Zbigniew Dauter

Molecular basis of agonism and antagonism in the oestrogen receptor.

Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery,. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17β-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 Å, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.

Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease

Chitin, the second most abundant polysaccharide on earth, is degraded by chitinases and chitobiases. The structure of Serratia marcescens chitobiase has been refined at 1.9 Å resolution. The mature protein is folded into four domains and its active site is situated at the C-terminal end of the central (βα)8-barrel. Based on the structure of the complex with the substrate disaccharide chitobiose, we propose an acid-base reaction mechanism, in which only one protein carboxylate acts as catalytic acid, while the nucleophile is the polar acetamido group of the sugar in a substrate-assisted reaction. The structural data lead to the hypothesis that the reaction proceeds with retention of anomeric configuration. The structure allows us to model the catalytic domain of the homologous hexosaminidases to give a structural rationale to pathogenic mutations that underlie Tay–Sachs and Sandhoff disease.

Novel approach to phasing proteins: derivatization by short cryo-soaking with halides

A quick (less than 1 min) soak of protein crystals in a cryo-solution containing bromide or iodide anions leads to incorporation of these anomalous scatterers into the ordered solvent region around the protein molecules. These halide anions provide a convenient way of phasing through their anomalous scattering signal: bromides using multiwavelength anomalous dispersion (MAD) and bromides and/or iodides using single-wavelength anomalous dispersion (SAD) or single isomorphous replacement with anomalous scattering (SIRAS) methods. This approach has been tested successfully on four different proteins and has been used to solve the structure of a new protein of molecular weight 30 kDa.

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

The majority of structural efforts addressing RNAs catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a λ-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures

The number of macromolecular structures deposited in the Protein Data Bank now exceeds 45 000, with the vast majority determined using crystallographic methods. Thousands of studies describing such structures have been published in the scientific literature, and 14 Nobel prizes in chemistry or medicine have been awarded to protein crystallographers. As important as these structures are for understanding the processes that take place in living organisms and also for practical applications such as drug design, many non‐crystallographers still have problems with critical evaluation of the structural literature data. This review attempts to provide a brief outline of technical aspects of crystallography and to explain the meaning of some parameters that should be evaluated by users of macromolecular structures in order to interpret, but not over‐interpret, the information present in the coordinate files and in their description. A discussion of the extent of the information that can be gleaned from the coordinates of structures solved at different resolution, as well as problems and pitfalls encountered in structure determination and interpretation are also covered.

Novel fold and capsid-binding properties of the |[lambda]|-phage display platform protein gpD

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage λ, was solved at 1.1 Å resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0.132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 Å resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

The Crystal Structure of a Major Dust Mite Allergen Der p 2, and its Biological Implications

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.

Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme

Here we report the solution and refinement at 1.9 Å resolution of the crystal structure of the Escherichia coli medium chain length acyl-CoA thioesterase II. This enzyme is a close homolog of the human protein that interacts with the product of the HIV-1 Nef gene, sharing 45% amino acid sequence identity with it. The structure of the E. coli thioesterase II reveals a new tertiary fold, a ‘double hot dog’, showing an internal repeat with a basic unit that is structurally similar to the recently described β-hydroxydecanoyl thiol ester dehydrase. The catalytic site, inferred from the crystal structure and verified by site directed mutagenesis, involves novel chemistry and includes Asp 204, Gln 278 and Thr 228, which synergistically activate a nucleophilic water molecule.

Data‐collection strategies

The optimal strategy for collecting X-ray diffraction data from macromolecular crystals is discussed. Two kinds of factors influencing the completeness of data are considered. The first are geometric, arising from the symmetry of the reciprocal lattice and from the experimental setup; they affect quantitatively the completeness of the measured set of reflections. The second concern the quality, or information content, of the recorded intensities of these measured reflections.

Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members.

414 nature structural biology ¥ volume 6 number 5 ¥ may 1999 Two enzyme types, penicillin V acylases (PVA) and penicillin G acylases (PGA), with distinct substrate preferences, account for all the enzymic industrial production of 6-aminopenicillanic acid 1,2. This b-lactam compound is then elaborated into a range of semi-synthetic penicillins. Although their industrial substrates are very similar, representative examples of the two enzyme types differ widely in molecular properties. PVA from Bacillus sphaericus is tetrameric with a monomer M r of 35,000 while PGA from Escherichia coli is a heterodimer of M r 90,000. Furthermore, they have no detectable sequence homology. These differences, which exist in spite of the similarity of their industrial substrates, provoked us to determine the crystal structure of PVA to establish the nature of its catalytic mechanism and to identify any biochemical and structural relationships with PGA and other Ntn (N-terminal nucleophile) hydrolases. The PVA molecule is a well-defined tetramer with 222 organization made up of two obvious dimers (A and D) and (B and C), which generate a flat disc-like assembly (Fig. 1a). The X-ray analysis revealed that the PVA monomer contains two central anti-parallel b-sheets above and below which is a pair of anti-parallel helices (Fig. 1b). There are two extensions , one from the upper pair of helices and the other at the C-terminal segment, that interact with other monomers in the tetramer and help stabilize it. The b-sheet and helix organization and connectivity are characteristic of members of the Ntn hydrolase family, which have an N-terminal catalytic residue that is often created by autocatalytic processing 3,4. In the PVA structure, cysteine was observed as the N-terminal residue, whereas the gene sequence predicts an N-terminal sequence of Met-Leu-Gly-Cys 5. This finding shows that three amino acids are processed from the precursor N-terminus to unmask a nucleophile with a free a-amino group. Since PVA is an Ntn hydro-lase, we can deduce that the N-terminal cysteine in PVA is the catalytic residue. The PVA and PGA enzymes thus share a distinctive structural core but are otherwise unrelated in primary sequence, including the active site residue. Both PGA and PVA have approximately the same angle (+30°) between the b-strands of the two b-sheets, which are decorated by the active site residues in Ntn hydro-lases. Using these b-sheets for structural alignment reveals that the catalytic regions of PVA and PGA overlap (Fig. 1c) with a root …