Molecular and cellular analysis of Grb2 SH3 domain mutants: interaction with Sos and dynamin.

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.

Efficacy of a novel metalloprotease inhibitor on botulinum neurotoxin B activity.

The novel inhibitor 7-N-phenylcarbamoylamino-4-chloro-3-propyloxyisocoumarin (ICD 1578) was tested for its ability to antagonize the zinc metalloprotease activity of botulinum toxin B (BoNT/B). The efficacy of this compound was tested in a cell-free system using a 50-mer synaptobrevin peptide as substrate. The peptide, designated as [Pya88] S 39-88, had a fluorescent amino acid analog, L-pyrenylalanine (Pya), substituted for the normal Phe88 of synaptobrevin-2. Cleavage by BoNT light chain yielded fragments of 38 and 11 amino acids, respectively. The smaller fragment, containing the Pya fluorophore, was readily separated and quantified by fluorescence spectroscopy at 377 nm. In the presence of 7-200 microM ICD 1578, cleavage of [Pya88] S 39-88 was progressively reduced (IC50 = 27.6 microM), and 100 microM ICD 1578 produced >95% inhibition. For comparison, captopril, a well-known zinc metalloprotease inhibitor, generated less than 10% inhibition at a concentration of 5 mM. ICD 1578 is the most potent antagonist of BoNT/B light chain thus far described.

Beta-amino-thiols inhibit the zinc metallopeptidase activity of tetanus toxin light chain.

Tetanus neurotoxin is a 150-kDa protein produced by Clostridium tetani, which causes the lethal spastic paralytic syndromes of tetanus by blocking inhibitory neurotransmitter release at central synapses. The toxin light chain (50 kDa) has a zinc endopeptidase activity specific for synaptobrevin, an essential component of the neuroexocytosis apparatus. Previous unsuccessful attempts to block the proteolytic activity of this neurotoxin with well-known inhibitors of other zinc proteases led us to study the design of specific inhibitors as a possible drug therapy to prevent the progressive evolution of tetanus following infection. Starting from the synaptobrevin sequence at the level of the cleavage site by tetanus neurotoxin (Gln76-Phe77), a thiol analogue of glutamine demonstrated inhibitory activities in the millimolar range. A structure-activity relationship performed with this compound led us to determine the requirement for the correct positioning of the thiol group, the primary amino group, and a carboxamide or sulfonamide group on the side chain. This resulted in the design of a beta-amino-(4-sulfamoylphenyl)glycine-thiol, the first significantly efficient inhibitor of tetanus neurotoxin with a Ki value of 35 +/- 5 microM.

Metallopeptidase inhibitors of tetanus toxin: A combinatorial approach.

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln76-Phe77). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. beta-Aminothiols mimicking Gln76 of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 microM range. These compounds have now been modified to interact with S' subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P1 side chain and natural amino acids in P1' and P2' positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P1' and P2' components, respectively. This led to new inhibitors of TeNt with Ki values in the 3-4 microM range. These molecules are the most potent inhibitors of TeNt described so far.

[Tetanus and botulinum toxins are zinc metallopeptidases: molecular mechanisms and inhibition of their neurotoxicity]. / Les toxines tétaniques et botuliniques sont des métallopeptidases à zinc: mécanismes moléculaires et inhibition de leur neurotoxicité.

The very high toxicity of tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) are related to their nature of zinc metallopeptidases able to selectively cleave small proteins involved in neurotransmitters exocytosis. At this time, there is no efficient and selective therapy towards tetanos and tobulism as well as protection against a possible spreading of the toxins. We have therefore investigated the minimum sequences of TeNT and BoNT substrates allowing an efficient and simple fluorescent dosage of the enzymatic activity to be developed. Using synaptobrevin (93 amino acids) as substrate of TeNT and several fragments synthesised by solid phase method, we have shown that the clostridial neurotoxins behave as allosteric-type enzymes. This is the first example in zinc metallopeptidases. Based on these results a strategy, including the use of combinatorial chemistry, was carried out issuing in the design of the first potent inhibitors of TeNT and BoNT.

