Quantitation of phosphohistidine in proteins in a mammalian cell line by 31P NMR.

There is growing evidence to suggest that phosphohistidines are present at significant levels in mammalian cells and play a part in regulating cellular activity, in particular signaling pathways related to cancer. Because of the chemical instability of phosphohistidine at neutral or acid pH, it remains unclear how much phosphohistidine is present in cells. Here we describe a protocol for extracting proteins from mammalian cells in a way that avoids loss of covalent phosphates from proteins, and use it to measure phosphohistidine concentrations in human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. Phosphohistidine is determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 15 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 µmol/g and 68 µmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. Phosphorylation is largely at the N3 (tele) position. Typical tryptic digest conditions result in loss of most of the phosphohistidine present, which may explain why the amounts reported here are greater than is generally seen using mass spectroscopy assays. The results further strengthen the case for a functional role of phosphohistidine in eukaryotic cells.

CORM-3 induces DNA damage through Ru(II) binding to DNA.

When the 'CO-releasing molecule-3', CORM-3 (Ru(CO)3Cl(glycinate)), is dissolved in water it forms a range of ruthenium complexes. These are taken up by cells and bind to intracellular ligands, notably thiols such as cysteine and glutathione, where the Ru(II) reaches high intracellular concentrations. Here, we show that the Ru(II) ion also binds to DNA, at exposed guanosine N7 positions. It therefore has a similar cellular target to the anticancer drug cisplatin, but not identical, because Ru(II) shows no evidence of forming intramolecular crossbridges in the DNA. The reaction is slow, and with excess Ru, intermolecular DNA crossbridges are formed. The addition of CORM-3 to human colorectal cancer cells leads to strand breaks in the DNA, as assessed by the alkaline comet assay. DNA damage is inhibited by growth media containing amino acids, which bind to extracellular Ru and prevent its entry into cells. We conclude that the cytotoxicity of Ru(II) is different from that of platinum, making it a promising development target for cancer therapeutics.

The accuracy of NMR protein structures in the Protein Data Bank.

The program ANSURR measures the accuracy of NMR structures by comparing rigidity obtained from experimental backbone chemical shifts and from structures. We report on ANSURR analysis of 7,000 PDB NMR ensembles within the Protein Data Bank, which can be found at ansurr.com. The accuracy of NMR structures progressively improved up until 2005, but since then, it has plateaued. Most structures have accurate secondary structure, but are generally too floppy, particularly in loops. Thus, there is a need for more experimental restraints in loops. Currently, the best predictors of accuracy are Ramachandran distribution and the number of NOE restraints per residue. The precision of structures within the ensemble correlates well with accuracy, as does the number of hydrogen bond restraints per residue. Structure accuracy is improved when other components (such as additional polypeptide chains or ligands) are included.

Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b.

Lysostaphin is a bacteriolytic enzyme targeting peptidoglycan, the essential component of the bacterial cell envelope. It displays a very potent and specific activity toward staphylococci, including methicillin-resistant Staphylococcus aureus. Lysostaphin causes rapid cell lysis and disrupts biofilms, and is therefore a therapeutic agent of choice to eradicate staphylococcal infections. The C-terminal SH3b domain of lysostaphin recognizes peptidoglycans containing a pentaglycine crossbridge and has been proposed to drive the preferential digestion of staphylococcal cell walls. Here we elucidate the molecular mechanism underpinning recognition of staphylococcal peptidoglycan by the lysostaphin SH3b domain. We show that the pentaglycine crossbridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH3b domain, thereby inducing a clustering of SH3b domains. We propose that this unusual binding mechanism allows synergistic and structurally dynamic recognition of S. aureus peptidoglycan and underpins the potent bacteriolytic activity of this enzyme.

Conformational exchange in the potassium channel blocker ShK.

