Moncef M. Ladjimi

Pierre and Marie Curie University - Paris 6, Lutetia Parisorum, Île-de-France, France

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Publications (25)85.62 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The self-association properties of the molecular chaperone HSC 70 have been assessed by analytical ultracentrifugation. Sedimentation velocity analysis indicates the presence of three species, whose proportions were dependent on protein concentration, but whose sedimentation coefficients, s 20, w, of 4.3 S, 6.6 S and 8.5 S did not vary with concentration, which is indicative of a slowly equilibrating system. Sedimentation equilibrium studies indicate a dissociation into monomers at low HSC 70 concentrations and an association into dimers and trimers at high concentrations. Multiple sets of sedimentation equilibrium data, obtained at various initial loading concentrations and rotor speeds, were adequately fitted to a single set of equilibrium constants by a monomerdimer-trimer association model in which the association constants for the monomer-dimer and dimer-trimer equilibrium. K1−2=1.1.·105 M−1 and K 2−3=0.9·105 M−1 respectively, were nearly identical. Interestingly, na isodesmic, indefinite type of association describes the data almost equally well with a single constant of 1.2·105 M−1. These results might have important implications for the chaperone function of HSC 70.
    08/2007: pages 1-6;
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    ABSTRACT: Data of this study showed that alphaD-alphaE helices and the conserved interdomain linker are two interfaces essential not only for the self-association but also for the functional properties of rat HSC70. Self-association which is a conserved property of HSP70 seems to be important for the activity of these proteins.
    Protein and Peptide Letters 02/2007; 14(8):761-5. · 1.74 Impact Factor
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    ABSTRACT: The hexahistidine is a fusion tag used for the isolation of proteins via an immobilized metal-ion affinity chromatography (IMAC). In the present study, we have purified and analyzed two constructs of the heat shock protein HSC70 in the presence or the absence of the His-tag (C30WT-His(+)/C30WT and C30DeltaL-His(+)/C30DeltaL). The oligomerization properties of the constructs were analyzed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AU). Results from SEC analysis indicated that the His-tag promotes the dimerization of C30DeltaL-His(+) but has no effect on the elution profile of C30WT-His(+), compared to their respective untagged forms C30DeltaL and C30WT. These observations were also confirmed by AU analysis which indicates that C30DeltaL is stabilized in the dimeric form in the presence of the His-tag. These results emphasize the need to remove the His-tag before structural characterization of some recombinant proteins.
    Journal of Chromatography B 01/2007; 844(2):328-34. · 2.69 Impact Factor
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    ABSTRACT: HSP70 from bacteria to man are known to self-associate to form multiple species suggesting that self-association is related to function. In order to determine the structural basis of HSP70 oligomerization, deletion mutants in the C-terminal domain of HSC70, a constitutive member of the HSP70 family, have been constructed and analyzed for their self-association properties by gel electrophoresis, size-exclusion chromatography and analytical ultracentrifugation. The results of this investigation indicate that, whereas deletion of the GGMP rich C-terminal extremity of HSC70, containing EEVD motif stabilizes the oligomeric species, deletions of either the aD-aE C-terminal helices or the inter-domain hydrophobic linker contribute to the stabilization of the monomeric form. Thus, two non-contiguous regions, located at both ends of the C-terminal domain of the protein, appear to form the contact interface in the oligomers and may interact in a dynamic fashion leading to the formation of several coexisting species.
    Archives de l'Institut Pasteur de Tunis 02/2006; 83(1-4):53-62.
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    ABSTRACT: Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and lambda phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for lambda phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed.
    Journal of Bacteriology 10/2004; 186(18):6248-53. · 2.69 Impact Factor
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    ABSTRACT: The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.
    Journal of Biological Chemistry 02/2002; 277(1):259-66. · 4.60 Impact Factor
  • Marion Velten, Bruno O. Villoutreix, Moncef M. Ladjimi
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    ABSTRACT: HSC70 interacting protein (HIP) is an essential cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor. To determine the quaternary structure and the gross conformation of the protein in solution, a wide array of biochemical and biophysical techniques has been used. Size-exclusion chromatography and sedimentation velocity indicate the presence of a single species with a Stokes radius, R(s), of 55 A and a sedimentation coefficient, s degrees (20,w), of 4.34 S. The combination of these data gives a molecular mass of 101 000 Da, a value close to that of the theoretical molecular mass of a dimer (87 090 Da). Sedimentation equilibrium, performed at various protein concentrations and rotor speeds, gives a molecular mass of 88 284 Da, almost in exact agreement with the molecular mass of a dimer. On the basis of these data, a frictional ratio f/f(0) of 1.6 is obtained, suggesting an elongated shape for the HIP dimer. Secondary structure predictions, supported by circular dichroism experiments, indicate that HIP is an almost all alpha-protein, able to form extended coiled coils. Using threading and comparative model building methods, a structural model of a segment of HIP involved in HSC70 binding has been constructed and potential sites of interaction between HIP and HSC70 are proposed on the basis of electrostatic as well as shape complementarity. Altogether, these results indicate that HIP is an elongated dimer, able to bind two HSC70 molecules through its TPR regions, and suggest the existence of a versatile binding site on HSC70 that may be involved in the interaction of the chaperone with the cochaperones or other interacting proteins.