High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity.

Botulinum neurotoxins are responsible for botulism, a flaccid muscular paralysis caused by inhibition of acetylcholine release at the neuromuscular junction. This occurs by cleavage of conserved proteins involved in exocytosis such as synaptobrevin by the zinc metallopeptidase activity of the light chain of some botulinum neurotoxins. Botulism, for which there is presently no therapy available, is a relatively widespread disease that may result in death. Consequently, the development of drugs able to inhibit the hydrolytic activity of these neurotoxins is of great interest. Design and screening of such inhibitors could be largely facilitated by using high-throughput assays. With this aim, a novel in vitro test for quantifying the proteolytic activity of botulinum type B neurotoxin was developed. The substrate is the 60--94 fragment of human synaptobrevin-1 which was modified by introduction of the fluorescent amino acid l-pyrenylalanine in position 74 and a p-nitrophenylalanyl residue as quenching group in position 77. The cleavage of Syb 60-94 [Pya(74), Nop(77)] by the toxin active chain occurs selectively between residues 76 and 77 as in the case of the unmodified synaptobrevin and is directly quantified by measuring the strong fluorescence of the formed metabolite Syb 60-76 [Pya(74)]. This is the easiest, quickest, and cheapest assay described to date for measuring the proteolytic activity of botulinum type B neurotoxin. It can be easily automated for high-throughput screening. Moreover, amounts of about 3.5 pg/ml of botulinum type B neurotoxin could be detected by this method.

Nociceptin/orphanin FQ metabolism: role of aminopeptidase and endopeptidase 24.15.

The endogenous opioid receptor-like1 (ORL1) ligand, nociceptin/orphanin FQ (FGGFTGARKSARKLANQ), a heptadecapeptide structurally resembling dynorphin A, has recently been identified. The wide distribution of ORL1 mRNA and nociceptin/orphanin FQ precursor in the CNS, particularly in the limbic system regions and in several areas known to be involved in pain perception, suggests that nociceptin/orphanin FQ is potentially endowed with various central functions. In general, activation and/or inactivation of regulatory peptides occur through the action of cell surface peptidases. The physiological mechanisms under which nociceptin/orphanin FQ is metabolized should lead to a better understanding of its physiological functions. Mouse brain cortical slices were incubated in medium containing the heptadecapeptide in the presence or in the absence of peptidase inhibitors. The critical sites of enzymatic cleavage are Phe1-Gly2, Ala7-Arg8, Ala11-Arg12, and Arg12-Lys13 bonds. The major role played by metallopeptidases was confirmed by the complete protection of metabolism in the presence of EDTA. Aminopeptidase N and endopeptidase 24.15 are the two main enzymes involved in nociceptin/orphanin FQ metabolism, whereas endopeptidase 24.11 (involved in enkephalin [YGGFM(L)] catabolism) does not appear critically involved in nociceptin/orphanin FQ metabolism. The physiological relevance of aminopeptidase N and endopeptidase 24.15 in the heptadecapeptide metabolism remains to be determined.

Solid-phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1-93 by tetanus toxin L chain.

