title: Deconstructing nucleotide binding activity of the Mdm2 RING domain.
abstract: Mdm2, a central negative regulator of the p53 tumor suppressor, possesses a Really Interesting New Gene (RING) domain within its C-terminus. In addition to E3 ubiquitin ligase activity, the Mdm2 RING preferentially binds adenine base nucleotides, and such binding leads to a conformational change in the Mdm2 C-terminus. Here, we present further biochemical analysis of the nucleotide-Mdm2 interaction. We have found that MdmX, an Mdm2 family member with high sequence homology, binds adenine nucleotides with similar affinity and specificity as Mdm2, suggesting that residues involved in nucleotide binding may be conserved between the two proteins and adenosine triphosphate (ATP) binding may have similar functional consequences for both Mdm family members. By generating and testing a series of proteins with deletions and substitution mutations within the Mdm2 RING, we mapped the specific adenine nucleotide binding region of Mdm2 to residues 429-484, encompassing the minimal RING domain. Using a series of ATP derivatives, we demonstrate that phosphate coordination by the Mdm2 P-loop contributes to, but is not primarily responsible for, ATP binding. Additionally, we have identified the 2' and 3' hydroxyls of the ribose and the C6 amino group of the adenine base moiety as being essential for binding.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Priest
authors.forename: Carol
authors.initials: C
authors.lastname: Prives
authors.forename: Masha V
authors.initials: MV
authors.lastname: Poyurovsky
mesh_terms: D000225:Adenine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000255:Adenosine Triphosphate; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D008274:Magnesium; D000225:Adenine