The mechanism for acetylcholine receptor inhibition by alpha-neurotoxins and species-specific resistance to alpha-bungarotoxin revealed by NMR

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta 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 beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.     

Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide

A method for the measurement of proton T(1)(rho) relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90 degrees excitation in a HOHAHA pulse sequence. The experiment is applied to alpha-bungarotoxin (alpha-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) alpha-subunit. A good correlation between high T(1)(rho) values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the alpha-BTX complex with an AChR peptide and the model for alpha-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor alpha-subunit is exhibited, while residues implicated in binding gamma- and delta-subunits retain their mobility. In addition, the quantitative T(1)(rho) measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.     

Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor alpha subunit: effects of cysteine/cystine modification and species-specific amino acid substitutions

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.     

Structural determinants within residues 180-199 of the rodent alpha 5 nicotinic acetylcholine receptor subunit involved in alpha-bungarotoxin binding

Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) alpha subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. We have previously defined alpha-bungarotoxin (alpha-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR alpha subunit, designated alpha 5 [McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 9816-9824], and between residues 181 and 200 of the chick neuronal alpha 7 and alpha 8 subunits [McLane, K. E., Wu, X., Schoepfer, R., Lindstrom, J., & Conti-Tronconi, B. M. (1991) J. Biol. Chem. (in press)]. These sequences are relatively divergent compared with the Torpedo and muscle nAChR alpha 1 alpha-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the alpha 5 sequence, we were interested in determining the critical amino acid residues for alpha-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat alpha 5(180-199) sequence were tested, using a competition assay, in which peptides compete for 125I-alpha-BTX binding with native Torpedo nAChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.     

Design and synthesis of peptides that bind alpha-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.     

Copper binding properties of a tau peptide associated with Alzheimer's disease studied by CD, NMR, and MALDI-TOF MS

We have previously reported the copper binding properties of R3 peptide (residues 318-335: VTSKCGSLGNIHHKPGGG, according to the longest tau protein) derived from the third repeat microtubule-binding domain of water-soluble tau protein. In this work, we have investigated copper binding properties of R2 peptide (residues 287-304: VQSKCGSKDNIKHVPGGG) derived from the second repeat region of tau protein. Similar to R3 peptide, R2 peptide also plays an important role in the formation of neurofibrillary tangles (NFTs) which is one of the two main biological characteristics of Alzheimer's disease (AD). Based on the copper binding properties of R2 peptide, the possible influences of the binding on the formation of NFTs were investigated. Results from circular dichroism (CD) spectra, nuclear magnetic resonance (NMR) spectroscopy, and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) suggest that the binding is pH-dependent and stoichiometry-determined. In addition, these results also reveal that R2 peptide adopts a monomeric alpha-helical structure in aqueous solutions at physiological pH after the addition of 1 mol equiv. of Cu2+. Since alpha-helix structure is responsible for the formation of paired helical filaments (PHFs) which aggregate into NFTs, it is hypothesized that Cu2+ induces R2 peptide to self-assemble into a PHFs-like structure. Hence, it is postulated that Cu2+ plays an important role in the aggregation of R2 peptide and tau protein and that copper binding to R2 peptide may be another possible involvement in AD.     

Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

Summary RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5β raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶-Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel β-strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5β.     

Identification of residues necessary for clonally specific recognition of a cytotoxic T cell determinant.

The residues in an influenza nucleoprotein (NP) cytotoxic T cell determinant necessary for cytotoxic T cell (CTL) recognition, were identified by assaying the ability of hybrid peptides to sensitize a target cell to lysis. The hybrid peptides were formed by substituting amino acids from one determinant (influenza NP 147-158) for the corresponding residues of a second peptide (HLA CW3 171-182) capable of binding to a common class I protein (H-2Kd). Six amino acids resulted in partial recognition; however, the presence of a seventh improved the potency of the peptide. Five of the six amino acids were shown to be required for recognition. The spacing of the six amino acids was consistent with the peptide adopting a helical conformation when bound. The importance of each amino acid in CTL recognition and binding to the restriction element was investigated further by assaying the ability of peptides containing point substitutions either to sensitize target cells or to compete with the natural NP sequence for recognition by CTL. The T cell response was much more sensitive to substitution than the ability of the peptide to bind the restriction element. Collectively the separate strategies identified an approximate conformation and orientation of the peptide when part of the complex and permitted a potential location in the MHC binding site to be identified. The model provides a rationalization for analogues which have previously been shown to exhibit greater affinity for the class I molecule and suggests that the binding site in major histocompatibility complex (MHC) class I molecules might have greater steric constraints that the corresponding area of class II proteins.     

