Joachim Behlke

Structure of the DLM-1-Z-DNA complex reveals a conserved family of Z-DNA-binding proteins.

The first crystal structure of a protein, the Zα high affinity binding domain of the RNA editing enzyme ADAR1, bound to left-handed Z-DNA was recently described. The essential set of residues determined from this structure to be critical for Z-DNA recognition was used to search the database for other proteins with the potential for Z-DNA binding. We found that the tumor-associated protein DLM-1 contains a domain with remarkable sequence similarities to ZαADAR. Here we report the crystal structure of this DLM-1 domain bound to left-handed Z-DNA at 1.85 Å resolution. Comparison of Z-DNA binding by DLM-1 and ADAR1 reveals a common structure-specific recognition core within the binding domain. However, the domains differ in certain residues peripheral to the protein–DNA interface. These structures reveal a general mechanism of Z-DNA recognition, suggesting the existence of a family of winged-helix proteins sharing a common Z-DNA binding motif.

Structural basis of oligomerization in the stalk region of dynamin-like MxA

The interferon-inducible dynamin-like myxovirus resistance protein 1 (MxA; also called MX1) GTPase is a key mediator of cell-autonomous innate immunity against pathogens such as influenza viruses. MxA partially localizes to COPI-positive membranes of the smooth endoplasmic reticulum–Golgi intermediate compartment. At the point of infection, it redistributes to sites of viral replication and promotes missorting of essential viral constituents. It has been proposed that the middle domain and the GTPase effector domain of dynamin-like GTPases constitute a stalk that mediates oligomerization and transmits conformational changes from the G domain to the target structure; however, the molecular architecture of this stalk has remained elusive. Here we report the crystal structure of the stalk of human MxA, which folds into a four-helical bundle. This structure tightly oligomerizes in the crystal in a criss-cross pattern involving three distinct interfaces and one loop. Mutations in each of these interaction sites interfere with native assembly, oligomerization, membrane binding and antiviral activity of MxA. On the basis of these results, we propose a structural model for dynamin oligomerization and stimulated GTP hydrolysis that is consistent with previous structural predictions and has functional implications for all members of the dynamin family.

Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

In addition to endocytosis-mediated cellular uptake, hydrophilic cell-penetrating peptides are able to traverse biological membranes in a non-endocytic mode termed transduction, resulting in immediate bioavailability. Here we analysed structural requirements for the non-endocytic uptake mode of arginine-rich cell-penetrating peptides, by a combination of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation. We demonstrate that the transduction efficiency of arginine-rich peptides increases with higher peptide structural rigidity. Consequently, cyclic arginine-rich cell-penetrating peptides showed enhanced cellular uptake kinetics relative to their linear and more flexible counterpart. We propose that guanidinium groups are forced into maximally distant positions by cyclization. This orientation increases membrane contacts leading to enhanced cell penetration.

The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton

Ahnak is a ubiquitously expressed giant protein of 5643 amino acids implicated in cell differentiation and signal transduction. In a recent study, we demonstrated the association of ahnak with the regulatory β2 subunit of the cardiac L‐type Ca2+ channel. Here we identify the most carboxyl‐terminal ahnak region (aa 5262–5643) to interact with recombinant β2a as well as with β2 and β1a isoforms of native muscle Ca2+ channels using a panel of GST fusion proteins. Equilibrium sedimentation analysis revealed Kd values of 55 ± 11 nM and 328 ± 24 nM for carboxyl‐terminal (aa 195–606) and amino‐terminal (aa 1–200) truncates of the β2a subunit, respectively. The same carboxylterminal ahnak region (aa 5262–5643) bound to G‐actin and cosedimented with F‐actin. Confocal microscopy of human left ventricular tissue localized the carboxylterminal ahnak portion to the sarcolemma including the T‐tubular system and the intercalated disks of cardiomyocytes. These results suggest that ahnak provides a structural basis for the subsarcolemmal cytoarchitecture and confers the regulatory role of the actin‐based cytoskeleton to the L‐type Ca2+ channel.—Hohaus, A., Person, V., Behlke, J., Schaper, J., Morano, I., Haase, H. The carboxyl‐terminal region of ahnak provides a link between cardiac L‐type Ca2+ channels and the actin‐based cytoskeleton. FASEB J. 16, 1205–1216 (2002)

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimers, Huntingtons, and Creutzfeldt-Jacobs. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.

Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β-adrenergic regulation

Defective L‐type Ca2+ channel (ICaL) regulation is one major cause for contractile dysfunction in the heart. The ICaL is enhanced by sympathetic nervous stimulation: via the activation of β‐adrenergic receptors, PKA phosphorylates the α1C(CaV1.2)‐ and β2‐channel subunits and ahnak, an associated 5643‐amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr‐ahnak on ICaL. Binding experiments with ahnak fragments (wild‐type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr‐ahnak critically affected both β2 subunit interaction and ICaL regulation. Binding affinity between ahnak‐C1 (aa 4646‐5288) and β2 subunit decreased by ≈50% after PKA phosphorylation or in the presence of Ile5236Thr‐ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA‐effects on ICaL increasing the amplitude by ≈60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr‐peptide and Ile5236Thr‐fragment (aa 5215‐5288) prevented specifically the further up‐regulation of ICaL by isoprenaline. Hence, we suggest the ahnak‐C1 domain serves as physiological brake on ICaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr‐ahnak fragments to increase ICaL. This genetic ahnak variant might cause individual differences in ICaL regulation upon physiological challenges or therapeutic interventions.—Haase, H., Alvarez, J., Petzhold, D., Doller, A., Behlke, J., Erdmann, J., Hetzer, R., Regitz‐Zagrosek, V., Vassort, G., Morano, I. Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β‐adrenergic regulation. FASEB J. 19, 1969–1977 (2005)

In vitro and in vivo stability of the epsilon2zeta2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system

Abstract Streptococcus pyogenes pSM19035-encoded (10.7 kDa) and ζ (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with ζ acting as toxin that kills plasmiddeprived cells and as an antitoxin that neutralises the activity of ζ. The and ζ proteins copurify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as 2ζ2 heterotetramer in solution. Cocrystals of the 2ζ2 complex contain and ζ in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the ζ protein has a significantly lower thermodynamic stability than the protein both in free state and in the complex. Proteolytic studies indicate that ζ protein is more stable in the the 2ζ2 complex than in the free state. In vivo studies reveal a short halflife of the antitoxin (~18min) and a long lifetime of the ζ toxin (>60min). When transcriptiontranslation of a plasmid containing the and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.

Supramolecular structure of the recombinant murine small heat shock protein hsp25

The size and shape of the recombinant murine small heat shock protein, hsp25, have been analyzed by hydrodynamic and electron microscopic methods. According to these studies recombinant hsp25 exists in large complexes with a sphere‐like shape and diameters of 15–18 nm. The molecular mass of these complexes amounts to about 730 kDa indicating that they are composed of about 32 monomers.

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis‐Golgi membrane. The crystal structure of human BET3 has been determined to 1.55‐Å resolution. BET3 adopts an α/β‐plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane‐associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein–protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.

Analysis of the thermodynamic non-ideality of proteins by sedimentation equilibrium experiments.

This paper presents a modified method to determine experimentally the second virial coefficient of protein solutions by sedimentation equilibrium experiments. The improvement is based on the possibility of fitting simultaneously up to seven radial concentration distribution curves of solutions with different loading concentrations. The possibility of precise determination of the second virial coefficient allows estimation of the net charge and the excluded volume of a monomeric protein. Application of the method is demonstrated for lysozyme and ovalbumin. In 0.1 M sodium acetate buffer, pH 4.5, the second virial coefficient of hen egg white lysozyme amounts to 24 +/- 1 ml/g. Analysis based on spherical particle theory yield an excluded volume of 3.5 ml/g and a charge dependent value of 20.5 ml/g which is induced by a net charge number of 14.1 +/- 1. Under low salt conditions self-association processes on lysozyme are unfavorable due to electrostatic repulsion. To overcome these repulsive contributions, either a shift to neutral pH or addition of at least 2% NaCl is necessary. In this way the charge dependent contribution decreases below the value responsible for the excluded volume and allows crystallization of the protein. Similar effects can be observed with ovalbumin. The high virial coefficient observed at pH 8.5 is induced by the high net charge number of 27 +/- 1.