Crystallization of Ethylene Plant Hormone Receptor-Screening for Structure.

The plant hormone ethylene is a key regulator of plant growth, development, and stress adaptation. Many ethylene-related responses, such as abscission, seed germination, or ripening, are of great importance to global agriculture. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs), which form dimers and higher-order oligomers in their functional state as determined by the binding of Cu(I), a cofactor to their transmembrane helices in the ER-Golgi endomembrane system. The molecular structure and signaling mechanism of the membrane-integral sensor domain are still unknown. In this article, we report on the crystallization of transmembrane (TM) and membrane-adjacent domains of plant ethylene receptors by Lipidic Cubic Phase (LCP) technology using vapor diffusion in meso crystallization. The TM domain of ethylene receptors ETR1 and ETR2, which is expressed in E. coli in high quantities and purity, was successfully crystallized using the LCP approach with different lipids, lipid mixtures, and additives. From our extensive screening of 9216 conditions, crystals were obtained from identical crystallization conditions for ETR1 (aa 1-316) and ETR2 (aa 1-186), diffracting at a medium-high resolution of 2-4 Å. However, data quality was poor and not sufficient for data processing or further structure determination due to rotational blur and high mosaicity. Metal ion loading and inhibitory peptides were explored to improve crystallization. The addition of Zn(II) increased the number of well-formed crystals, while the addition of ripening inhibitory peptide NIP improved crystal morphology. However, despite these improvements, further optimization of crystallization conditions is needed to obtain well-diffracting, highly-ordered crystals for high-resolution structural determination. Overcoming these challenges will represent a major breakthrough in structurally determining plant ethylene receptors and promote an understanding of the molecular mechanisms of ethylene signaling.

Crystal search - feasibility study of a real-time deep learning process for crystallization well images.

To avoid the time-consuming and often monotonous task of manual inspection of crystallization plates, a Python-based program to automatically detect crystals in crystallization wells employing deep learning techniques was developed. The program uses manually scored crystallization trials deposited in a database of an in-house crystallization robot as a training set. Since the success rate of such a system is able to catch up with manual inspection by trained persons, it will become an important tool for crystallographers working on biological samples. Four network architectures were compared and the SqueezeNet architecture performed best. In detecting crystals AlexNet accomplished a better result, but with a lower threshold the mean value for crystal detection was improved for SqueezeNet. Two assumptions were made about the imaging rate. With these two extremes it was found that an image processing rate of at least two times, but up to 58 times in the worst case, would be needed to reach the maximum imaging rate according to the deep learning network architecture employed for real-time classification. To avoid high workloads for the control computer of the CrystalMation system, the computing is distributed over several workstations, participating voluntarily, by the grid programming system from the Berkeley Open Infrastructure for Network Computing (BOINC). The outcome of the program is redistributed into the database as automatic real-time scores (ARTscore). These are immediately visible as colored frames around each crystallization well image of the inspection program. In addition, regions of droplets with the highest scoring probability found by the system are also available as images.

The structure of the Aquifex aeolicus MATE family multidrug resistance transporter and sequence comparisons suggest the existence of a new subfamily.

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H+ or Na+ to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family.

The fine art of integral membrane protein crystallisation.

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation.

A standardized technique for high-pressure cooling of protein crystals.

Cryogenic temperatures slow down secondary radiation damage during data collection from macromolecular crystals. In 1973, cooling at high pressure was identified as a method for cryopreserving crystals in their mother liquor [Thomanek et al. (1973). Acta Cryst. A29, 263-265]. Results from different groups studying different crystal systems indicated that the approach had merit, although difficulties in making the process work have limited its widespread use. Therefore, a simplified and reliable technique has been developed termed high-pressure cooling (HPC). An essential requirement for HPC is to protect crystals in capillaries. These capillaries form part of new sample holders with SPINE standard dimensions. Crystals are harvested with the capillary, cooled at high pressure (220 MPa) and stored in a cryovial. This system also allows the usage of the standard automation at the synchrotron. Crystals of hen egg-white lysozyme and concanavalin A have been successfully cryopreserved and yielded data sets to resolutions of 1.45 and 1.35 Å, respectively. Extensive work has been performed to define the useful working range of HPC in capillaries with 250 µm inner diameter. Three different 96-well crystallization screens that are most frequently used in our crystallization facility were chosen to study the formation of amorphous ice in this cooling setup. More than 89% of the screening solutions were directly suitable for HPC. This achievement represents a drastic improvement for crystals that suffered from cryoprotection or were not previously eligible for cryoprotection.

Development of a Thermofluor assay for stability determination of membrane proteins using the Na(+)/H(+) antiporter NhaA and cytochrome c oxidase.

Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

Zinc and ATP binding of the hexameric AAA-ATPase PilF from Thermus thermophilus: role in complex stability, piliation, adhesion, twitching motility, and natural transformation.

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.

The ESFRI Instruct Core Centre Frankfurt: automated high-throughput crystallization suited for membrane proteins and more.

Structure determination of membrane proteins and membrane protein complexes is still a very challenging field. To facilitate the work on membrane proteins the Core Centre follows a strategy that comprises four labs of protein analytics and crystal handling, covering mass spectrometry, calorimetry, crystallization and X-ray diffraction. This general workflow is presented and a capacity of 20% of the operating time of all systems is provided to the European structural biology community within the ESFRI Instruct program. A description of the crystallization service offered at the Core Centre is given with detailed information on screening strategy, screens used and changes to adapt high throughput for membrane proteins. Our aim is to constantly develop the Core Centre towards the usage of more efficient methods. This strategy might also include the ability to automate all steps from crystallization trials to crystal screening; here we look ahead how this aim might be realized at the Core Centre.

