Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.

Mycobacterium tuberculosis TMP kinase (TMPK(Mtub)) represents a promising target for developing drugs against tuberculosis because the configuration of its active site is unique in the TMPK family. To help elucidate the phosphorylation mechanism employed by this enzyme, structural changes occurring upon binding of substrates and subsequent catalysis were investigated by protein crystallography. Six new structures of TMPK(Mtub) were solved at a resolution better than 2.3A, including the first structure of an apo-TMPK, obtained by triggering catalysis in a crystal of a TMPK(Mtub)-TMP complex, which resulted in the release of the TDP product. A series of snapshots along the reaction pathway is obtained, revealing the closure of the active site in going from an empty to a fully occupied state, suggestive of an induced-fit mechanism typical of NMPKs. However, in TMPK(Mtub) the LID closure couples to the binding with an unusual location for a magnesium ion coordinating TMP in the active site. Our data suggest strongly that this ion is required for catalysis, acting as a clamp, possibly in concert with Arg95, to neutralise electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions. The 3'-hydroxyl moiety of TMP, critical to metal stabilisation, appears to be a target of choice for the design of potent inhibitors. On the other hand, the usual NTP-bound magnesium is not seen in our structures and Arg14, a P-loop residue unique to TMPK(Mtub), may take over its role. Therefore, TMPK(Mtub) seems to have swapped the use of a metal ion as compared with e.g. human TMPK. Finally, TTP was observed in crystals of TMPK(Mtub), locked by Arg14, thus providing a structural explanation for the observed inhibitory effect of TTP putatively involved in a mechanism of feedback regulation of the enzymatic activity.

A molecular movie at 1.8 A resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds.

The photocycle of the bacterial blue-light photoreceptor, photoactive yellow protein, was stimulated by illumination of single crystals by a 7 ns laser pulse. The molecular events were recorded at high resolution by time-resolved X-ray Laue diffraction as they evolved in real time, from 1 ns to seconds after the laser pulse. The complex structural changes during the photocycle at ambient temperature are displayed in a movie of difference electron density maps relative to the dark state. The step critical to entry into the photocycle is identified as flipping of the carbonyl group of the 4-hydroxycinnamic acid chromophore into an adjacent, hydrophobic environment rather than the concomitant isomerization about the double bond of the chromophore tail. The structural perturbation generated at the chromophore propagates throughout the entire protein as a light-induced "protein quake" with its "epicenter" at the carbonyl moiety of the chromophore.

Protein conformational relaxation and ligand migration in myoglobin: a nanosecond to millisecond molecular movie from time-resolved Laue X-ray diffraction.

A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 micros X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation involving the E and F helices is evident from the data at a 1 ns time delay. The photodissociated CO molecules are detected at two locations: at a distal pocket docking site and at the Xe 1 binding site in the proximal pocket. The population by CO of the primary, distal site peaks at a 1 ns time delay and decays to half the peak value in 70 ns. The secondary, proximal docking site reaches its highest occupancy of 20% at approximately 100 ns and has a half-life of approximately 10 micros. At approximately 100 ns, all CO molecules are accounted for within the protein: in one of these two docking sites or bound to the heme. Thereafter, the CO molecules migrate to the solvent from which they rebind to deoxymyoglobin in a bimolecular process with a second-order rate coefficient of 4.5 x 10(5) M(-1) s(-1). Our results also demonstrate that structural changes as small as 0.2 A and populations of CO docking sites of 10% can be detected by time-resolved X-ray diffraction.

Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue. Cloning, sequencing and expression of the gene coding for Sulfolobus solfataricus superoxide dismutase.

The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al-Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol. 286, 189--205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH(2)SO(2)F), a protease inhibitor routinely added during the preparation of cell-free extracts, showed that the modification was caused by PhCH(2)SO(2)F. Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH(2)SO(2)F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD.

Spectroscopic characterization of bacteriorhodopsin's L-intermediate in 3D crystals cooled to 170 K.

Spectra are presented from a single 3D microcrystal of bacteriorhodopsin (bR) cooled to 170 K under various illumination conditions. This set is necessary and sufficient to assign the relevant crystal reference spectra. A spectral decomposition of the difference spectrum obtained following the trapping protocol of Royant et al. (2000) (Nature 406, 645-648) is given, confirming that the low temperature L-intermediate was the species that dominated the structural rearrangements previously reported. Smaller contributions from the K and M spectral intermediates are also quantified. Mechanistic insights derived from the X-ray structures of the early bR intermediates are discussed.

Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin.

