Richard Kahn

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

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Publications (118)537.89 Total impact

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    ABSTRACT: A set of seven caged gadolinium complexes were used as vectors for introducing the chelated Gd(3+) ion into protein crystals in order to provide strong anomalous scattering for de novo phasing. The complexes contained multidentate ligand molecules with different functional groups to provide a panel of possible interactions with the protein. An exhaustive crystallographic analysis showed them to be nondisruptive to the diffraction quality of the prepared derivative crystals, and as many as 50% of the derivatives allowed the determination of accurate phases, leading to high-quality experimental electron-density maps. At least two successful derivatives were identified for all tested proteins. Structure refinement showed that the complexes bind to the protein surface or solvent-accessible cavities, involving hydrogen bonds, electrostatic and CH-π interactions, explaining their versatile binding modes. Their high phasing power, complementary binding modes and ease of use make them highly suitable as a heavy-atom screen for high-throughput de novo structure determination, in combination with the SAD method. They can also provide a reliable tool for the development of new methods such as serial femtosecond crystallography.
    Acta Crystallographica Section D Biological Crystallography 06/2014; 70(Pt 6):1506-1516. · 12.67 Impact Factor
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    ABSTRACT: Gene expression in bacteria is regulated at the level of transcription initiation, a process driven by σ factors. The regulation of σ factor activity proceeds from the regulation of their cytoplasmic availability, which relies on specific inhibitory proteins called anti-σ factors. With anti-σ factors regulating their availability according to diverse cues, ExtraCytoplasmic Function σ factors (σ(ECF)) form a major signal transduction system in bacteria. Here, structure:function relationships have been characterized in an emerging class of minimal-size, transmembrane anti-σ factors, using CnrY from Cupriavidus metallidurans CH34 as a model. This study reports the 1.75 Å resolution structure of CnrY cytosolic domain in complex with CnrH, its cognate σ(ECF) and identifies a small hydrophobic knob in CnrY as the major determinant of this interaction in vivo. Unsuspected structural similarity with the molecular switch regulating the general stress response in α-proteobacteria unravels a new class of anti-σ factors targeting σ(ECF). Members of this class carry out their function via a 30-residue stretch that displays helical propensity but no canonical structure on its own.
    Journal of Molecular Biology 04/2014; · 3.91 Impact Factor
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    ABSTRACT: Incorporating in a non-covalent manner lanthanide derivatives into protein crystals has shown to be of prime interest for X-ray crystallography, insofar as these versatile compounds can co-crystallize with proteins through supramolecular interactions, in addition to being strong anomalous scatterers for anomalous-based diffraction techniques. In this paper, the selective affinity of tris-dipicolinate lanthanide complexes for cationic amino-acid residues is explored, using a panel of experimental (X-ray diffraction, NMR titration) and theoretical methods that provides access to an accurate description of the interaction process.
    Physical Chemistry Chemical Physics 09/2013; · 4.20 Impact Factor
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    ABSTRACT: New trisdipicolinic acid-lanthanide complexes are reported as phasing agents for X-ray crystallography of proteins. It is demonstrated that CuAAC modifications allow protein co-crystallization with low concentration of lanthanide complexes leading to an accurate structure determination.
    Chemical Communications 11/2012; · 6.38 Impact Factor
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    ABSTRACT: Circumventing radiation damage remains a major problem for X‐ray macromolecular crystallography. Analysis of diffraction data collected from normal‐sized cryocooled lysozyme crystals shows that the dose required to collect a data set of prescribed resolution and signal‐to‐noise ratio, assuming an ideally efficient detector, decreases with increasing photon energy in the investigated 6.5–33 keV range. For example, the data collection efficiency is increased by a factor of ∼8 from 8 to 33 keV. Monte Carlo simulations on lysozyme crystals in the range 5–80 keV, taking into account electron escape from samples of different size, also show a positive effect of high energy (albeit less pronounced than in experiments), especially for micrometre‐sized samples, and suggest that the optimum energy range is ∼24–41 keV, depending on crystal size. The importance of counting pixel detectors with a good efficiency at high energy is underlined. Macromolecular crystallography beamlines should be modified, or purposely designed, in order to benefit from higher‐energy radiation through reduction of global radiation damage, better data accuracy and extension of phasing by anomalous dispersion.
    Journal of Applied Crystallography 08/2012; 45(4). · 3.34 Impact Factor
