Jürgen Kuhlmann

The Palmitoylation Machinery Is a Spatially Organizing System for Peripheral Membrane Proteins

Reversible S-palmitoylation of cysteine residues critically controls transient membrane tethering of peripheral membrane proteins. Little is known about how the palmitoylation machinery governs their defined localization and function. We monitored the spatially resolved reaction dynamics and substrate specificity of the core mammalian palmitoylation machinery using semisynthetic substrates. Palmitoylation is detectable only on the Golgi, whereas depalmitoylation occurs everywhere in the cell. The reactions are not stereoselective and lack any primary consensus sequence, demonstrating that substrate specificity is not essential for de-/repalmitoylation. Both palmitate attachment and removal require seconds to accomplish. This reaction topography and rapid kinetics allows the continuous redirection of mislocalized proteins via the post-Golgi sorting apparatus. Unidirectional secretion ensures the maintenance of a proper steady-state protein distribution between the Golgi and the plasma membrane, which are continuous with endosomes. This generic spatially organizing system differs from conventional receptor-mediated targeting mechanisms and efficiently counteracts entropy-driven redistribution of palmitoylated peripheral membrane proteins over all membranes.

Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue : implications for nuclear transport

The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-β-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran·GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran·GppNHp resembles the canonical Ras·GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular ‘embrace’ enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran·GTP from importin-β-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein–protein interaction for a Ras-related protein.

Structural Basis for Guanine Nucleotide Exchange on Ran by the Regulator of Chromosome Condensation (RCC1)

RCC1 (regulator of chromosome condensation), a beta propeller chromatin-bound protein, is the guanine nucleotide exchange factor (GEF) for the nuclear GTP binding protein Ran. We report here the 1.8 A crystal structure of a Ran*RCC1 complex in the absence of nucleotide, an intermediate in the multistep GEF reaction. In contrast to previous structures, the phosphate binding region of the nucleotide binding site is perturbed only marginally, possibly due to the presence of a polyvalent anion in the P loop. Biochemical experiments show that a sulfate ion stabilizes the Ran*RCC1 complex and inhibits dissociation by guanine nucleotides. Based on the available structural and biochemical evidence, we present a unified scenario for the GEF mechanism where interaction of the P loop lysine with an acidic residue is a crucial element for the overall reaction.

Photochemical Surface Patterning by the Thiol-Ene Reaction†

The immobilization of proteins on solid substrates while controlling the size and dimensions of the generated patterns is increasingly relevant in biotechnology. Site-specific immobilization and thus control over the orientation of proteins is particularly important because, as opposed to nonspecific adsorption, it generates homogeneous surface coverage and accessibility to the active site of the protein. Consequently, different types of bioorthogonal reactions have been developed to attach proteins site-specifically to surfaces and to control protein patterning. Herein, we report the photochemical coupling of olefins to thiols to generate a stable thioether bond for the covalent surface patterning of proteins and small molecules. This reaction has been applied previously in solution for carbohydrate and peptide coupling. The thiol-ene photoreaction proceeds at close to visible wavelengths (l = 365–405 nm) and in buffered aqueous solutions. As a result of its specificity for olefins, this photoreaction can be considered to be bioorthogonal, unlike other photochemical methods used previously for protein immobilization. To adopt the thiol-ene reaction for the immobilization of biomolecules, surfaces functionalized with thiols and biomolecules derivatized with olefins were prepared (Figure 1). Polyamidoamine (PAMAM) dendrimers were attached covalently to silicon oxide surfaces. An aminocaproic acid spacer was attached to the dendrimers to create distance from the surface. Cystamine was coupled to the spacer, and subsequent reduction of the disulfide yielded the desired thiolterminated surfaces. A liquid layer of terminal-olefinfunctionalized molecules dissolved in ethylene glycol was spread onto these wafers, which were then covered immediately with a photomask. Subsequent irradiation of the surfaces through the photomask led to patterning with adducts of covalently attached thioethers. To establish the method, we photochemically attached the biotin derivative 1 to a thiol-functionalized surface as described above (Figure 1). After the removal of unreacted biotin molecules, the surface was incubated with Cy5-labeled streptavidin (SAv) to produce a SAv-patterned surface. Fluorescence images of the resulting surface (Figure 1) demonstrated that lateral gradients and patterns with micrometer-sized features (5–100 mm) over areas of centimeters in width (Figure 1A) were readily accessible. Figure 1B,C and the fluorescence-intensity profile in Figure 1D show that the patterns have a well-defined shape and are homogeneous over large distances. When prolonged sonication (4 h) and stringent washing were carried our after irradiation, SAv patterns with similar fluorescence intensities were observed, whereas control experiments with biotin that lacked the olefin linker showed no distinctive SAv patterns. These results indicate that the covalent attachment of biotin to the surface occurs specifically through the proposed thiol-ene reaction and that the nonspecific adsorption of biotin is insignificant. Figure 1E shows that the amount of material immobilized can be modified by changing the irradiation time. The procedure reproducibly requires a short irradiation time of 60 s to yield sufficient surface coverage for fabricating dense SAv patterns. To obtain homogeneous fluorescence signals of the patterns, the starting concentration of the solution that is drop cast onto the surface is also important. When the solution of 1 was diluted (to 1 mm), the Cy5-fluorescence intensity decreased considerably. Further dilution (below 500 mm) resulted eventually in disrupted SAv patterns. The application of more concentrated solutions of 1 (> 20 mm) resulted in the saturation of the fluorescence intensity of the SAv patterns. This behavior corresponds well with the effects observed upon varying the irradiation time. Longer irradi[*] Dr. D. N sse, Dr. H. Schroeder, Dr. R. Wacker, Prof. Dr. C. M. Niemeyer Faculty of Chemistry Biological-Chemical Microstructuring Technical University of Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax: (+49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de