ShK is a 35-residue disulfide-linked polypeptide produced by the sea anemone Stichodactyla helianthus, which blocks the potassium channels Kv1.1 and Kv1.3 with pM affinity. An analogue of ShK has been developed that blocks Kv1.3 > 100 times more potently than Kv1.1, and has completed Phase 1b clinical trials for the treatment of autoimmune diseases such as psoriasis and rheumatoid arthritis. Previous studies have indicated that ShK undergoes a conformational exchange that is critical to its function, but this has proved difficult to characterise. Here, we have used high hydrostatic pressure as a tool to increase the population of the alternative state, which is likely to resemble the active form that binds to the Kv1.3 channel. By following changes in chemical shift with pressure, we have derived the chemical shift values of the low- and high-pressure states, and thus characterised the locations of structural changes. The main difference is in the conformation of the Cys17-Cys32 disulfide, which is likely to affect the positions of the critical Lys22-Tyr23 pair by twisting the 21-24 helix and increasing the solvent exposure of the Lys22 sidechain, as indicated by molecular dynamics simulations.

Structural Investigation into the Threading Intercalation of a Chiral Dinuclear Ruthenium(II) Polypyridyl Complex through a B-DNA Oligonucleotide.

Herein we report the separation of the three stereoisomers of the DNA light-switch compound [{Ru(bpy)2}2(tpphz)]4+ (tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2″-h:2‴,3‴-j]phenazine) by column chromatography and the characterization of each stereoisomer by X-ray crystallography. The interaction of these compounds with a DNA octanucleotide d(GCATATCG).d(CGATATGC) has been studied using NMR techniques. Selective deuteration of the bipyridyl rings was needed to provide sufficient spectral resolution to characterize structures. NMR-derived structures for these complexes show a threading intercalation binding mode with slow and chirality-dependent rates. This represents the first solution structure of an intercalated bis-ruthenium ligand. Intriguingly, we find that the binding site selectivity is dependent on the nature of the stereoisomer employed, with Λ RuII centers showing a better intercalation fit.

Structure and function of a bacterial Fasciclin I Domain Protein elucidates function of related cell adhesion proteins such as TGFBIp and periostin.

Fasciclin I (FAS1) domains have important roles in cell adhesion, which are not understood despite many structural and functional studies. Examples of FAS1 domain proteins include TGFBIp (ßig-h3) and periostin, which function in angiogenesis and development of cornea and bone, and are also highly expressed in cancer tissues. Here we report the structure of a single-domain bacterial fasciclin I protein, Fdp, in the free-living photosynthetic bacterium Rhodobacter sphaeroides, and show that it confers cell adhesion properties in vivo. A binding site is identified which includes the most highly conserved region and is adjacent to the N-terminus. By mapping this onto eukaryotic homologues, which all contain tandem FAS1 domains, it is concluded that the interaction site is normally buried in the dimer interface. This explains why corneal dystrophy mutations are concentrated in the C-terminal domain of TGFBIp and suggests new therapeutic approaches.

Intra- and intermolecular effects on 1H chemical shifts in a silk model Peptide determined by high-field solid state 1H NMR and empirical calculations.

A combination of solid state 1H NMR chemical shift measurements and empirical chemical shift calculations has been used to interpret 1H solid state chemical shifts of a model peptide (Ala-Gly)15 for the crystalline domain of Bombyx mori silk fibroin in silk I and silk II structures, including a treatment of both intra- and intermolecular arrangements. Silk I and silk II are the structures of silk fibroin before and after spinning, respectively. Two peaks with equal intensity were observed for the amide protons of (AG)15 in silk I, whereas only one broad peak was observed for silk II, reflecting a difference of 1.1 ppm in Ala HN shift between silk I and silk II, but a difference of only 0.2 ppm in Gly HN shift. Chemical shift calculations predicted chemical shifts that are in good agreement with the experimental observations and showed that the origin of these chemical shift differences was predominantly the magnetic anisotropy effect from the C=O bond that hydrogen bonds with HN, which has a more favorable geometry for Ala HN in silk II than for the other HN. This result shows that we could distinguish between proton chemical shift effects arising from intermolecular interactions and those from intramolecular interactions by combining observation of the solid state 1H NMR chemical shift and empirical chemical shift calculation.

Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy.