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    ABSTRACT: Bacterial UDP-sugar dehydrogenases are part of the biosynthesis pathway of extracellular polysaccharides. These compounds act as important virulence factors by protecting the cell from opsonophagocytosis and complement-mediated killing. In Staphylococcus aureus, the protein Cap5O catalyzes the oxidation of UDP-N-acetyl-mannosamine to UDP-N-acetyl-mannosaminuronic acid. Cap5O is crucial for the production of serotype 5 capsular polysaccharide that prevents the interaction of bacteria with both phagocytic and nonphagocytic eukaryotic cells. However, details of its catalytic mechanism remain unknown. We thus crystallized Cap5O and solved the first structure of an UDP-N-acetyl-mannosamine dehydrogenase. This study revealed that the catalytic cysteine makes a disulfide bond that has never been observed in other structurally characterized members of the NDP-sugar dehydrogenase family. Biochemical and mutagenesis experiments demonstrated that the formation of this disulfide bridge regulates the activity of Cap5O. We also identified two arginine residues essential for Cap5O activity. Previous data suggested that Cap5O is activated by tyrosine phosphorylation, so we characterized the phosphorylation site and examined the underlying regulatory mechanism.
    Journal of Biological Chemistry 03/2011; 286(19):17112-21. · 4.65 Impact Factor
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    ABSTRACT: CnrX is the metal sensor and signal modulator of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34 that is involved in the setup of cobalt and nickel resistance. We have determined the atomic structure of the soluble domain of CnrX in its Ni-bound, Co-bound, or Zn-bound form. Ni and Co ions elicit a biological response, while the Zn-bound form is inactive. The structures presented here reveal the topology of intraprotomer and interprotomer interactions and the ability of metal-binding sites to fine-tune the packing of CnrX dimer as a function of the bound metal. These data suggest an allosteric mechanism to explain how the complex is switched on and how the signal is modulated by Ni or Co binding. These results provide clues to propose a model for signal propagation through the membrane in the complex.
    Journal of Molecular Biology 03/2011; 408(4):766-79. · 3.91 Impact Factor
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    ABSTRACT: Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined.
    Journal of Synchrotron Radiation 01/2011; 18(1):31-6. · 2.19 Impact Factor
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    ABSTRACT: Lanthanoid ions exhibit extremely large anomalous X-ray scattering at their L(III) absorption edge. They are thus well suited for anomalous diffraction experiments. A novel class of lanthanoid complexes has been developed that combines the physical properties of lanthanoid atoms with functional chemical groups that allow non-covalent binding to proteins. Two structures of large multimeric proteins have already been determined by using such complexes. Here the use of the luminescent europium tris-dipicolinate complex [Eu(DPA)(3)](3-) to solve the low-resolution structure of a 444 kDa homododecameric aminopeptidase, called PhTET1-12s from the archaea Pyrococcus horikoshii, is reported. Surprisingly, considering the low resolution of the data, the experimental electron density map is very well defined. Experimental phases obtained by using the lanthanoid complex lead to maps displaying particular structural features usually observed in higher-resolution maps. Such complexes open a new way for solving the structure of large molecular assemblies, even with low-resolution data.
    Journal of Synchrotron Radiation 01/2011; 18(1):74-8. · 2.19 Impact Factor
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    ABSTRACT: Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species. The S. ruber enzyme is active at the high physiological concentrations of KCl but, unlike typical halo-adapted enzymes, remains folded and active at low salt concentrations. Structural aspects of the protein, including acidic residues at the surface, solvent-exposed hydrophobic surface, and buried hydrophobic surface, place it between the typical halo-adapted and non-halo-adapted proteins. The enzyme lacks inter-subunit ion-binding sites often seen in halo-adapted enzymes. These observations permit us to suggest an evolutionary pathway that is highlighted by subtle trade-offs to achieve an optimal compromise among solubility, stability, and catalytic activity.
    Journal of Molecular Biology 09/2010; 404(3):493-505. · 3.91 Impact Factor
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    ABSTRACT: Tris-dipicolinate lanthanide complexes were used to prepare derivative crystals of six proteins: hen egg-white lysozyme, turkey egg-white lysozyme, thaumatin from Thaumatococcus daniellii, urate oxidase from Aspergillus flavus, porcine pancreatic elastase and xylanase from Trichoderma reesei. Diffraction data were collected using either synchrotron radiation or X-rays from a laboratory source. In all cases, the complex turned out to be bound to the protein and the phases determined using the anomalous scattering of the lanthanide led to high-quality electron-density maps. The binding mode of the complex was characterized from the refined structures. The lanthanide tris-dipicolinate was found to bind through interactions between carboxylate groups of the dipicolinate ligands and hydrogen-bond donor groups of the protein. In each binding site, one enantiomeric form of the complex is selected from the racemic solution according to the specific site topology. For hen egg-white lysozyme and xylanase, derivative crystals obtained by cocrystallization belonged to a new monoclinic C2 crystal form that diffracted to high resolution.
