was then subjected to a Sephacryl S-100 HR filtration column (1×50 cm) equilibrated with BPES buffer. Gel filtration profile of the CTP-TMR binding to HMGA2Δ95–108 was monitored by a graph of OD556 versus elution volume (A) and a 15% SDS PAGE gel (B). Lanes 1 to 8 of the SDS-PAGE gel (B) correspond to the fractions 1 to 8 labeled in panel a. Free HMGA2Δ95–108 and the CTP-TMR were eluted at 22 and 30 ml respectively in the column.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130478.g006: The CTP-TMR and HMGA2Δ95–108 co-eluting in gel-filtration chromatography.The CTP-TMR was prepared as described under “Materials and Methods” and incubated with HMGA2Δ95–108 at 24°C for 30 min in BPES buffer. The CTP-TMR and HMGA2Δ95–108 mixture was then subjected to a Sephacryl S-100 HR filtration column (1×50 cm) equilibrated with BPES buffer. Gel filtration profile of the CTP-TMR binding to HMGA2Δ95–108 was monitored by a graph of OD556 versus elution volume (A) and a 15% SDS PAGE gel (B). Lanes 1 to 8 of the SDS-PAGE gel (B) correspond to the fractions 1 to 8 labeled in panel a. Free HMGA2Δ95–108 and the CTP-TMR were eluted at 22 and 30 ml respectively in the column.
Mentions: We next investigated what factors are critical to HMGA2 homodimer formation. One possible factor is electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus. We therefore made a mutant HMGA2Δ95–108 that lacks the negatively charged C-terminus, and reasoned that it should not form homodimers. Indeed, our EDC chemical cross-linking experiments showed that HMGA2Δ95–108 could not form homodimers (compare lanes 2 & 3 with lanes 5 & 6 of Fig 5). These results suggest that the negatively charged C-terminus is required for the dimer formation. We then, used tetramethylrhodamine-5-maleimide (TMR) to label a 14 amino acid residue C-terminal peptide (the CTP) of HMGA2 to produce the CTP-TMR. The CTP-TMR was incubated with HMGA2Δ95–108 and subjected to a pre-equilibrated gel filtration column. Fig 6 shows the elution profile of the gel filtration experiment. Our results demonstrated that the CTP-TMR was co-eluted with HMGA2Δ95–108. Interestingly, there are two co-elution peaks (Fig 6). Possibly, the first peak represents two CTP-TMR molecules binding to one HMGA2Δ95–108 and the second peak represents one CTP-TMR molecule binding to one HMGA2Δ95–108. An alternative possibility would be that the first peak contains one molecule of the CTP-TMR binding to two molecules of HMGA2Δ95–108 and the second peak corresponds to one molecule of the CTP-TMR binding to one molecule of HMGA2∆95–108. Further studies are required to determine the binding stoichiometry.

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