Steven J. Freedman

Structural basis for recruitment of CBP/p300 byhypoxia-inducible factor-1α

Adaptation to hypoxia is mediated by transactivation of hypoxia-responsive genes by hypoxia-inducible factor-1 (HIF-1) in complex with the CBP and p300 transcriptional coactivators. We report the solution structure of the cysteine/histidine-rich 1 (CH1) domain of p300 bound to the C-terminal transactivation domain of HIF-1α. CH1 has a triangular geometry composed of four α-helices with three intervening Zn2+-coordinating centers. CH1 serves as a scaffold for folding of the HIF-1α C-terminal transactivation domain, which forms a vise-like clamp on the CH1 domain that is stabilized by extensive hydrophobic and polar interactions. The structure reveals the mechanism of specific recognition of p300 by HIF-1α, and shows how HIF-1α transactivation is regulated by asparagine hydroxylation.

Identification of the phospholipid binding site in the vitamin K-dependent blood coagulation protein factor IX.

The blood coagulation and regulatory proteins that contain γ-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to γ-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1–11 of the Gla domain. In the presence of Ca2+, Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

NMR structures for the membrane binding gla domain of blood coagulation factor IX

Publisher Summary This chapter describes the nuclear magnetic resonance (NMR) structures for the membrane binding Gla domain of the blood coagulation factor IX. Factor IX is a vitamin K-dependent protein. At the amino terminus of factor IX, the Gla domain mediates phospholipid recognition by liganding calcium ions and the consequent formation of a phospholipid binding site. The Ca(II)-induced conformational transition that leads to phospholipid binding is between a relatively disordered metal-free structure and a compact Ca(II)-bound structure. The structure of the Mg(II) bound peptide is the same as the Ca(II) bound form, except for residues 1–11 that are disordered. Comparison of the apo, Mg(II) and Ca(II) structures reveals a feature required for phospholipid binding by Factor IX. The chapter investigates the apo, Mg(II), and Ca(II) forms of the peptide using nuclear magnetic resonance (NMR) spectroscopy. The structures of the Gla domain delineate the region of factor IX that interacts with a lipid bilayer. The chapter also explains the sequential conformational transition model proposed using conformation-specific antibodies.