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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

Chemical cross-linking of HMGA2 and HMGA2Δ95–108 with EDC.Chemical cross-linking reactions with EDC in MES buffer were performed as described under “Materials and Methods.” Cross-linked protein samples were analyzed by electrophoresis in a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. Lane 1 contained HMGA2Δ95–108 in the absence of EDC; lanes 2 and 3 contained 40 μM HMGA2Δ95–108 in the presence of 2 mM EDC; lane 4 contained HMGA2 in the absence of EDC; lanes 5 and 6 contained 40 μM HMGA2 in the presence of 2 mM EDC; lane 7 contained molecular standards.
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pone.0130478.g005: Chemical cross-linking of HMGA2 and HMGA2Δ95–108 with EDC.Chemical cross-linking reactions with EDC in MES buffer were performed as described under “Materials and Methods.” Cross-linked protein samples were analyzed by electrophoresis in a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. Lane 1 contained HMGA2Δ95–108 in the absence of EDC; lanes 2 and 3 contained 40 μM HMGA2Δ95–108 in the presence of 2 mM EDC; lane 4 contained HMGA2 in the absence of EDC; lanes 5 and 6 contained 40 μM HMGA2 in the presence of 2 mM EDC; lane 7 contained molecular standards.

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)

Chemical cross-linking of HMGA2 and HMGA2Δ95–108 with EDC.Chemical cross-linking reactions with EDC in MES buffer were performed as described under “Materials and Methods.” Cross-linked protein samples were analyzed by electrophoresis in a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. Lane 1 contained HMGA2Δ95–108 in the absence of EDC; lanes 2 and 3 contained 40 μM HMGA2Δ95–108 in the presence of 2 mM EDC; lane 4 contained HMGA2 in the absence of EDC; lanes 5 and 6 contained 40 μM HMGA2 in the presence of 2 mM EDC; lane 7 contained molecular standards.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130478.g005: Chemical cross-linking of HMGA2 and HMGA2Δ95–108 with EDC.Chemical cross-linking reactions with EDC in MES buffer were performed as described under “Materials and Methods.” Cross-linked protein samples were analyzed by electrophoresis in a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. Lane 1 contained HMGA2Δ95–108 in the absence of EDC; lanes 2 and 3 contained 40 μM HMGA2Δ95–108 in the presence of 2 mM EDC; lane 4 contained HMGA2 in the absence of EDC; lanes 5 and 6 contained 40 μM HMGA2 in the presence of 2 mM EDC; lane 7 contained molecular standards.
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