A wide variety of mechanisms are used to generate a proton-motive potential across cell membranes, a function lying at the heart of bioenergetics. Bacteriorhodopsin, the simplest known proton pump, provides a paradigm for understanding this process. Here we report, at 2.1 A resolution, the structural changes in bacteriorhodopsin immediately preceding the primary proton transfer event in its photocycle. The early structural rearrangements propagate from the protein's core towards the extracellular surface, disrupting the network of hydrogen-bonded water molecules that stabilizes helix C in the ground state. Concomitantly, a bend of this helix enables the negatively charged primary proton acceptor, Asp 85, to approach closer to the positively charged primary proton donor, the Schiff base. The primary proton transfer event would then neutralize these two groups, cancelling their electrostatic attraction and facilitating a relaxation of helix C to a less strained geometry. Reprotonation of the Schiff base by Asp 85 would thereby be impeded, ensuring vectorial proton transport. Structural rearrangements also occur near the protein's surface, aiding proton release to the extracellular medium.

The crystallography beamline I711 at MAX II.

A new X-ray crystallographic beamline is operational at the MAX II synchrotron in Lund. The beamline has been in regular use since August 1998 and is used both for macro- and small molecule diffraction as well as powder diffraction experiments. The radiation source is a 1.8 T multipole wiggler. The beam is focused vertically by a bendable mirror and horizontally by an asymmetrically cut Si(111) monochromator. The wavelength range is 0.8-1.55 A with a measured flux at 1 A of more than 10(11) photons s(-1) in 0.3 mm x 0.3 mm at the sample position. The station is currently equipped with a Mar345 imaging plate, a Bruker Smart 1000 area CCD detector and a Huber imaging-plate Guinier camera. An ADSC 210 area CCD detector is planned to be installed during 2000.

Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: analysis of structure and thermostability.

The crystal structure of superoxide dismutase (SOD) from the hyper thermophile Sulfolobus solfataricus has been determined at 2.3 A resolution by molecular replacement and refined to a crystallographic R-factor of 16.8 % (Rfree 19.8 %). The crystals belong to the space group C2 (a=76.3 A, b=124.3 A, c=60.3 A, beta=128.8 degrees) with two identical monomers in the asymmetric unit. The monomer has a molecular weight of 24 kDa and consists of 210 amino acid residues of which 205 are visible in the electron density map. The overall fold of the monomer of S. solfataricus SOD is similar to that of the other known Fe or Mn-SODs. S. solfataricus SOD forms a very compact tetramer of a type similar to that of SOD from the hyperthermophile Aquifex pyrophilus. Both structures show an elevated number of inter-subunit ion-pairs compared with the mesophilic SOD from Mycobacterium tuberculosis and the thermophilic SOD from Thermus thermophilus. However, in contrast to the A. pyrophilus SOD structure, the number of intra-subunit ion-pairs as well as inter- subunit hydrogen bonds is not higher than in the compared mesophilic and thermophilic SOD structures. The electron density also revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site. Its involvement in the specific activity of the enzyme is discussed.

Energy transduction on the nanosecond time scale: early structural events in a xanthopsin photocycle.

Photoactive yellow protein (PYP) is a member of the xanthopsin family of eubacterial blue-light photoreceptors. On absorption of light, PYP enters a photocycle that ultimately transduces the energy contained in a light signal into an altered biological response. Nanosecond time-resolved x-ray crystallography was used to determine the structure of the short-lived, red-shifted, intermediate state denoted [pR], which develops within 1 nanosecond after photoelectronic excitation of the chromophore of PYP by absorption of light. The resulting structural model demonstrates that the [pR] state possesses the cis conformation of the 4-hydroxyl cinnamic thioester chromophore, and that the process of trans to cis isomerization is accompanied by the specific formation of new hydrogen bonds that replace those broken upon excitation of the chromophore. Regions of flexibility that compose the chromophore-binding pocket serve to lower the activation energy barrier between the dark state, denoted pG, and [pR], and help initiate entrance into the photocycle. Direct structural evidence is provided for the initial processes of transduction of light energy, which ultimately translate into a physiological signal.

Photolysis of the carbon monoxide complex of myoglobin: nanosecond time-resolved crystallography.

The biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.

Feasibility and Realization of Single-Pulse Laue Diffraction on Macromolecular Crystals at ESRF.

Laue diffraction patterns with an exposure time of ca 60 ps have been acquired at the European Synchrotron Radiation Facility (ESRF) on protein crystals by using the single-bunch mode of the storage ring. A 10 ns laser pulse initiating photodissociation was synchronized with the X-ray pulse. The potential for a quantitative detection of conformational changes in proteins on the nanosecond timescale with this technique is demonstrated using the example of carbonmonoxymyoglobin, from simulations and real data. The instrumental aspects of the experiment (highly intense X-ray beam, fast shutter system, Laue camera, detector, laser apparatus and synchronization technique) are emphasized.