    Biochemistry 02/2000; 39(2):307-15. · 3.19 Impact Factor
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    ABSTRACT: Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.
    European Journal of Biochemistry 02/1999; 259(1-2):379-84. · 3.58 Impact Factor
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    C Purcarea, G Hervé, M M Ladjimi, R Cunin
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    ABSTRACT: The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae. Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.
    Journal of Bacteriology 08/1997; 179(13):4143-57. · 2.69 Impact Factor
  • N Benaroudj, B Fouchaq, M M Ladjimi
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    ABSTRACT: We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.
    Journal of Biological Chemistry 04/1997; 272(13):8744-51. · 4.60 Impact Factor
  • N Benaroudj, F Triniolles, M M Ladjimi
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    ABSTRACT: In a previous study, we showed that the molecular chaperone HSC70 self-associates in solution in a reversible and likely unlimited fashion. Here, we examine the influence of nucleotides, nucleotide analogs, peptides, and unfolded proteins on the self-association properties of this protein. Whereas in the presence of ADP, HSC70 exists as a slow, concentration- and temperature-dependent monomer-oligomer equilibrium, in the presence of ATP, the protein is essentially monomeric, indicating that ATP shifts this equilibrium toward the monomer by stabilizing the monomer. Dissociation of oligomers into monomers is also obtained with the slowly hydrolyzable ATP analogs, adenosine 5'-O-(thiotriphosphate) and 5'-adenylyl-beta,gamma-imidodiphosphate, or the complex between ADP and the phosphate analog, BeF3, indicating that binding but not hydrolysis of ATP is necessary and sufficient for the stabilization of HSC70 monomer. Furthermore, binding of short peptides or permanently unfolded proteins to the peptide binding site of HSC70 promotes the dissociation of oligomers into monomers, suggesting that protein substrates are able to compete with HSC70 for the same binding site. Because the release of peptides or unfolded proteins from HSC70 has also been shown to require ATP binding, these results indicate that dissociation of oligomers is controlled by a mechanism similar to that of release of protein substrates and suggest that binding of HSC70 to itself occurs via the peptide binding site and mimics binding of HSC70 to protein substrates.
    Journal of Biological Chemistry 09/1996; 271(31):18471-6. · 4.60 Impact Factor
  • Luc Fetler, Patrice Vachette, Guy Herve, Moncef M. Ladjimi
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    ABSTRACT: The quaternary structural change associated with the homotropic cooperative interactions in Escherichia coli aspartate transcarbamylase (ATCase) is accompanied by various tertiary structural modifications; the most notable one involves the 240s loop formed by residues 230--245 of the catalytic chain. In order to monitor local conformational changes in this region by fluorescence spectroscopy, Tyr-240 has been replaced by a Trp residue, in a mutant enzyme, in which both naturally occurring Trp residues in positions 209 and 284 of the catalytic chains had previously been substituted by Phe residues. This F209F284W240-ATCase still displays homotropic cooperativity for aspartate and undergoes the same T to R quaternary structure change as does the wild-type enzyme. Upon binding of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate, the fluorescence emission spectrum of this mutant shows a red shift directly proportional to the fraction of catalytic sites occupied by this compound, a maximum value of 4 nm being attained when all six active sites are ligated. An identical shift is observed with the catalytic subunits of this modified enzyme, when all three active sites are occupied. In contrast, the quaternary structural change of the F209F284W240-ATCase, monitored by small-angle X-ray scattering, is complete when only four out of six catalytic sites are occupied. Thus, the 240s loop adopts its final conformation only when the neighboring active site is bound.
    Biochemistry 01/1996; 34(48):15654-60. · 3.19 Impact Factor
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    ABSTRACT: The self-association properties of the molecular chaperone HSC70 have been analyzed by a wide range of biochemical and biophysical techniques. Nondenaturing gel electrophoresis and cross-linking studies show the presence of multiple species going from monomer to at least trimer. Size-exclusion chromatography gives two overlapping peaks, a major one corresponding to species having the molecular mass of monomer (70 kDa) and a minor broad one corresponding to species with a molecular mass range of 150-300 kDa. Progressive dilution of the protein leads to an increase in the size of the monomer peak at the expense of that of the oligomeric peak, thus indicating a concentration-dependent chemical equilibrium. Sedimentation velocity reveals the presence of three species, whose proportions were dependent on concentration, but whose sedimentation coefficients, s20,w, of 4.3, 6.6, and 8.5 S did not vary with concentration, indicative of a slowly equilibrating system. Sedimentation equilibrium studies confirmed these results and showed a dissociation into monomers at low concentrations and an association into dimers and trimers at high concentrations. The multiple sedimentation equilibrium datasets, obtained at various initial loading concentrations as well as different rotor speeds, were fitted to a single set of equilibrium constants by a monomer-dimer-trimer association model in which the association constants for the monomer-dimer and dimer-trimer equilibrium were respectively K1-2 = 1.1 x 10(5) M-1 and K2-3 = 0.9 x 10(5) M-1. Interestingly, an isodesmic, indefinite type of association describes the data almost equally well with a single constant of 1.2 x 10(5) M-1. (ABSTRACT TRUNCATED AT 250 WORDS)