BACKGROUND: The green tea flavonoid, epigallocatechin gallate (EGCG), has been proposed to have an anti-HIV-1 effect by preventing the binding of HIV-1 glycoprotein (gp) 120 to the CD4 molecule on T cells. OBJECTIVE: To demonstrate that EGCG binds to the CD4 molecule at the gp120 attachment site and inhibits gp120 binding at physiologically relevant levels, thus establishing EGCG as a potential therapeutic treatment for HIV-1 infection. METHODS: Nuclear magnetic resonance spectroscopy was used to examine the binding of EGCG and control, (-)-catechin, to CD4-IgG2 (PRO 542). Gp120 binding to human CD4+ T cells was analyzed by flow cytometry. RESULTS: Addition of CD4 to EGCG produced a linear decrease in nuclear magnetic resonance signal intensity from EGCG but not from the control, (-)-catechin. In saturation transfer difference experiments, addition of 5.8 micromol/L CD4 to 310 micromol/L EGCG produced strong saturation at the aromatic rings of EGCG, but identical concentrations of (-)-catechin produced much smaller effects, implying EGCG/CD4 binding strong enough to reduce gp120/CD4 binding substantially. Molecular modeling studies suggested a binding site for EGCG in the D1 domain of CD4, the pocket that binds gp120. Physiologically relevant concentrations of EGCG (0.2 micromol/L) inhibited binding of gp120 to isolated human CD4+ T cells. CONCLUSION: We have demonstrated clear evidence of high-affinity binding of EGCG to the CD4 molecule with a Kd of approximately 10 nmol/L and inhibition of gp120 binding to human CD4+ T cells. CLINICAL IMPLICATIONS: Epigallocatechin gallate has potential use as adjunctive therapy in HIV-1 infection.

The solution structure of the PufX polypeptide from Rhodobacter sphaeroides.

PufX organizes the photosynthetic reaction centre-light harvesting complex 1 (RC-LH1) core complex of Rhodobacter sphaeroides and facilitates quinol/quinone exchange between the RC and cytochrome bc(1) complexes. The structure of PufX in organic solvent reveals two hydrophobic helices flanked by unstructured termini and connected by a helical bend. The proposed location of basic residues and tryptophans at the membrane interface orients the C-terminal helix along the membrane normal, with the GXXXG motifs in positions unsuitable as direct drivers of dimerisation of the RC-LH1 complex. The N-terminal helix is predicted to extend approximately 40 Anggstrom along the membrane interface.

Noncovalent cross-linking of casein by epigallocatechin gallate characterized by single molecule force microscopy.

Interaction of the tea polyphenol epigallocatechin gallate (EGCG) with beta-casein in milk affects the taste of tea and also affects the stability of the tea and the antioxidant ability of the EGCG. In addition, interaction of polyphenols with the chemically similar salivary proline-rich proteins is largely responsible for the astringency of tea and red wine. With the use of single molecule force microscopy, we demonstrate that the interaction of EGCG with a single casein molecule is multivalent and leads to reduction in the persistence length of casein as calculated using the wormlike chain model and a reduction in its radius of gyration. The extra force required to stretch casein in the presence of EGCG is largely entropic, suggesting that multivalent hydrophobic interactions cause a compaction of the casein micelle.

Molecular model for astringency produced by polyphenol/protein interactions.

Polyphenols are responsible for the astringency of many beverages and foods. This is thought to be caused by the interaction of polyphenols with basic salivary proline-rich proteins (PRPs). It is widely assumed that the molecular origin of astringency is the precipitation of PRPs following polyphenol binding and the consequent change to the mucous layer in the mouth. Here, we use a variety of biophysical techniques on a simple model system, the binding of beta-casein to epigallocatechin gallate (EGCG). We show that at low EGCG ratios, small soluble polydisperse particles are formed, which aggregate to form larger particles as EGCG is added. There is an initial compaction of the protein as it binds to the polyphenol, but the particle subsequently increases in size as EGCG is added because of the incorporation of EGCG and then to aggregation and precipitation. These results are shown to be compatible with what is known of astringency in foodstuffs.

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation.

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 microM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.