A 93-residue peptide corresponding to the cytosolic domain of a human vesicle associated membrane protein (VAMP or synaptobrevin) has been prepared by solid-phase peptide synthesis in order to investigate the proteolytic activity of the tetanus toxin light chain (TeTx L chain). This protein has been recently reported to inactivate the neuronal rat synaptobrevin II by proteolysis. We show in this study that the synthetic human synaptobrevin II 1-93 (Syb II 1-93) as well as an N-terminus-shortened 69-residue peptide (Syb II 25-93) were cleaved selectively at the Gln76-Phe77 peptide bond by TeTx L chain while shorter peptides were not. A Michaelis constant Km = 192 +/- 2 microM and a catalytic constant kcat = 0.5 min-1 were found for the 93-residue peptide. A neutral optimum pH for the cleavage rate, an inhibition by preincubation of the toxin with well known nonspecific inhibitors of metallopeptidases as well as a zinc-dependent enzyme activity suggest that TeTx belongs to the zinc endopeptidase family. Moreover an activation by reducing agents and an inhibition by cysteine-modifying chemical reagents indicate a critical thiol dependency. Among several specific inhibitors of zinc endopeptidases tested, none could inhibit TeTx L chain even at high concentration. Structural studies by 600-MHz 1H-NMR showed that in water or dimethylsulfoxide the peptide Syb II 1-93 and shorter fragments did not present well defined conformations. Nevertheless protein-protein interactions have been shown for the peptides Syb II 1-93 and 25-93 but not for Syb II 51-93, a fragment not cleaved by TeTx L chain.

Inhibition of neurotransmitter release by synthetic proline-rich peptides shows that the N-terminal domain of vesicle-associated membrane protein/synaptobrevin is critical for neuro-exocytosis.

Tetanus toxin and clostridial neurotoxins type B, D, F, and G inhibit intracellular Ca(2+)-dependent neurotransmitter release via the specific proteolytic cleavage of vesicle-associated membrane protein (VAMP)/synaptobrevin, a highly conserved 19-kDa integral protein of the small synaptic vesicle membrane. This results in the release of the larger part of the cytosolic domain of this synaptic protein into the cytoplasm. Microinjection of synthetic peptides corresponding to this fragment into identified presynaptic neurons of Aplysia californica led to a potent, long lasting, and dose-dependent inhibition (approximately 50% at 10 MicroM) of acetylcholine release, probably by hindering endogenous VAMP/synaptobrevin from interacting with synaptic proteins involved in exocytosis. Structure activity studies showed that this effect is confined to the N-terminal domain of VAMP/synaptobrevin isoform II and is related to the presence of a proline-rich motif (PGGPXGX3PP or PAAPXGX3PP). At higher concentrations, the inhibitory effect was lower and only transient, suggesting that the N-terminal proline-rich domain of VAMP/synaptobrevin plays opposing roles in neurotransmitter release very likely by interacting with different synaptic proteins. This probably occurs by disruption of the recently reported in vitro VAMP-synaptophysin interaction that involves the N-terminal domain of VAMP II and was proposed to hinder synatophysin-related formation of a fusion pore. The observed recovery of neurotransmitter release following injection of high concentration of N-terminal fragments of VAMP II brings a strong in vivo support to this hypothesis. The minimum active peptide GPGGPQGGMQPPREQS could be used for rationally designing potent synthetic blockers of neurotransmission.

Efficient solid-phase synthesis of Vpr from HIV-1 using low quantities of uniformly 13C-, 15N-labeled amino acids for NMR structural studies.

The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in the human immunodeficiency virus, type 1 (HIV-1) life cycle, including cell-cycle arrest at the G2/M check point, promotion of the HIV-1 preintegration complex for nuclear transport, induction of apoptosis and transcriptional activation of a variety of viral and cellular promoters. Preliminary 1H NMR experiments performed on Vpr fragments showed the presence of several helical regions. However, the assignment of many protons in the amide region of the complete sequence of Vpr proved to be impossible due to the overlap of multiple NOE cross peaks. Moreover, because of its cytotoxicity, it is difficult to produce large quantities of 15N- and 13C-labeled Vpr using molecular biology approaches. Therefore, the solid-phase peptide synthesis of (1-96)Vpr, labeled at 22 selected positions, using recently commercially available uniformly 13C-, 15N-labeled fmoc amino acids, has been optimized to produce large quantities (104 mg, 15% yield) of pure compound, while minimizing the quantity of labeled amino acids used for each coupling. As expected two-dimensional heteronuclear NMR experiments performed with this protein allowed the unequivocal assignments of all the proton signals. This study shows that introduction of few labeled 13C/15N labeled amino acids in selected positions facilitates the determination of structure solution of small protein accessible by solid-phase peptide synthesis, and could allow dynamic studies of their conformational behavior to be carried out.