    Acta Crystallographica Section D Biological Crystallography 07/2010; 66(Pt 7):762-9. · 12.67 Impact Factor
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    ABSTRACT: High-pressure molecular biophysics is a developing field for three main reasons. Pressure has a unique potential, in particular for the exploration of the energy landscape of biomolecules. Progress in instrumentation has extended the range of biophysical techniques under pressure and often relaxed technical constraints on sample confinement. Two high-resolution structural methods are now available at high pressure, NMR and macromolecular crystallography (HPMX). We describe materials and methods of HPMX, now a full-fledged technique taking advantage of purposely-built diamond-anvil cells, ultra-short wavelength synchrotron radiation and improved crystal-loading procedures. KeywordHigh pressure-crystallography-diamond anvil cell-macromolecules
    06/2010: pages 591-601;
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    ABSTRACT: A new pneumatic diamond anvil cell has been constructed, generating continuous pressure and temperature variations in the range 0–2.5 GPa and 293–393 K. The cell is designed mainly for high-pressure macromolecular crystallography and should facilitate pressure and temperature annealing of the sample. The analysis is reported of several diffraction data sets of tetragonal hen egg-white lysozyme crystals loaded either in the new cell or in a currently used membrane-based cell. These experiments were performed on beamline FIP-BM30A at the ESRF, Grenoble, a macromolecular crystallography beamline on a bending magnet. Cells were handled and automatically centred by a six-axis robotic arm that was used as a goniometer for data collection by the oscillation method.
    Journal of Applied Crystallography 06/2010; 43(4):762-768. · 3.34 Impact Factor
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    ABSTRACT: The 2 A resolution crystal structure of bovine erythrocyte Cu,Zn superoxide dismutase (CuZnSOD) has been determined by X-ray diffraction at high pressure (0.57 GPa) and room temperature. At 0.57 GPa the secondary, tertiary and quaternary structures are similar to other previously determined bovine erythrocyte CuZnSOD structures. Nevertheless, pressure has a localized impact on the atomic coordinates of C(alpha) atoms and on side chains. The compression of the crystal and of the protein backbone is anisotropic. This anisotropy is discussed, taking into account intermolecular contacts and protein conformation. Pressure perturbation highlights the more flexible zones in the protein such as the electrostatic loop. At 0.57 GPa, a global shift of the dimetallic sites in both subunits and changes in the oxidation state of Cu were observed. The flexibility of the electrostatic loop may be useful for the interaction of different metal carriers in the copper-uptake process, whereas the flexibility of the metal sites involved in the activity of the protein could contribute to explaining the ubiquitous character of CuZnSODs, which are found in organisms living in very different conditions, including the deep-sea environment. This work illustrates the potential of combining X-ray crystallography with high pressure to promote and stabilize higher energy conformational substates.
    Acta Crystallographica Section D Biological Crystallography 06/2010; 66(Pt 6):654-63. · 12.67 Impact Factor
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    ABSTRACT: Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.
    Biophysical Journal 05/2010; 98(10):2365-73. · 3.67 Impact Factor
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    ABSTRACT: Different polymorphs of rasburicase, a recombinant urate oxidase enzyme (Uox) from Aspergillus flavus, were obtained as a series of polycrystalline precipitates. Different crystallization protocols were followed in which the salt type, pH and polyethylene glycol 8000 (PEG 8000) concentration were varied. The related crystalline phases were characterized by means of high-resolution synchrotron X-ray powder diffraction. In all cases, Uox complexed with the inhibitor 8-azaxanthine (AZA) was not altered from its robust orthorhombic I222 phase by variation of any of the factors listed above. However, in the absence of AZA during crystallization ligand-free Uox was significantly affected by the type of salt, resulting in different crystal forms for the four salts tested: sodium chloride, potassium chloride, ammonium chloride and ammonium sulfate. Remarkable alterations of some of these phases were observed upon gradual increase of the exposure time of the sample to the synchrotron beam in addition to variation of the PEG 8000 concentration. When Uox was crystallized in Tris buffer or pure water in the absence of salt, a distinct polymorph of orthorhombic symmetry (P2(1)2(1)2) was obtained that was associated with significantly altered lattice dimensions in comparison to a previously reported isosymmetrical structure. The latter form of Uox exhibits enhanced stability to variation of pH and PEG 8000 concentration accompanied by minor modifications of the unit-cell dimensions in the ranges under study. Accurate lattice parameters were extracted for all crystalline phases. This study reveals the rich phase diagram of Uox, a protein of high pharmaceutical importance, which is associated with an enhanced degree of polymorphism. The outcome of our analysis verifies previously reported results as well as demonstrating polymorphs that have altered unit-cell dimensions with respect to known structural models.