Assessment of GABARAP self-association by its diffusion properties.

Gamma-aminobutyric acid type A receptor-associated protein (GABARAP) belongs to a family of small ubiquitin-like adaptor proteins implicated in intracellular vesicle trafficking and autophagy. We have used diffusion-ordered nuclear magnetic resonance spectroscopy to study the temperature and concentration dependence of the diffusion properties of GABARAP. Our data suggest the presence of distinct conformational states and provide support for self-association of GABARAP molecules. Assuming a monomer-dimer equilibrium, a temperature-dependent dissociation constant could be derived. Based on a temperature series of (1)H(15)N heteronuclear single quantum coherence nuclear magnetic resonance spectra, we propose residues potentially involved in GABARAP self-interaction. The possible biological significance of these observations is discussed with respect to alternative scenarios of oligomerization.

Comparative modeling of human NSF reveals a possible binding mode of GABARAP and GATE-16.

Vesicular trafficking is an important homeostatic process in eukaryotic cells which critically relies on membrane fusion. One of the essential components of the universal membrane fusion machinery is NSF (N-ethylmaleimide-sensitive factor), a large hexameric ATPase involved in disassembly of SNARE (soluble NSF attachment protein receptor) complexes. To improve our understanding of this sophisticated molecular machine, we have modeled the structure of the NSF hexamer in two alternative assemblies. Our data suggest a mechanistic concept of the operating mode of NSF which helps to explain the functional impact of post-translational modifications and mutations reported previously. Furthermore, we propose a binding site for the ubiquitin-like proteins GABARAP and GATE-16, which is supported by experimental evidence, yielding a complex with favorable surface complementarity.

Structural characterization of GABARAP-ligand interactions.

The GABA(A) receptor-associated protein (GABARAP) plays an important role in intracellular trafficking of several proteins. It undergoes a C-terminal lipidation process that enables anchoring in the cytosolic leaflet of cellular membranes. While the three-dimensional structure of GABARAP itself has been determined, structural investigation of complexes with its interaction partners has just commenced. Studies with indole derivatives revealed that GABARAP features two hydrophobic binding sites (hp1 and hp2). These also play an essential role in complex formation with the native ligand calreticulin. Furthermore, a model of hexameric N-ethylmaleimide-sensitive factor (NSF) suggests that binding of GABARAP to this molecular machine may involve a similar site. Since hp1 and hp2 are highly conserved throughout the GABARAP family, the relevance of the structural data presented here is likely to extend to GABARAP homologues.

Nix directly binds to GABARAP: a possible crosstalk between apoptosis and autophagy.

Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail. The gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein GABARAP belongs to a family of proteins implicated in intracellular transport events and was shown to be associated to autophagic processes. Using a phage display screening against the target protein GABARAP, we identified the proapoptotic protein Nix/Bnip3L to be a potential GABARAP ligand. In vitro binding studies, pull-down analysis, coimmunoprecipitation assays and colocalization studies confirmed a direct interaction of both proteins in mammalian cells.

Structural framework of the GABARAP-calreticulin interface--implications for substrate binding to endoplasmic reticulum chaperones.

The 4-aminobutyrate type A receptor-associated protein (GABARAP) is a versatile adaptor protein that plays an important role in intracellular vesicle trafficking, particularly in neuronal cells. We have investigated the structural determinants underlying the interaction of GABARAP with calreticulin using spectroscopic and crystallographic techniques. Specifically, we present the crystal structure of GABARAP in complex with its major binding epitope on the chaperone. Molecular modeling of a complex containing full-length calreticulin suggests a novel mode of substrate interaction, which may have functional implications for the calreticulin/calnexin family in general.

Ligand binding mode of GABAA receptor-associated protein.

The gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein is a versatile adaptor protein playing an important role in intracellular vesicle trafficking, particularly in neuronal cells. We present the X-ray structure of the soluble form of human GABA(A) receptor-associated protein complexed with a high-affinity synthetic peptide at 1.3 A resolution. The data shed light on the probable binding modes of key interaction partners, including the GABA(A) receptor and the cysteine protease Atg4. The resulting models provide a structural background for further investigation of the unique biological properties of this protein.

An indole-binding site is a major determinant of the ligand specificity of the GABA type A receptor-associated protein GABARAP.

The role of tryptophan as a key residue for ligand binding to the ubiquitin-like modifier GABA(A) receptor associated protein (GABARAP) was investigated. Two tryptophan-binding hydrophobic patches were identified on the conserved face of the GABARAP structure by NMR spectroscopy and molecular docking. GABARAP binding of indole and indole derivatives, including the free amino acid tryptophan was quantified. The two tryptophan binding sites can be clearly distinguished by mapping the NMR spectroscopy-derived residue-specific apparent dissociation constant, K(d), onto the three-dimensional structure of GABARAP. The biological relevance of tryptophan-binding pockets of GABARAP was supported by a highly conserved tryptophan residue in the GABARAP binding region of calreticulin, clathrin heavy chain, and the gamma2 subunit of the GABA(A) receptor. Replacement of tryptophan by alanine abolished ligand binding to GABARAP.