A small domain of CBP/p300 binds diverse proteins: solution structure and functional studies.

The transcriptional coactivators CBP and p300 are critical regulators of metazoan gene expression. They associate with many different DNA-bound transcription factors through small, conserved domains. We have identified a compactly folded 46 residue domain in CBP and p300, the IRF-3 binding domain (IBiD), and we have determined its structure by NMR. It has a helical framework containing an apparently flexible polyglutamine loop that participates in ligand binding. Spectroscopic data indicate that induced folding accompanies association of IBiD with its partners, which exhibit no evident sequence similarities. We demonstrate the significance both in vitro and in vivo of interactions between IBiD and a number of diverse partners. Thus, IBiD is an important contributor to signal integration by CBP and p300.

Application of automated NOE assignment to three-dimensional structure refinement of a 28 kDa single-chain T cell receptor.

An automated procedure for NOE assignment and three-dimensional structure refinement is presented. The input to the procedure consists of (1) an ensemble of preliminary protein NMR structures, (2) partial sequence-specific assignments for the protein and (3) the positions and volumes of unassigned NOESY cross peaks. Chemical shifts for unassigned side chain protons are predicted from the preliminary structures. The chemical shifts and unassigned NOESY cross peaks are input to an automated procedure for NOE assignment and structure calculation (ARIA) [Nilges et al. (1997) J. Mol. Biol., 269, 408-422]. ARIA is optimized for the task of structure refinement of larger proteins. Errors are filtered to ensure that sequence-specific assignments are reliable. The procedure is applied to the 27.8 kDa single-chain T cell receptor (scTCR). Preliminary NMR structures, nearly complete backbone assignments, partial assignments of side chain protons and more than 1300 unassigned NOESY cross peaks are input. Using the procedure, the resonant frequencies of more than 40 additional side chain protons are assigned. Over 400 new NOE cross peaks are assigned unambiguously. Distances derived from the automatically assigned NOEs improve the precision and quality of calculated scTCR structures. In the refined structures, a hydrophobic cluster of side chains on the scTCR surface that binds major histocompatibility complex (MHC)/antigen is revealed. It is composed of the side chains of residues from three loops and stabilizes the conformation of residues that interact with MHC.

Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor.

Using NMR spectroscopy, we determined the solution structure of a single-chain T-cell receptor (scTCR) derived from the major histocompatibility complex (MHC) class II-restricted D10 TCR. The conformations of complementarity-determining regions (CDRs) 3beta and 1alpha and surface properties of 2alpha are different from those of related class I-restricted TCRs. We infer a conserved orientation for TCR V(alpha) domains in complexes with both class I and II MHC-peptide ligands, which implies that small structural variations in V(alpha) confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond time scale) provides insight into immune recognition and selection mechanisms.

Small angle x-ray scattering studies of magnetically oriented lipid bilayers.

Magnetically oriented lipid/detergent bilayers are potentially useful for studies of membrane-associated molecules and complexes using x-ray scattering and nuclear magnetic resonance (NMR). To establish whether the system is a reasonable model of a phospholipid bilayer, we have studied the system using x-ray solution scattering to determine the bilayer thickness, interparticle spacing, and orientational parameters for magnetically oriented lipid bilayers. The magnetically orientable samples contain the phospholipid L-alpha-dilauroylphosphatidylcholine (DLPC) and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) in a 3:1 molar ratio in 70% water (w/v) and are similar to magnetically orientable samples used as NMR media for structural studies of membrane-associated molecules. A bilayer thickness of 30 A was determined for the DLPC/CHAPSO particles, which is the same as the bilayer thickness of pure DLPC vesicles, suggesting that the CHAPSO is not greatly perturbing the lipid bilayer. These data, as well as NMR data on molecules incorporated in the oriented lipid particles, are consistent with the sample consisting of reasonably homogeneous and well dispersed lipid particles. Finally, the orientational energy of the sample suggests that the size of the cooperatively orienting unit in the samples is 2 x 10(7) phospholipid molecules.

13C NMR studies of wheat germ agglutinin interactions with N-acetylglucosamine at a magnetically oriented bilayer surface.

