Identification of the binding site on S100B protein for the actin capping protein CapZ.

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.

The solution structure of the bovine S100B protein dimer in the calcium-free state.

BACKGROUND: S100B (S100beta) is a member of the S100 family of small calcium-binding proteins: members of this family contain two helix-loop-helix calcium-binding motifs and interact with a wide range of proteins involved mainly in the cytoskeleton and cell proliferation. S100B is a neurite-extension factor and levels of S100B are elevated in the brains of patients with Alzheimer's disease or Down's syndrome: the pattern of S100B overexpression in Alzheimer's disease correlates with the pattern of neuritic-plaque formation. Identification of a growing class of S100 proteins and the likely neurochemical importance of S100B make the determination of the structure of S100B of interest. RESULTS: We have used NMR to determine the structure of the reduced S100B homodimer in the absence of calcium. Each monomer consists of a four-helix bundle, arranged in the dimer in an antiparallel fashion. The fourth helix of each monomer runs close to the equivalent helix of the other monomer for almost its full length, extending the hydrophobic core through the interface. The N-terminal, but not the C-terminal, calcium-binding loop is similar to the equivalent loop in the monomeric S100 protein calbindin and is in a conformation ready to bind calcium. CONCLUSIONS: The novel dimer structure reported previously for calcyclin (S100A6) is the common fold for the dimeric S100B proteins. Calcium binding to the C-terminal calcium-binding loop would be expected to require a conformational change, which might provide a signal for activation. The structure suggests regions of the molecule likely to be involved in interactions with effector molecules.

Nuclear magnetic resonance assignments and secondary structure of bovine S100 beta protein.

S100 beta is a neurite extension factor and has been implicated in Alzheimer's disease and Down's syndrome. It belongs to a group of low molecular weight calcium-binding proteins containing the helix-loop-helix calcium binding motif. The structure of only one S100 protein, calbindin D9k, which has the lowest sequence similarity to the other members of the S100 group has been determined. We report the NMR assignments and secondary structure of calcium-free S100 beta. The secondary structure is similar to that of calbindin D9k, determined using NMR, except that there is clear evidence for an additional well ordered 5-residue alpha-helix in S100 beta.

Changes in the structure of bovine phospholipase A2 upon micelle binding.

Phospholipase A2 (PLA2) is a calcium-dependent enzyme which hydrolyses the 2-acyl ester bond of phospholipids. The extracellular PLA2s are activated by as much as 10000-fold on binding to micelles or vesicles of substrate, possibly due to a conformational change induced in the enzyme. We have studied the complex of bovine pancreatic PLA2 with micelles of SDS by ultracentrifugation, equilibrium dialysis, microcalorimetry, fluorescence and n.m.r. spectroscopy. Ultracentrifugation and equilibrium dialysis measurements showed that on average 1.28 (+/- 0.17) PLA2 molecules and 26.4 (+/- 3.1) SDS molecules are involved in the complex and that there is a rapid equilibrium between micellar species containing one or more enzyme monomers. The estimated heat of formation of the complex, measured calorimetrically as the heat released when PLA2 was injected into excess 10 mM SDS, was 162.3 +/- 1.5) kJ/mol [38.8 (+/- 0.35) kcal/mol] of PLA2 added. The fluorescence of the single tryptophan at position 3 in the N-terminal helix of the protein increases when PLA2 binds to SDS micelles, indicating that this part of the protein is in a more hydrophobic environment in the complex. The structural changes in PLA2 on addition of [2H25]SDS were monitored using n.m.r. spectroscopy. The overall structure of the protein is unchanged, but changes in nuclear Overhauser effects (NOEs) were observed for residues in the N-terminal helix, at the active site region and in a lysine-rich region near the C-terminus. The NOE changes at the N-terminus indicate that this portion of the protein molecule adopts a more ordered, helical conformation when bound to a micelle. We suggest that these conformational changes could be the mechanism by which the enzyme becomes activated in the presence of aggregated substrate.

31P-NMR study of brain phospholipid structures in vivo.

31P-NMR has been used extensively for the study of cytosolic small molecule phosphates in vivo and phospholipid structures in vitro. We present in this paper a series of studies of the brain by 31P-NMR, both in vivo and in extracts, showing the information that can be derived about phospholipids. 31P-NMR spectra of mouse brain at 73 mHz are characterised by almost a complete absence of the large phosphodiester peak in comparison to equivalent spectra at 32 mHz. Proton decoupled spectra in vivo, and spectra of extracts, show that the phosphodiester peak observed in 32 mHz spectra in vivo is mainly due to phospholipid bilayers. Homogenates of quaking and control mouse brains, and of bovine grey matter, show another narrower phosphodiester peak possibly from small phospholipid vesicles. This peak is increased in intensity in the affected mice. These experiments demonstrate the presence of three major components contributing to the phosphodiester resonance: bilayer phospholipids, more mobile phospholipids, and the freely soluble cytosolic molecules glycerophosphocholine and glycerophosphoethanolamine. These NMR methods for non-invasive investigation of phospholipid structures in the brain might be extended to studies of patients with membrane involved diseases such as multiple sclerosis.

Spin-spin relaxation of the phosphodiester resonance in the 31P NMR spectrum of human brain. The determination of the concentrations of phosphodiester components.

The phosphodiester peak in 31P nuclear magnetic resonance spectra of human brain in vivo is often the most prominent feature of the spectrum. We have demonstrated that this resonance exhibits bi-exponential spin-spin relaxation, giving relaxation times of 2 and 10 ms. We interpret this in terms of the two components which make up the peak, bilayer lipids and the small cytosolic phosphates glycerophosphoethanolamine and glycerophosphocholine. Using the relaxation times and the relative peak heights of the two components we have also been able to quantitate the concentration of the bilayer lipids as 20-40 times that of ATP.