New Conformations of Acylation Adducts of Inhibitors of ß-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to ß-lactam antibiotics due to the production of the extended spectrum ß-lactamase BlaC. ß-Lactam/ß-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the ß-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other ß-lactamases are discussed.

Structure of ScpC, a virulence protease from Streptococcus pyogenes, reveals the functional domains and maturation mechanism.

Group A Streptococcus (GAS; Streptococcus pyogenes) causes a wide range of infections, including pharyngitis, impetigo, and necrotizing fasciitis, and results in over half a million deaths annually. GAS ScpC (SpyCEP), a 180-kDa surface-exposed, subtilisin-like serine protease, acts as an essential virulence factor that helps S. pyogenes evade the innate immune response by cleaving and inactivating C-X-C chemokines. ScpC is thus a key candidate for the development of a vaccine against GAS and other pathogenic streptococcal species. Here, we report the crystal structures of full-length ScpC wild-type, the inactive mutant, and the ScpC-AEBSF inhibitor complex. We show ScpC to be a multi-domain, modular protein consisting of nine structural domains, of which the first five constitute the PR + A region required for catalytic activity. The four unique C-terminal domains of this protein are similar to collagen-binding and pilin proteins, suggesting an additional role for ScpC as an adhesin that might mediate the attachment of S. pyogenes to various host tissues. The Cat domain of ScpC is similar to subtilisin-like proteases with significant difference to dictate its specificity toward C-X-C chemokines. We further show that ScpC does not undergo structural rearrangement upon maturation. In the ScpC-inhibitor complex, the bound inhibitor breaks the hydrogen bond between active-site residues, which is essential for catalysis. Guided by our structure, we designed various epitopes and raised antibodies capable of neutralizing ScpC activity. Collectively, our results demonstrate the structure, maturation process, inhibition, and substrate recognition of GAS ScpC, and reveal the presence of functional domains at the C-terminal region.

Ankyrin repeats as a dimerization module.

Legionella pneumophila is a pathogen, causing severe pneumonia in humans called Legionnaires' disease. AnkC (LegA12) is a poorly characterized 495-residue effector protein conserved in multiple Legionella species. Here, we report the crystal structure of a C-terminally truncated AnkC (2-384) at 3.2 Å resolution. The structure shows seven ankyrin repeats (ARs) with unique structural features. AnkC forms a dimer along the outer surface of loops between ARs. The dimer exists both in the crystal form and in solution, as shown by analytical ultracentrifugation. This is the first example of ARs as a dimerization module as opposed to solely a protein interaction domain. In addition, a novel α-helix insert between AR3-AR4 is positioned across the surface opposite the ankyrin groove. Sequence conservation suggests that the ankyrin groove of AnkC is a functional site that interacts with binding targets. This ankyrin domain structure is an important step towards a functional characterization of AnkC.

Phosphate Promotes the Recovery of Mycobacterium tuberculosis ß-Lactamase from Clavulanic Acid Inhibition.

The rise of multi- and even totally antibiotic resistant forms of Mycobacterium tuberculosis underlines the need for new antibiotics. The pathogen is resistant to ß-lactam compounds due to its native serine ß-lactamase, BlaC. This resistance can be circumvented by administration of a ß-lactamase inhibitor. We studied the interaction between BlaC and the inhibitor clavulanic acid. Our data show hydrolysis of clavulanic acid and recovery of BlaC activity upon prolonged incubation. The rate of clavulanic acid hydrolysis is much higher in the presence of phosphate ions. A specific binding site for phosphate is identified in the active site pocket, both in the crystalline state and in solution. NMR spectroscopy experiments show that phosphate binds to this site with a dissociation constant of 30 mM in the free enzyme. We conclude that inhibition of BlaC by clavulanic acid is reversible and that phosphate ions can promote the hydrolysis of the inhibitor.

Structural and functional characterization of the alanine racemase from Streptomyces coelicolor A3(2).

The conversion of l-alanine (L-Ala) into d-alanine (D-Ala) in bacteria is performed by pyridoxal phosphate-dependent enzymes called alanine racemases. D-Ala is an essential component of the bacterial peptidoglycan and hence required for survival. The Gram-positive bacterium Streptomyces coelicolor has at least one alanine racemase encoded by alr. Here, we describe an alr deletion mutant of S. coelicolor which depends on D-Ala for growth and shows increased sensitivity to the antibiotic d-cycloserine (DCS). The crystal structure of the alanine racemase (Alr) was solved with and without the inhibitors DCS or propionate, at 1.64 Å and 1.51 Å resolution, respectively. The crystal structures revealed that Alr is a homodimer with residues from both monomers contributing to the active site. The dimeric state of the enzyme in solution was confirmed by gel filtration chromatography, with and without L-Ala or d-cycloserine. The activity of the enzyme was 66 ± 3 U mg-1 for the racemization of L- to D-Ala, and 104 ± 7 U mg-1 for the opposite direction. Comparison of Alr from S. coelicolor with orthologous enzymes from other bacteria, including the closely related d-cycloserine-resistant Alr from S. lavendulae, strongly suggests that structural features such as the hinge angle or the surface area between the monomers do not contribute to d-cycloserine resistance, and the molecular basis for resistance therefore remains elusive.