A sensitive and rapid fluorescence-based assay for determination of tetanus toxin peptidase activity.

The light chain of tetanus toxin (TeNT-L chain), endowed with a zinc metalloendopeptidase activity, cleaves specifically the vesicle-associated membrane protein (VAMP), also called synaptobrevin, at a single peptide bond (Gln76-Phe77), resulting in the blockade of neuroexocytosis. The 50-mer synaptobrevin peptide S 39-88, synthesized by solid-phase peptide synthesis, was determined to be the minimum substrate of TeNT still notably hydrolyzed by TeNT-L chain. In this peptide, Tyr88 was substituted by the highly fluorescent amino acid (L) pyrenylalanine (Pya) which was synthesized in good yields by an enantioselective method. The fluorescent substrate [Pya88] S 39-88 was cleaved four times more rapidly by TeNT-L chain than S 39-88 (kcat/Km = 9635 and 2455 M-1.min-1, respectively). One of the two metabolites formed by the action of TeNT L chain, [Pya88] S 77-88, was easily separated from the substrate in one step using Sep-Pak Vac C18 cartridges and its concentration quantified by fluorescence. This novel enzymatic assay, which could be easily extended to other clostridial neurotoxins, is a major improvement in term of sensitivity and time saving, compared to currently used methods (SDS-PAGE, HPLC). It lends itself readily to automation for large-scale screening of selective and potent inhibitors of these neurotoxins which remain to be developed.

Cooperative exosite-dependent cleavage of synaptobrevin by tetanus toxin light chain.

The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln76-Phe77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH2 and COOH terminus of synaptobrevin have shown that the sequence 39-88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH2- and COOH-terminal peptides of synaptobrevin, S 27-55 (S1) and S 82-93 (S2), to the synaptobrevin fragment S 56-81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S1 and S2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.

Solution structure of GAP SH3 domain by 1H NMR and spatial arrangement of essential Ras signaling-involved sequence.

Src homology 3 (SH3) domains are found in numerous cytoplasmic proteins involved in intracellular signal transduction. We used 2-D 1H NMR to determine the structure of the SH3 domain of the guanosine triphosphatase-activating protein (GAP), an essential component of the Ras signaling pathway. The structure of the GAP SH3 domain (275-350) was found to be a compact beta-barrel made of six antiparallel beta-strands arranged in two roughly perpendicular beta-sheets with the acidic residues located at the surface of the protein. The Trp317, Trp319, Thr321 and Leu323 residues belonging to the sequence (317-326), which was shown to be essential for Ras signaling, formed two nearby lipophilic bulges followed by a hydrophilic domain (Arg324-Asp326). These structural data could be used to characterize the still unidentified downstream components of GAP, which are involved in Ras signaling, and to rationally design inhibitors of this pathway.

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8.

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.

Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent.

The zinc fingers of retroviral gag nucleocapsid proteins (NC) are required for the specific packaging of the dimeric RNA genome into virions. In vitro, NC proteins activate both dimerization of viral RNA and annealing of the replication primer tRNA onto viral RNA, two reactions necessary for the production of infectious virions. In this study the role of the zinc finger of Moloney murine leukemia virus (MoMuLV) NCp10 in RNA binding and annealing activities was investigated through modification or replacement of residues involved in zinc coordination. These alterations did not affect the ability of NCp10 to bind RNA and promote RNA annealing in vitro, despite a complete loss of zinc affinity. However mutation of two conserved lysine residues adjacent to the finger motif reduced both RNA binding and annealing activities of NCp10. These findings suggest that the complexed NC zinc finger is not directly involved in RNA-protein interactions but more probably in a zinc dependent conformation of NC protein modulating viral protein-protein interactions, essential to the process of viral RNA selection and virion assembly. Then the NC zinc finger may cooperate to select the viral RNA genome to be packaged into virions.