Light-Driven Water Splitting for (Bio-)Hydrogen Production: Photosystem 2 as the Central Part of a Bioelectrochemical Device

Abstract To establish a semiartificial device for (bio-)hydrogen production utilizing photosynthetic water oxidation, we report on the immobilization of a Photosystem 2 on electrode surfaces. For this purpose, an isolated Photosystem 2 with a genetically introduced His tag from the cyanobacterium Thermosynechococcus elongatus was attached onto gold electrodes modified with thiolates bearing terminal Ni(II)-nitrilotriacetic acid groups. Surface enhanced infrared absorption spectroscopy showed the binding kinetics of Photosystem 2, whereas surface plasmon resonance measurements allowed the amount of protein adsorbed to be quantified. On the basis of these data, the surface coverage was calculated to be 0.29 pmol protein cm−2, which is in agreement with the formation of a monomolecular film on the electrode surface. Upon illumination, the generation of a photocurrent was observed with current densities of up to 14 μA cm−2. This photocurrent is clearly dependent on light quality showing an action spectrum similar to an isolated Photosystem 2. The achieved current densities are equivalent to the highest reported oxygen evolution activities in solution under comparable conditions.

The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain

Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein–protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (KD) of 8.1×10−9 M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as β-catenin playing a major role in the regulation of cell division, migration and cell adhesion.

Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling

ABSTRACT The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser259, a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS259 crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer259/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.

B- and C-RAF Display Essential Differences in Their Binding to Ras.The isotype-specific N terminus of B-RAF facilitates RAS binding

Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

Preparation of Biomolecule Microstructures and Microarrays by Thiol–ene Photoimmobilization

A mild, fast and flexible method for photoimmobilization of biomolecules based on the light‐initiated thiol–ene reaction has been developed. After investigation and optimization of various surface materials, surface chemistries and reaction parameters, microstructures and microarrays of biotin, oligonucleotides, peptides, and MUC1 tandem repeat glycopeptides were prepared with this photoimmobilization method. Furthermore, MUC1 tandem repeat glycopeptide microarrays were successfully used to probe antibodies in mouse serum obtained from vaccinated mice. Dimensions of biomolecule microstructures were shown to be freely controllable through photolithographic techniques, and features down to 5 μm in size covering an area of up to 75×25 mm were created. Use of a confocal laser microscope with a UV laser as UV‐light source enabled further reduction of biotin feature size opening access to nanostructured biochips.

Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis

The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.