Tali Scherf

The acquisition of multidimensional NMR spectra within a single scan

A scheme enabling the complete sampling of multidimensional NMR domains within a single continuous acquisition is introduced and exemplified. Provided that an analytes signal is sufficiently strong, the acquisition time of multidimensional NMR experiments can thus be shortened by orders of magnitude. This could enable the characterization of transient events such as proteins folding, 2D NMR experiments on samples being chromatographed, bring the duration of higher dimensional experiments (e.g., 4D NMR) into the lifetime of most proteins under physiological conditions, and facilitate the incorporation of spectroscopic 2D sequences into in vivo imaging investigations. The protocol is compatible with existing multidimensional pulse sequences and can be implemented by using conventional hardware; its performance is exemplified here with a variety of homonuclear 2D NMR acquisitions.

Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells.

Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.

A Beta-Hairpin Structure in a 13-mer Peptide that Binds Alpha-Bungarotoxin with High Affinity and Neutralizes its Toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

The Mechanism for Acetylcholine Receptor Inhibition by α-Neurotoxins and Species-Specific Resistance to α-Bungarotoxin Revealed by NMR

Abstract The structure of a peptide corresponding to residues 182–202 of the acetylcholine receptor α1 subunit in complex with α-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in α-bungarotoxin binding. One face of the long β hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the β hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two α-bungarotoxin molecules shows that this pocket is occupied by the conserved α-neurotoxin residue R36, which forms cation-π interactions with both α W149 and γ W55/ δ W57 of the receptor and mimics acetylcholine.

Design and synthesis of peptides that bind α-bungarotoxin with high affinity

Abstract Background: α-Bungarotoxin (α-BTX) is a highly toxic snake venom α-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind α-BTX with high affinity, and inhibit its interaction with AChR with an IC 50 of 2 nM. The design of these peptides was based on a lead peptide with an IC 50 of 3×10 −7 M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. Results: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of α-BTX with the lead peptide, as well as structure–function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC 50 values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against α-BTX lethality. Conclusions: Synthetic peptides with high affinity to α-BTX may be used as potential lead compounds for developing effective antidotes against α-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

The Conformation and Orientation of a 27-Residue CCR5 Peptide in a Ternary Complex with HIV-1 gp120 and a CD4-Mimic Peptide.

Interaction of CC chemokine receptor 5 (CCR5) with the human immunodeficiency virus type 1 (HIV-1) gp120/CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of two to four tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied both in its free form and in a ternary complex with deglycosylated gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27), which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by 2-fold the length of Nt-CCR5s known structure. Two-dimensional saturation transfer experiments and measurement of relaxation times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4, while the C-terminal segment of Nt-CCR5(1-27) points toward the target cell membrane, reflecting an Nt-CCR5 orientation that differs by 180° from that of a previous model. A gp120 site that could accommodate (CCR5)Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated (CCR5)Tyr14 with (gp120)Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion, furthering our understanding of CCR5 recognition by HIV-1.

The Binding Site of Acetylcholine Receptor

Abstract: Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) α‐subunit for the analysis of the binding site of the AChR. A 13‐mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by α‐bungarotoxin (α‐BTX) from a phage‐display peptide library. The solution structure of a complex between this library‐lead peptide and α‐BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure‐function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to α‐BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high‐affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of α‐BTX to the AChR with an IC50 of 2 nM. The solution and crystal structures of complexes of α‐BTX with HAP were solved, demonstrating that the HAP fits snugly to α‐BTX and adopts a β‐hairpin conformation. The X‐ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that α‐BTX wraps around the receptor binding‐site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand‐binding site. Our proposed model explains the strong antagonistic activity of α‐BTX and accommodates much of the biochemical data on the mode of interaction of α‐BTX with the AChR.

NMR mapping of RANTES surfaces interacting with CCR5 using linked extracellular domains

Chemokines constitute a large family of small proteins that regulate leukocyte trafficking to the site of inflammation by binding to specific cell‐surface receptors belonging to the G‐protein‐coupled receptor (GPCR) superfamily. The interactions between N–terminal (Nt‐) peptides of these GPCRs and chemokines have been studied extensively using NMR spectroscopy. However, because of the lower affinities of peptides representing the three extracellular loops (ECLs) of chemokine receptors to their respective chemokine ligands, information concerning these interactions is scarce. To overcome the low affinity of ECL peptides to chemokines, we linked two or three CC chemokine receptor 5 (CCR5) extracellular domains using either biosynthesis in Escherichia coli or chemical synthesis. Using such chimeras, CCR5 binding to RANTES was followed using 1H‐15N‐HSQC spectra to monitor titration of the chemokine with peptides corresponding to the extracellular surface of the receptor. Nt‐CCR5 and ECL2 were found to be the major contributors to CCR5 binding to RANTES, creating an almost closed ring around this protein by interacting with opposing faces of the chemokine. A RANTES positively charged surface involved in Nt‐CCR5 binding resembles the positively charged surface in HIV‐1 gp120 formed by the C4 and the base of the third variable loop of gp120 (V3). The opposing surface on RANTES, composed primarily of β2–β3 hairpin residues, binds ECL2 and was found to be analogous to a surface in the crown of the gp120 V3. The chemical and biosynthetic approaches for linking GPCR surface regions discussed herein should be widely applicable to the investigation of interactions of extracellular segments of chemokine receptors with their respective ligands.

Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind alpha-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the alpha-BTX/AChR system yielded 13-mer peptides that bind to alpha-BTX with high affinity and are potent inhibitors of alpha-BTX binding to AChR (IC(50) of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel beta-hairpin structure when bound to alpha-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.

NMR observation of interactions in the combining site region of an antibody using a spin-labeled peptide antigen and NOESY difference spectroscopy.

A spin‐labeled peptide antigen (TEMPO‐VEVPGSQHIDSQ) was used to measure NOESY difference spectra that show interactions in the binding site region of the Fab fragment of the anti‐cholera toxin peptide antibody TE33. In addition to identification of peptide‐Fab interactions and interactions within the bound peptide, these difference spectra show well‐resolved cross peaks due to interactions within the large Fab fragment (50 kDa). These difference spectra indicate that the conformational changes in the Fab upon peptide binding are confined to the combining site region of the antibody. The NOESY difference spectra of selectively deuterated Fab molecules were used in combination with HOHAHA measurements to assign the interactions to amino acid type and to identify the interactions within the Fab as either inter‐ or intraresidue interactions. The assignment of interactions within the Fab to corresponding aromatic residues in the Fab sequence was facilitated by an earlier NMR‐derived model calculated on the basis of NOE restraints on Fab‐peptide and intra‐boundpeptide distances. The new restraints on distances within the Fab, combined with the previously obtained restraints, were used to generate a refined NMR‐derived model for the TE33‐peptide complex.—Scherf, T., Hiller, R., Anglister, J. NMR observation of interactions in the combining site region of an antibody using a spin‐labeled peptide antigen and NOESY difference spectroscopy. FASEB J. 9, 120‐126 (1995)