    Acta Crystallographica Section D Biological Crystallography 05/2010; 66(Pt 5):539-48. · 12.67 Impact Factor
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    ABSTRACT: Modern macromolecular crystallography has the potential, together with NMR, to provide an accurate description of structural changes produced by pressure in a macromolecular system. In particular, promoting and trapping higher-energy conformations of biological significance is of particular interest to explore functional mechanisms. An overview of this technique is given, including instrumentation and selected applications. This method is now full-fledged and would deserve more interest from the structural biology community.
    High Pressure Research 04/2010; 30:100-103. · 0.90 Impact Factor
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    ABSTRACT: CzcE is a periplasmic protein from Cupriavidus metallidurans CH34 that can bind four copper atoms per dimer. We have crystallized the apo form of the protein and determined its structure at 1.85 A resolution. Three Cu atoms were localized by soaking apo-CzcE crystals into a CuCl(2) solution. We identified His24 as a Cu(II) ligand in each protomer and Asp100 as a key residue for Cu binding at the interface of the dimer. The role of these amino acids was confirmed by site-directed mutagenesis and UV-visible spectroscopy. The fourth Cu atom was not located. The oxidized form of CzcE contains four Cu(II) atoms, while the reduced form contains four Cu(I) atoms. Average coordination spheres of four N or O atoms for Cu(II) and of one N or O atom and two S atoms for Cu(I) were determined by X-ray absorption spectroscopy. As there is no evidence for preformed metal-binding sites in apo-CzcE, we suggest that different conformational changes occurred upon Cu(II) or Cu(I) binding. These changes were further demonstrated by digestion experiments that gave different proteolysis patterns depending not only on the presence of the metal but also on its speciation. The ability of CzcE to bind copper and to adapt its conformation to different copper oxidation states could be related to a role in copper sensing for this protein.
    Biochemistry 03/2010; 49(9):1913-22. · 3.38 Impact Factor
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    ABSTRACT: Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr. For further information see http://journals.iucr.org/services/authorrights.html Many research topics in condensed matter research, materials science and the life sci-ences make use of crystallographic methods to study crystalline and non-crystalline mat-ter with neutrons, X-rays and electrons. Articles published in the Journal of Applied Crys-tallography focus on these methods and their use in identifying structural and diffusion-controlled phase transformations, structure–property relationships, structural changes of defects, interfaces and surfaces, etc. Developments of instrumentation and crystallo-graphic apparatus, theory and interpretation, numerical analysis and other related sub-jects are also covered. The journal is the primary place where crystallographic computer program information is published. The compressibility of several nucleic acid and globular protein crystals has been investigated by high-pressure macromolecular crystallography. Further, crystal structures at four different pressures allowed the determination of the intrinsic compressibility versus pressure of d(GGTATACC) 2 and hen egg-white lysozyme. For lysozyme, the values for the intrinsic molecular compressibility at atmospheric pressure and the nonlinearity index were 0.070 GPa À1 and 8.15, respectively. On the basis of two crystal structures at atmospheric and high pressure, similar, albeit less complete, information was derived for d(CGCGAATTCGCG) 2 and bovine erythrocyte Cu,Zn superoxide dismutase. Using these data and accurate calculations of the solvent-excluded volume, the apparent solvent compressibility in the crystalline state was determined as a function of pressure and compared with results from a simple model that assumes invariant unit-cell content, with the conclusion that solvent compres-sibility was abnormal for three out of the five crystals investigated. Experimental results suggest that macromolecular crystals submitted to high pressure may have a variable unit-cell mass due to solvent exchange with the surrounding pool, as already observed in other hydrated crystals such as zeolites.
    Journal of Applied Crystallography 01/2010; 43(43):407-416. · 3.34 Impact Factor

Publication Stats

1k Citations
537.89 Total Impact Points

Institutions

  • 2014
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 1970–2014
    • Institut de Biologie Structurale (IBS)
      Lutetia Parisorum, Île-de-France, France
  • 2011–2012
    • Cea Leti
      Grenoble, Rhône-Alpes, France
  • 2005–2011
    • SOLEIL synchrotron
      Gif, Île-de-France, France
  • 2010
    • Pierre and Marie Curie University - Paris 6
      Lutetia Parisorum, Île-de-France, France
  • 2005–2009
    • Institute of Physical Chemistry Rocasolano
      Madrid, Madrid, Spain
  • 2006
    • Université de Caen Basse-Normandie
      Caen, Lower Normandy, France
  • 1978–2000
    • Université Paris-Sud 11
      Orsay, Île-de-France, France
    • Université Paris-Est Créteil Val de Marne - Université Paris 12
      Créteil, Île-de-France, France
  • 1977–1982
    • CERN
      Genève, Geneva, Switzerland