Yvonne Thielmann

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 γ-aminobutyric acid type A (GABAA) 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, pulldown 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 GABAA 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, Kd, 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 GABAA receptor. Replacement of tryptophan by alanine abolished ligand binding to GABARAP.

Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus: ROLE IN COMPLEX STABILITY, PILIATION, ADHESION, TWITCHING MOTILITY, AND NATURAL TRANSFORMATION

Background: Thermus PilF is essential for pilus biogenesis and transformation. Results: Zinc binding is essential for PilF complex stability, piliation, adhesion, and twitching motility. Conclusion: PilF complex thermostability is essential for piliation but not for natural transformation. Significance: This work highlights the role of zinc and ATP in AAA-ATPase stability and provides evidence that T4P and the DNA translocator are distinct systems. 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.

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. Proteins 2009.

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 1H15N 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.

MPI tray: a versatile crystallization plate for membrane proteins

High-throughput crystallization of biological macromolecules is usually performed on multi-well plates, the design of which needs to address different and sometimes conflicting requirements. In this regard, handling of membrane proteins presents a particular challenge owing to the common use of detergents with associated effects on surface tension. Reported here is the design of a new crystallization plate, termed the MPI tray, which is optimized for UV and visible imaging with membrane protein samples. Following basic considerations regarding geometry and material, the surface properties of the plate were subjected to extensive analysis and modification in order to improve the performance in a robotic environment. An electrostatic surface potential was identified as the major problem affecting the automated setup of experiments, and it was found that treatment of the crystallization plate with ethanol is effective in removing this potential.

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.