Mycobacterium abscessus l,d-Transpeptidases Are Susceptible to Inactivation by Carbapenems and Cephalosporins but Not Penicillins.

As a growing number of clinical isolates of Mycobacterium abscessus are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of M. abscessus are synthesized by nonclassical transpeptidases, namely, the l,d-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of ß-lactams determines their activity against Mycobacterium tuberculosis Here, we studied the interactions of ß-lactams with two l,d-transpeptidases in M. abscessus, namely, LdtMab1 and LdtMab2, and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of ß-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with ß-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible M. abscessus and clinical isolates that are resistant to most antibiotics, which suggests that dual-ß-lactam therapy has potential for the treatment of M. abscessus Finally, we solved the first crystal structure of an M. abscessus l,d-transpeptidase, LdtMab2, and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and ß-lactams, which provide an insight into the molecular basis for the relative efficacy of different ß-lactams against M. abscessus.

Non-classical transpeptidases yield insight into new antibacterials.

Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the ß-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of ß-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for ß-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A ß-Lactamase with a Monobactam Substrate.

The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum ß-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M ß-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum ß-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A ß-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

Exploring the Mechanism of ß-Lactam Ring Protonation in the Class A ß-lactamase Acylation Mechanism Using Neutron and X-ray Crystallography.

The catalytic mechanism of class A ß-lactamases is often debated due in part to the large number of amino acids that interact with bound ß-lactam substrates. The role and function of the conserved residue Lys 73 in the catalytic mechanism of class A type ß-lactamase enzymes is still not well understood after decades of scientific research. To better elucidate the functions of this vital residue, we used both neutron and high-resolution X-ray diffraction to examine both the structures of the ligand free protein and the acyl-enzyme complex of perdeuterated E166A Toho-1 ß-lactamase with the antibiotic cefotaxime. The E166A mutant lacks a critical glutamate residue that has a key role in the deacylation step of the catalytic mechanism, allowing the acyl-enzyme adduct to be captured for study. In our ligand free structures, Lys 73 is present in a single conformation, however in all of our acyl-enzyme structures, Lys 73 is present in two different conformations, in which one conformer is closer to Ser 70 while the other conformer is positioned closer to Ser 130, which supports the existence of a possible pathway by which proton transfer from Lys 73 to Ser 130 can occur. This and further clarifications of the role of Lys 73 in the acylation mechanism may facilitate the design of inhibitors that capitalize on the enzyme's native machinery.

The structure of Toho1 ß-lactamase in complex with penicillin reveals the role of Tyr105 in substrate recognition.

The role of the conserved residue Tyr105 in class A ß-lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 ß-lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring-opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site. In addition to the trapped penicillin, however, are two additional intact ring-closed penicillin molecules, captured by the enzyme through noncovalent interactions at the edge of the active site.

Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography.

A practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy.

The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using ß2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.

The absence of tertiary interactions in a self-assembled DNA crystal structure.

DNA is a highly effective molecule for controlling nanometer-scale structure. The convenience of using DNA lies in the programmability of Watson-Crick base-paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky-ended cohesion. Recently, the tensegrity triangle motif has been used to self-assemble three-dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine-PO(4) tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation.

From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal.

We live in a macroscopic three-dimensional (3D) world, but our best description of the structure of matter is at the atomic and molecular scale. Understanding the relationship between the two scales requires a bridge from the molecular world to the macroscopic world. Connecting these two domains with atomic precision is a central goal of the natural sciences, but it requires high spatial control of the 3D structure of matter. The simplest practical route to producing precisely designed 3D macroscopic objects is to form a crystalline arrangement by self-assembly, because such a periodic array has only conceptually simple requirements: a motif that has a robust 3D structure, dominant affinity interactions between parts of the motif when it self-associates, and predictable structures for these affinity interactions. Fulfilling these three criteria to produce a 3D periodic system is not easy, but should readily be achieved with well-structured branched DNA motifs tailed by sticky ends. Complementary sticky ends associate with each other preferentially and assume the well-known B-DNA structure when they do so; the helically repeating nature of DNA facilitates the construction of a periodic array. It is essential that the directions of propagation associated with the sticky ends do not share the same plane, but extend to form a 3D arrangement of matter. Here we report the crystal structure at 4 A resolution of a designed, self-assembled, 3D crystal based on the DNA tensegrity triangle. The data demonstrate clearly that it is possible to design and self-assemble a well-ordered macromolecular 3D crystalline lattice with precise control.

The many faces of radiation-induced changes.

During diffraction experiments even cryo-cooled protein crystals can be significantly damaged due to chemical and physical changes induced by absorbed X-ray photons. The character and scale of the observed effects depend strongly on the temperature and the composition of crystals. The absorption of radiation energy results in incremental regular changes to the crystal structure, making its impact on the process of solving the structure strongly correlated with other experimental variables. An understanding of all the dependencies is still limited and does not allow for a precise prediction of the outcome of a particular diffraction experiment. Results are presented of diffraction experiments performed under different experimental conditions. The influence of temperature and crystal composition on different characteristics of radiation damage is analyzed. The observed effects are discussed in terms of their impact on data processing and phasing procedures.

The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results.

The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5-0.6 A wavelength) with fluxes up to 8-18 x 10(12) photons s(-1) (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-maximum) at the sample position can be varied from 2.2 mm x 1.0 mm (horizontal x vertical, unfocused) to 0.083 mm x 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a kappa-geometry goniometer and beamline control software allows optimal strategies to be adopted in protein crystallographic experiments, thus maximizing the chances of their success. A large-area mosaic 3 x 3 CCD detector allows high-quality diffraction data to be measured rapidly to the crystal diffraction limits. The beamline layout and the X-ray optical and endstation components are described in detail, and the results of representative crystallographic experiments are presented.

Temperature and cryoprotectant influence secondary quinone binding position in bacterial reaction centers.

We have determined the first de novo position of the secondary quinone QB in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, QB occupies primarily the proximal binding site. In contrast, our room temperature structure showed that QB is largely in the distal position. Both data sets were collected in dark-adapted conditions. We estimate that the occupancy of the QB site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far-reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.