The Principal Neutralizing Determinant of HIV-1IIIB: Conformation of the Peptide Bound to a Neutralizing Antibody Studied by 2D-NMR

RP135 is a 24-residue peptide corresponding to the principal neutralizing determinant of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. We have studied the conformation of RP135 in complex with a neutralizing antibody 0.5 raised against gp120 by 2D NMR spectroscopy. The antigenic determinant recognized by this antibody was mapped using a combination of HOHAHA and ROESY measurements, in which resonances of the Fab and the tightly bound peptide residues are eliminated and the mobile residues of the bound peptide are sequentially assigned. We found that residues Ser⁶ - Thr¹⁹ are part of the epitope, while Lys⁵ and Ile²⁰ are at its boundaries. Difference spectroscopy was applied to study the conformation of the bound peptide representing the epitope within the 52 kDa of the Fab complex. Specific residues of the peptide were deuterated or replaced and the difference between the NOESY spectrum of the complex with the unlabeled residue and the NOESY spectrum of the complex with the modified residue revealed the interactions of the labeled residue both within the peptide and with the Fab fragment. A total of 122 distance restraints derived from the difference spectra enabled the calculation of the structure of the bound peptide. The peptide forms a 10-residue loop, while the two segments flanking this loop interact extensively with each other and possibly form anti-parallel -strands. The loop conformation could be observed due to the unusual large size (17 residues) of the antigenic determinant recognized by 0.5.     

Antibodies against an alpha-bungarotoxin-binding peptide of the alpha-subunit of the acetylcholine receptor

Polyclonal and monoclonal antibodies were raised against a peptide comprising residues 173-204 of the alpha-subunit of the acetylcholine receptor. The polyclonal and pooled monoclonal antibodies inhibited up to 50% of 125I-alpha-bungarotoxin binding to peptide 173-204. Some of the antibodies recognized native receptor but did not significantly affect alpha-bungarotoxin binding. Epitope mapping revealed that the antibodies are directed against residues 183-194 indicating this region is a major determinant of toxin binding. This region is most likely conformationally constrained in the native receptor.     

NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).     

Mutational analysis of the C-domain in nonribosomal peptide synthesis.

The initial condensation event in the nonribosomal biosynthesis of the peptide antibiotics gramicidin S and tyrocidine A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-alpha-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

Conformational analysis of gamma' peptide (410-427) interactions with thrombin anion binding exosite II

Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this serine protease. Approximately 7-15% of the fibrinogen gamma chain exists as the highly anionic gamma' variant (408VRPEHPAETEY(S)DSLY(S)PEDDL427). This segment has been demonstrated to target thrombin ABE-II and can accommodate sites of phosphorylation in place of sulfonation without sacrificing binding affinity. The present work employed 1D and 2D solution NMR to characterize the structural features of the bound gamma' peptide (410-427) and to evaluate the requirement of sulfonation for effective thrombin interaction. The results indicate the gamma' residues 414-427 make significant contact with the enzyme, a beta-turn exists between residues 422-425 in the presence of thrombin, and there is a large cluster of through-space interactions involving residues 418-422. Effective contact with ABE-II requires the presence of at least one phosphotyrosine residue with Y(P)422 being the more important player. Hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS was implemented to examine the location of the gamma' peptide-thrombin interface and to screen for changes in solvent exposure at distant sites. The HDX results demonstrate that the gamma' peptide interacts with or is in close proximity to thrombin residues R93, R97, R173, and R175. The binding of the gamma' peptide also protects other regions of thrombin from deuterium exchange. Affected regions include segments of ABE-I, the autolysis loop, the edge of the active site region, and the A-chain. Finally, thrombin forms a ternary complex with the gamma' peptide and PPACK, generating an enzyme whose solvent-exposed regions are even further stabilized from HDX.     

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-CCR5's 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 ^CCR5Y3 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 ^CCR5Tyr14 with ^gp120Arg440 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.     

Recognition of DNA by p53 core domain and location of intermolecular contacts of cooperative binding

We present an analysis by NMR of a 58 kDa complex of the core domain of the tumour suppressor p53 with DNA that complements and extends the crystal structure analysis. Binding of specific DNA caused significant chemical shifts of residues on the DNA-binding interface that translated into the beta-sheet of the protein. Binding of non-specific DNA caused weak but qualitatively the same shifts, corresponding to weaker binding interactions. The observed chemical shift differences correlate with frequency of cancer-inducing mutations, suggesting that the affected residues contribute to the stability of p53 core domain-DNA complex. We also identified two affected regions on the surface of the protein: helix 1 (residues V173-C182) plus G244 and residues L114-T118, which may represent a dimerisation interface.