Investigation of zinc-binding affinities of Moloney murine leukemia virus nucleocapsid protein and its related zinc finger and modified peptides.

Nucleocapsid proteins of retroviruses are small basic, nucleic acid-binding proteins with either one or two "Cys-His" boxes, which have been shown to be involved in genomic RNA dimerization, encapsidation, and replication primer tRNA annealing to the initiation site for reverse transcription. The nucleocapsid (NC) protein of Moloney murine leukemia virus (MoMuLV NCp10) is made up of 56 residues with one Cys-His motif. The Zn(2+)-binding affinities and induced conformational changes of NCp10 were investigated by following the fluorescence of Trp 35 located in the Cys-His domain. At pH 7.5, NCp10 was shown to bind Zn2+ at a 1 : 1 ratio with a very high apparent binding constant of 1.2 (+/- 0.3).10(13)M-1. A similar apparent binding constant was obtained for a 19-residue peptide encompassing the Cys-His box, designated the "zinc finger motif," indicating that it contains most if not all the information to bind Zn2+ tightly. Changing Trp 35 to Phe in the peptide did not affect the Zn2+ affinity, indicating that Trp 35 is not crucial for Zn2+ binding. On the contrary, replacing Cys 29 by Ser, the chemical modification or oxidation of the three Cys sharply reduced Zn2+ affinity, confirming the essential role of Cys in Zn2+ binding. In addition, fluorescence and energy transfer data suggested that Zn2+ binding modifies the Trp 35 environment but not its solvent exposure, and increases the average distance between Tyr 28 and Trp 35 by about 2 A. These data suggest that Zn2+ binding to retroviral NC protein is biologically relevant.

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus.

The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX2CysX4HisX4Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH > or = 5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX2CysX4HEsX4Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz 1H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747 +/- 0.209 A for backbone atoms and 1.709 +/- 0.187 A when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr28, Trp35, Lys37, Lys41 and Lys42 residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.

NMR structure of the N-terminal SH3 domain of GRB2 and its complex with a proline-rich peptide from Sos.

GRB2 is a small adaptor protein of 217 amino acids comprising one SH2 domain surrounded by two SH3 domains. GRB2 couples receptor tyrosine kinase activation to Ras signalling by interacting, through its SH3 domains, to the carboxy-terminal proline-rich regions of the guanine nucleotide exchange factor Sos. Here we report the synthesis and solution structure of the amino-terminal SH3 domain of GRB2 and of its more stable Ser 32 mutant. 1H NMR analysis of the complex between the Ser-32-GRB2-N-SH3 domain and the proline-rich peptide VPPPVPPRRR, derived from h-Sos, shows that relative to the SH3 peptide complexes described for PI3K, Fyn and Abl, the proline-rich peptide in this complex binds in the opposite orientation.

Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding.

Regulatory factor X (RFX) proteins are transcriptional activators that recognize X-boxes (DNA of the sequence 5'-GTNRCC(0-3N)RGYAAC-3', where N is any nucleotide, R is a purine and Y is a pyrimidine) using a highly conserved 76-residue DNA-binding domain (DBD). DNA-binding defects in the protein RFX5 cause bare lymphocyte syndrome or major histocompatibility antigen class II deficiency. RFX1, -2 and -3 regulate expression of other medically important gene products (for example, interleukin-5 receptor alpha chain, IL-5R alpha). Fusions of the ligand-binding domain of the oestrogen receptor with the DBD of RFX4 occur in some human breast tumours. Here we present a 1.5 A-resolution structure of two copies of the DBD of human RFX1 (hRFX1) binding cooperatively to a symmetrical X-box. hRFX1 is an unusual member of the winged-helix subfamily of helix-turn-helix proteins because it uses a beta-hairpin (or wing) to recognize DNA instead of the recognition helix typical of helix-turn-helix proteins. A new model for interactions between linker histones and DNA is proposed.

DNA binding properties of a chemically synthesized DNA binding domain of hRFX1.

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound. Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.