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Numerous transcription factors interact with the basal transcriptional machinery through the transcriptional co-activators p300 and CREB-binding protein (CBP). The Zn(2+)-binding cysteine/histidine-rich 1 (CH1) domain of p300/CBP binds many of these transcription factors, including hypoxia-inducible factor (HIF). We studied the structural and biophysical properties of the p300 CH1 domain alone and bound to the HIF-1 alpha C-terminal transactivation domain (TAD) to understand the diverse binding properties of CH1. The Zn(2+)-bound CH1 domain (CH1-Zn(2+)) and the HIF-1 alpha TAD-CH1 complex (CH1-Zn(2+)-HIF-1 alpha) are similarly helical, whereas metal-free CH1 is mostly random coil. CH1-Zn(2+) undergoes noncooperative thermal denaturation, does not have a near-UV elliptical signal, and binds the hydrophobic fluorophore ANS. In contrast, the CH1-Zn(2+)-HIF-1 alpha complex undergoes cooperative thermal denaturation, does produce a near-UV signal, and does not bind ANS. Addition of Zn(2+) ions to metal-free CH1 produced one conformational change, and subsequent addition of a HIF-1 alpha TAD peptide induced a second conformational change as detected by intrinsic tryptophan fluorescence spectroscopy. The NMR (1)H-(15)N HSQC spectrum of CH1-Zn(2+) exhibits few poorly dispersed peaks with broad line widths. Removal of metal ions produces more poorly dispersed peaks with sharper line widths. Addition of a HIF-1 alpha TAD peptide to CH1-Zn(2+) produces many well-dispersed peaks with sharp line widths. Taken together, these data support three conformational states for CH1, including an unstructured metal-free domain, a partially structured Zn(2+)-bound domain with molten globule characteristics, and a stable, well-ordered HIF-1 alpha TAD-CH1 complex.

Leukocyte rolling in the vasculature is mediated by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Since cell-cell interaction mediated by P-selectin and PSGL-1 is cooperative and complex, we have developed a model system to examine the binding of P-selectin to PSGL-1 in a soluble system. Equilibrium binding analyses were performed with truncated forms of soluble human P-selectin and dimeric PSGL-1, both lacking the transmembrane domain and both produced in Chinese hamster ovary (CHO) cells. Soluble PSGL-1 (sPSGL-1), which contains no tryptophan residues and exhibits no intrinsic fluorescence, was harvested from CHO cells cotransfected with either fucosyltransferase III (sPSGL-1/Fuc-TIII) or fucosyltransferase VII (sPSGL-1/Fuc-TVII). Both fucosylation isoforms of sPSGL-1 bound to sP-selectin. The interaction of sP-selectin and sPSGL-1 was studied by monitoring changes in the intrinsic fluorescence of sP-selectin upon binding to sPSGL-1. Binding of sPSGL-1 to sP-selectin in the presence of calcium caused an increase in tryptophan fluorescence that could be reversed by the addition of ethylenediaminetetraacetic acid (EDTA). The fluorescence enhancement of sP-selectin by sPSGL-1 was used to generate binding isotherms, and these data were fitted to a bimolecular binding model. The binding constant, Kd, for the binding of sPSGL-1/Fuc-TIII and sPSGL-1/Fuc-TVII to sP-selectin was 3 +/- 2 nM and 80 +/- 44 nM, respectively. Monomeric sP-selectin bound to dimeric sPSGL-1 with a 2:1 stoichiometry. In a system in which both protein species are soluble and lack transmembrane domains, these results indicate high-affinity interaction between P-selectin and PSGL-1. Furthermore, the fucosylation pattern of PSGL-1 can affect its affinity for P-selectin. These binding constants can be used to explore models of cell adhesion in flow systems.

A genetic algorithm (GA) successfully identified the calcium positions in the crystal structure of bovine prothrombin fragment 1 bound with calcium ions (bf1/Ca). The same protocol was then used to determine the calcium positions in a closely related fragment, the Gla domain of coagulation factor IX, the structure of which had previously been determined by NMR spectroscopy in the presence of calcium ions. The most frequently occurring low-energy structure found by GA was used as the starting structure for a molecular dynamics refinement. The molecular dynamics simulation was performed using explicit water and the Particle-Mesh Ewald method to accommodate the long-range electrostatic forces. While the overall conformation of the NMR structure was preserved, significant refinement is apparent when comparing the simulation average structure with its NMR precursor in terms of the N-terminal (Tyr1-N) network, the total number of hydrogen bonds, the calcium ion coordinations, and the compactness of the structure. It is likely that the placement of calcium ions in the protein is critical for refinement. The calcium ions apparently induce structural changes during the course of the simulation that result in a more compact structure.