The orientation of synthetic 13C-labeled glycolipid receptors and their interaction with the plant lectin wheat germ agglutinin have been studied in an oriented membrane system using NMR spectroscopy. A series of 2-[1,2-13C2]acetamido-2-deoxy-beta-D-glucopyranosides were synthesized with between zero and four hydrophilic ethoxy units between the headgroup and an alkyl chain which anchors the receptors in the bilayers. The chemical shift anisotropy of the 13C carbonyl and a 13C-13C dipolar coupling between the labeled carbons provide information about the orientation and dynamics of the receptor headgroup in oriented membrane systems. It was found that the headgroups of the receptors with two, three, or four ethoxy units appeared isotropic when incorporated in the oriented bilayers, but those of the receptors with zero or one ethoxy units were significantly ordered by the bilayers. The average orientations consistent with measured spectral parameters were determined for the receptors with zero and one ethoxy units and were found to coincide with low-energy conformations from molecular modeling. When the plant lectin wheat germ agglutinin was added to the sample, only the receptors with two, three, or four ethoxy units separating the headgroup from the alkyl chain showed evidence of binding by the lectin. Although the 13C-labeled resonances broadened when the protein bound, no changes in dipolar couplings or chemical shift anisotropies could be detected, suggesting that the motion of the headgroup was slowed by protein binding, but average orientation and overall order changed little. Competition studies demonstrated that none of the lectin/receptor complexes are more stable than the complex of the lectin and N-acetylglucosamine in solution. These results suggest that the membrane does not stabilize the interactions of wheat germ agglutinin with these cell-surface receptors. Furthermore, molecular modeling demonstrates that the zero- and one-spacer receptors may not bind wheat germ agglutinin because the orientations of the N-acetyl groups in these receptors would result in significant steric contacts between the lectin/receptor complex and the membrane.

Application of neural networks to automated assignment of NMR spectra of proteins.

Simulated neural networks are described which aid the assignment of protein NMR spectra. A network trained to recognize amino acid type from TOCSY data was trained on 148 assigned spin systems from E. coli acyl carrier proteins (ACPs) and tested on spin systems from spinach ACP, which has a 37% sequence homology with E. coli ACP and a similar secondary structure. The output unit corresponding to the correct amino acid is one of the four most activated units in 83% of the spin systems tested. The utility of this information is illustrated by a second network which uses a constraint satisfaction algorithm to find the best fit of the spin systems to the amino acid sequence. Application to a stretch of 20 amino acids in spinach ACP results in 75% correct sequential assignment. Since the output of the amino acid type identification network can be coupled with a variety of sequential assignment strategies, the approach offers substantial potential for expediting assignment of protein NMR spectra.

Synthesis and characterization of a 13C-labeled alpha-mannosyl glycolipid analog from [13C]glucose.

A method is described for the synthesis of a [13C]alpha-mannosyl glycolipid analog from [13C]glucose. After acetylation and reduction of glucose (U-13C6, 30%) to tri-O-acetyl-D-glucal (U-13C6, 30%), addition of the nucleophile 2-[2-[2-[2-(tetradecyloxy)ethoxy]ethoxy]ethoxy]- ethanol (tetra-decyltetraglycol) yields the rearrangement product alpha-tetradecyltetraglycol 2,3-dideoxyl-4,6-di-O-acetyl-D-gluco- pyranoside (U-13C6, 30%). The rearrangement product is oxidized with osmium tetroxide to produce tetradecyltetraglycol alpha-mannoside (U-13C6, 30%). The interaction of the glycolipid with the plant lectin concanavalin A is characterized by a vesicle agglutination assay.

Torsion angle analysis of glycolipid order at membrane surfaces.

A method is presented for determining the average glycosidic torsion angles and motion about those angles for a glycolipid headgroup at a model membrane surface. Dipolar and quadrupolar coupling constants were previously collected on the headgroup of beta-dodecyl glucoside embedded in phospholipid/detergent bilayers which orient in a magnetic field (Sanders, C.R., and J.H. Prestegard. 1991. J. Am. Chem. Soc. 113:1987-1996). These observables are expressed as averages of second order spherical harmonics, and Wigner rotation matrices are used here to transform the spherical harmonics from the laboratory frame to a set of frames which allow motional averaging to be described as the result of simple bond rotations. Euler angles corresponding to rotations about glycosidic torsion angles phi and psi are chosen to best reproduce experimental coupling constants, using models which have varying degrees of motional averaging. These models include a rigid headgroup, axially symmetric headgroup motion, and independent motion about each torsion angle in a square well potential. The square well model proves to be significantly better than the rigid model in reproducing experimental observations and it offers a more physically meaningful description of motion than the axially symmetric model. The structures obtained, assuming a square well potential, are compared to potential energy maps for the glycolipid torsional angles to illustrate the need for inclusion of the membrane interface in energetic modeling of glycolipid conformations.