    Biochemistry 12/1995; 34(46):15282-90. · 3.19 Impact Factor
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    ABSTRACT: Aspartate transcarbamylase from Escherichia coli is stimulated by ATP and feedback-inhibited by CTP and UTP. Previous work allowed the identification of the hydrophobic interface between the two domains of the regulatory chain as a structural element specifically involved in the transmission of the ATP regulatory signal toward the catalytic sites. The present work describes the identification of a cluster of amino acid interactions at an interface between the regulatory chains and the catalytic chains of the enzyme as another structural feature involved in the transmission of the ATP regulatory signal but not in those of CTP and UTP. These interactions involve residues 146 to 149 of the regulatory chain and residues 242 to 245 of the catalytic chain. Perturbations of these interactions also alter to various extents the co-operativity between the catalytic sites for aspartate binding. These findings are in agreement with the idea that the primary effect of ATP might consist, in part, of a modulation of the stability of the interfaces between regulatory and catalytic subunits, thereby facilitating the T to R transition induced by aspartate binding, as was put forward in two recently proposed models, the "effector modulated transition" model and the "nucleotide perturbation" model. This does not exclude that this cluster of interactions could also act as a relay to transmit the ATP regulatory signal to the catalytic sites according to the previously proposed "primary-secondary effects" model.
    Journal of Molecular Biology 03/1995; 246(1):132-43. · 3.96 Impact Factor
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    ABSTRACT: The regulatory chain of E. coli aspartate transcarbamylase (E.C. is folded into two domains. The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively. The zinc domain ensures the contact with the catalytic chains. The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain. This structural feature mediates the transmission of the ATP regulatory signal. In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process. The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP. The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP. A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains.
    Journal of Molecular Biology 10/1994; 242(2):139-49. · 3.96 Impact Factor
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    ABSTRACT: In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequences so far, suggesting that this region is interacting with another domain of the multifunctional protein.
    Proteins Structure Function and Bioinformatics 08/1994; 19(3):230-43. · 2.92 Impact Factor
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    ABSTRACT: The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 11 of cell culture. Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.
    European Journal of Biochemistry 05/1994; 221(1):121-8. · 3.58 Impact Factor
  • Biochemical Society Transactions 03/1993; 21(1):195-8. · 3.24 Impact Factor
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    ABSTRACT: Aspartate transcarbamylase (EC contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.
    Biochemistry 01/1993; 31(49):12504-13. · 3.19 Impact Factor
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    ABSTRACT: Aspartate transcarbamoylase (EC is extensively studied as a model for cooperativity and allostery. This enzyme shows cooperativity between the catalytic sites, and its activity is feedback inhibited by CTP and activated by ATP. These regulatory processes involve several interfaces between catalytic and regulatory chains as well as between domains within these two types of chains. As far as the regulatory chain is concerned, its two domains are in contact through a hydrophobic interface, in which a tyrosine residue is inserted in a pocket involving two leucine residues of the allosteric domain and a valine and a leucine residue of the zinc domain. To probe the possible implication of this hydrophobic core in the CTP and ATP regulatory effect, the tyrosine was replaced by a phenylalanine through oligonucleotide-directed mutagenesis. Interestingly, the resulting mutant shows a complete inversion of the ATP effect; it is now inhibited by ATP instead of being activated by this nucleotide triphosphate. This mutant remains normally sensitive to the feedback inhibitor CTP. This result shows that the hydrophobic interface between the two domains of the regulatory chain plays an important role in the discrimination between the regulatory signals promoted by the two allosteric effectors.
    Proceedings of the National Academy of Sciences 11/1991; 88(20):9180-3. · 9.81 Impact Factor

Publication Stats

331 Citations
85.62 Total Impact Points


  • 1996–2002
    • Pierre and Marie Curie University - Paris 6
      Lutetia Parisorum, Île-de-France, France
  • 1997
    • Vlaams Instituut voor Biotechnologie
      Gand, Flanders, Belgium
  • 1996–1997
    • Laboratoire d'Enzymologie et Biochimie Structurales
      Gif, Île-de-France, France
  • 1991
    • Free University of Brussels
      • Laboratorium voor Erfelijkheidsleer en Microbiologie
      Bruxelles, Brussels Capital Region, Belgium
  • 1986
    • Université Libre de Bruxelles
      • Laboratory of Microbiology
      Brussels, BRU, Belgium