P-selectin is a multidomain adhesion protein on the surface of activated platelets and endothelial cells that functions in the recruitment of leukocytes to the site of inflammation. The amino-terminal lectin and EGF domains constitute the ligand recognition unit. We have produced a synthetic 40-residue P-selectin EGF domain (P-sel:EGF) to examine the structure and function of this domain independent of P-selectin. The peptide was folded in vitro and exhibited the same disulfide bonding pattern as other EGF-like domains. P-sel:EGF did not inhibit P-selectin-mediated cellular adhesion assays, indicating that the lectin domain is also required. We undertook the study of the P-selectin EGF by 1H NMR to determine its structure independent of the lectin domain and to compare its structure to that of E-selectin determined crystallographically [Graves et al. (1994) Nature 367, 532]. Although the binding of P-selectin to its carbohydrate ligand is calcium dependent, and some EGF domains have calcium binding sites, addition of calcium had no effect on the NMR spectrum or on the pH-induced changes. Nearly complete resonance assignments were made from 2D 1H NMR spectra at pH 6.0. Two sections of antiparallel beta-sheet were identified on the basis of the pattern of long-range NOEs, 3JHN alpha coupling constants, and slowly exchanging amides. The solution structure of the peptide backbone was determined using distance geometry and simulated annealing calculations. The backbone RMSD to the geometric average for 19 final structures is 0.64 +/- 0.17 A. The resulting fold closely resembles that of other EGF-like peptides, including the E-selectin EGF domain (RMSD approximately 1.08 A). However, compared to the E-selectin EGF structure which also contains the lectin domain, some residues from 1-11 are less ordered, and novel contacts occur between the amino terminus and the core beta-sheet. Despite marked structural homology of the selectin polypeptide backbones, the selectin EGF surfaces show unique distributions of charged residues, a feature that likely correlates to the functional differences.

We have determined the Ca(II)-bound structure of factor IX, residues 1-47, by nuclear magnetic resonance (NMR) spectroscopy. The amino-terminal 47 residues include the gamma-carboxyglutamic acid-rich and aromatic amino acid stack domains, and this region is responsible for Ca(II)-dependent phospholipid binding in factor IX. Protons in the 1-47 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. A total of 851 distance restraints and 57 torsion angle restraints were used to generate 17 final structures by distance geometry and simulated annealing methods. The backbone RMSD to the geometric average is 0.6 +/- 0.1 A. The Ca(II)-bound structure is substantially more ordered with increased helical content compared to the apo-factor IX (1-47) structure. The global fold is similar to the crystal structure of the Ca(II)-bound Gla domain of prothrombin fragment I from residues 12 to 47 (RMSD approximately 1.3 A), but the backbone conformation differs in the first 11 residues, particularly between residues 3 and 6. The amino-terminal nine Gla residues are oriented to the interior of the protein and suggest an internal Ca(II) binding pocket. The carboxyl-terminal three Gla residues are exposed to solvent. The majority of hydrophobic residues are required to stabilize a globular core in the carboxyl-terminal three-quarters of the molecule. However, a hydrophobic surface patch in the amino-terminal region may represent a phospholipid binding site in factor IX.

The gamma-carboxylase recognition site in the propeptide of profactor IX signals the gamma-carboxylation of specific glutamic acid residues in the adjacent Gla domain during factor IX biosynthesis. To study posttranslational processing of the vitamin K-dependent blood coagulation factors and the properties of processing intermediates, we have isolated an incompletely processed factor IX species, profactor IX, from the medium of heterologous mammalian cells expressing the human factor IX cDNA. Profactor IX was purified by sequential immunoaffinity chromatography using antibodies specific for the propeptide and antibodies specific for the well-carboxylated factor IX species. This purified profactor IX preparation was fully gamma-carboxylated and contained the N-terminal propeptide, but it exhibited no factor IX procoagulant activity. Profactor IX was not cleaved following incubation with factor XIa. In contrast to mature factor IX, profactor IX did not demonstrate Ca(II)-dependent binding to acidic phospholipid vesicles, nor can the membrane binding surface be expressed, as detected by antibodies specific for this epitope. The propeptide of profactor IX can be removed in vitro by a specific endopeptidase, furin/PACE, yielding factor IX, which can be converted to fully active factor IXa by factor XIa and which binds normally to acidic phospholipid vesicles. These results indicate that fully gamma-carboxylated profactor IX is biologically inactive due to the presence of the propeptide.