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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 HMGA2 into homodimers 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 molecular standards; lane 2 contained HMGA2 in the absence of EDC; lanes 3 to 6 contained, respectively, 29, 39, 58, and 116 μM HMGA2 in the presence of 2 mM EDC. M, monomer; D, dimer; T3, trimer; T4, tetramer.
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pone.0130478.g002: Chemical cross-linking HMGA2 into homodimers 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 molecular standards; lane 2 contained HMGA2 in the absence of EDC; lanes 3 to 6 contained, respectively, 29, 39, 58, and 116 μM HMGA2 in the presence of 2 mM EDC. M, monomer; D, dimer; T3, trimer; T4, tetramer.

Mentions: The oligomeric state of HMGA2 was first analyzed using EDC chemical cross-linking experiments. EDC is a zero-length cross-linker that reacts with closely contacted carboxyl and amino groups. For proteins, the carboxyl groups come from the side chains of Glu and Asp residues or from the unmodified C-terminus; the amino groups come from the side chains of Lys residue or the unmodified N-terminus. HMGA2 has 11 Glu and 1 Asp residues of which seven are located in the C-terminus (Fig 1A). It also has 13 Lys residues whose amino groups on the side chain can be cross-linked to the carboxyl groups by EDC. Fig 2 is a typical EDC chemical cross-linking experiment in which EDC efficiently cross-linked HMGA2 into homodimers (compare lanes 3–6 with lane 2, approximately 60–70% of HMGA2 was cross-linked into dimers). These results suggest that some residues between the subunits of HMGA2 homodimers are in very close proximity to one another. Interestingly, after the cross-linking reaction, the protein samples still had a significant amount of monomers that migrated faster than the protein in the control lane (compare lane 2 with lanes 3–6 of Fig 2). These results suggest that the negatively charged C-terminus may also interact with other parts of HMGA2 in the same subunit. When the HMGA2 concentration was increased in the cross-linking reactions, other homo-oligomers were also formed (Lanes 4 to 6 of Fig 2; they were assumed to be trimers or tetramers based on migration rates on the SDS-PAGE gels). Ionic strength has no apparent effects on the EDC chemical cross-linking reactions (data not shown). Similar results were obtained using disuccinimidyl suberate (DSS) and dimethyl suberimidate (DMS) as chemical cross-linking reagents that cross-link two primary amines (data not shown). However, the cross-linking efficiency of DSS and DMS is significantly lower than that of EDC. A cysteineless mutant protein, HMGA2C41G, which cannot form a disulfide bond, was also efficiently cross-linked into homodimers by EDC (data not shown).


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 HMGA2 into homodimers 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 molecular standards; lane 2 contained HMGA2 in the absence of EDC; lanes 3 to 6 contained, respectively, 29, 39, 58, and 116 μM HMGA2 in the presence of 2 mM EDC. M, monomer; D, dimer; T3, trimer; T4, tetramer.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130478.g002: Chemical cross-linking HMGA2 into homodimers 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 molecular standards; lane 2 contained HMGA2 in the absence of EDC; lanes 3 to 6 contained, respectively, 29, 39, 58, and 116 μM HMGA2 in the presence of 2 mM EDC. M, monomer; D, dimer; T3, trimer; T4, tetramer.
Mentions: The oligomeric state of HMGA2 was first analyzed using EDC chemical cross-linking experiments. EDC is a zero-length cross-linker that reacts with closely contacted carboxyl and amino groups. For proteins, the carboxyl groups come from the side chains of Glu and Asp residues or from the unmodified C-terminus; the amino groups come from the side chains of Lys residue or the unmodified N-terminus. HMGA2 has 11 Glu and 1 Asp residues of which seven are located in the C-terminus (Fig 1A). It also has 13 Lys residues whose amino groups on the side chain can be cross-linked to the carboxyl groups by EDC. Fig 2 is a typical EDC chemical cross-linking experiment in which EDC efficiently cross-linked HMGA2 into homodimers (compare lanes 3–6 with lane 2, approximately 60–70% of HMGA2 was cross-linked into dimers). These results suggest that some residues between the subunits of HMGA2 homodimers are in very close proximity to one another. Interestingly, after the cross-linking reaction, the protein samples still had a significant amount of monomers that migrated faster than the protein in the control lane (compare lane 2 with lanes 3–6 of Fig 2). These results suggest that the negatively charged C-terminus may also interact with other parts of HMGA2 in the same subunit. When the HMGA2 concentration was increased in the cross-linking reactions, other homo-oligomers were also formed (Lanes 4 to 6 of Fig 2; they were assumed to be trimers or tetramers based on migration rates on the SDS-PAGE gels). Ionic strength has no apparent effects on the EDC chemical cross-linking reactions (data not shown). Similar results were obtained using disuccinimidyl suberate (DSS) and dimethyl suberimidate (DMS) as chemical cross-linking reagents that cross-link two primary amines (data not shown). However, the cross-linking efficiency of DSS and DMS is significantly lower than that of EDC. A cysteineless mutant protein, HMGA2C41G, which cannot form a disulfide bond, was also efficiently cross-linked into homodimers by EDC (data not shown).

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