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular ß-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ß-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-ß-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through ß-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

UV damage endonuclease employs a novel dual-dinucleotide flipping mechanism to recognize different DNA lesions.

Repairing damaged DNA is essential for an organism's survival. UV damage endonuclease (UVDE) is a DNA-repair enzyme that can recognize and incise different types of damaged DNA. We present the structure of Sulfolobus acidocaldarius UVDE on its own and in a pre-catalytic complex with UV-damaged DNA containing a 6-4 photoproduct showing a novel 'dual dinucleotide flip' mechanism for recognition of damaged dipyrimidines: the two purines opposite to the damaged pyrimidine bases are flipped into a dipurine-specific pocket, while the damaged bases are also flipped into another cleft.

Multivariate estimation of substructure amplitudes for a single-wavelength anomalous diffraction experiment.

To determine a substructure from single-wavelength anomalous diffraction (SAD) data using Patterson or direct methods, the substructure-factor amplitude (|Fa|) is first estimated. Currently, the absolute value of the Bijvoet difference is widely used as an estimate of |Fa| values for SAD data. Here, an equation is derived from multivariate statistics and tested that takes into account the correlation between the observed positive (F+) and negative (F-) Friedel pairs and Fa along with measurement errors in the observed data. The multivariate estimation of |Fa| has been implemented in a new program, Afro. Results on over 180 test cases show that Afro provides a higher correlation to the final substructure-factor amplitudes (calculated from the refined, final substructures) than the Bijvoet differences and improves the robustness of direct-methods substructure detection.

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato.

Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a ß-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.

Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1.

Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility.

CCP4 Cloud for structure determination and project management in macromolecular crystallography.

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links between experimental observations and the final results deposited in the PDB. In this article, CCP4 Cloud, a new front-end of the CCP4 software suite, is presented which mitigates these effects by providing an online, cloud-based environment for crystallographic computation. CCP4 Cloud was developed for the efficient delivery of computing power, database services and seamless integration with web resources. It provides a rich graphical user interface that allows project sharing and long-term storage for structure-solution projects, and can be linked to data-producing facilities. The system is distributed with the CCP4 software suite version 7.1 and higher, and an online publicly available instance of CCP4 Cloud is provided by CCP4.

Reduction of density-modification bias by ß correction.

Density modification often suffers from an overestimation of phase quality, as seen by escalated figures of merit. A new cross-validation-based method to address this estimation bias by applying a bias-correction parameter 'ß' to maximum-likelihood phase-combination functions is proposed. In tests on over 100 single-wavelength anomalous diffraction data sets, the method is shown to produce much more reliable figures of merit and improved electron-density maps. Furthermore, significantly better results are obtained in automated model building iterated with phased refinement using the more accurate phase probability parameters from density modification.

Recent advances in the CRANK software suite for experimental phasing.

For its first release in 2004, CRANK was shown to effectively detect and phase anomalous scatterers from single-wavelength anomalous diffraction data. Since then, CRANK has been significantly improved and many more structures can be built automatically with single- or multiple-wavelength anomalous diffraction or single isomorphous replacement with anomalous scattering data. Here, the new algorithms that have been developed that have led to these substantial improvements are discussed and CRANK's performance on over 100 real data sets is shown. The latest version of CRANK is freely available for download at http://www.bfsc.leidenuniv.nl/software/crank/ and from CCP4 (http://www.ccp4.ac.uk/).

REFMAC5 for the refinement of macromolecular crystal structures.

This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Šcan be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, `jelly-body' restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.

Overview of the CCP4 suite and current developments.

The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.

Multivariate phase combination improves automated crystallographic model building.

Density modification is a standard technique in macromolecular crystallography that can significantly improve an initial electron-density map. To obtain optimal results, the initial and density-modified map are combined. Current methods assume that these two maps are independent and propagate the initial map information and its accuracy indirectly through previously determined coefficients. A multivariate equation has been derived that no longer assumes independence between the initial and density-modified map, considers the observed diffraction data directly and refines the errors that can occur in a single-wavelength anomalous diffraction experiment. The equation has been implemented and tested on over 100 real data sets. The results are dramatic: the method provides significantly improved maps over the current state of the art and leads to many more structures being built automatically.

A multivariate likelihood SIRAS function for phasing and model refinement.

A likelihood function based on the multivariate probability distribution of all observed structure-factor amplitudes from a single isomorphous replacement with anomalous scattering experiment has been derived and implemented for use in substructure refinement and phasing as well as macromolecular model refinement. Efficient calculation of a multidimensional integration required for function evaluation has been achieved by approximations based on the function's properties. The use of the function in both phasing and protein model building with iterative refinement was essential for successful automated model building in the test cases presented.

The Max-Inf2/Lorentz Center workshop on New algorithms in macromolecular crystallography and electron microscopy.

The resolution gap between macromolecular crystallography and electron microscopy continues to decrease. Recent advances in specimen preparation, instrumentation and computational power have allowed accurate structure determination of larger macromolecular complexes by crystallography and/or by electron microscopy on cryovitrified samples. New possibilities in structural biology have opened up and new challenges are faced to further reduce the resolution gap. A workshop at the Lorentz Center, Leiden, The Netherlands, which took place in May 2008, was organized to push further the limits of both complementary techniques through improved computational methods.

Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease.

The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.