Matthias Frech

Ubiquitin protein ligase Nedd4 binds to connexin43 by a phosphorylation-modulated process

Connexin43 is degraded by the proteasomal as well as the lysosomal pathway with ubiquitin playing a role in both degradation pathways. So far, no ubiquitin protein ligase has been identified for any of the connexins. By using pull-down assays, here we show binding of a ubiquitin protein ligase, Nedd4, to the C-terminus of connexin43. This observation was confirmed in vivo by coimmunoprecipitation and immunofluorescence, showing colocalization of Nedd4 and connexin43. Binding of Nedd4 to its interaction partners is generally carried out by its WW domains. Our results indicate that the interaction with connexin43 occurs through all three WW domains of Nedd4. Furthermore, whereas WW1 and WW2 domains mainly interact with the unphosphorylated form of connexin43, WW3 binds phosphorylated and unphosphorylated forms equally. In addition, using the surface plasmon resonance approach we show that only the WW2 domain binds to the PY motif located at the C-terminus of connexin43. Suppression of Nedd4 expression with siRNA resulted in an accumulation of gap junction plaques at the plasma membrane, suggesting an involvement of the ubiquitin protein ligase Nedd4 in gap junction internalization.

Specific Interaction of a Novel Foamy Virus Env Leader Protein with the N-Terminal Gag Domain

ABSTRACT Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa.

Features of the Env leader protein and the N-terminal Gag domain of feline foamy virus important for virus morphogenesis

Previous studies have shown that foamy virus (FV) particle budding, especially the involvement of the viral env glycoprotein is different from that of other (ortho) retroviruses: the N-terminal Env leader protein Elp is a constituent of released FV particles. A defined sequence in Elp required for particle budding binds to the MA domain of Gag. To extend these findings, we show that feline FV Elp is a membrane-anchored protein with the N-terminus located inside the particle. Thus, the internal/cytoplasmic domain of Elp has the correct topology for interacting with Gag during budding. In addition to Elp, an Elp-related protein of about 9 kDa was shown to be virion associated and is probably generated by cellular signal peptidases. Besides the function of Elp binding, the N-terminal domain of Gag was shown to be required for proper localization of feline FV Gag to the cytoplasm and the perinuclear/nuclear region.

Structure of the leech protein saratin and characterization of its binding to collagen

The leech protein Saratin from Hirudo medicinalis prevents thrombocyte aggregation by interfering with the first binding step of the thrombocytes to collagen by binding to collagen. We solved the three-dimensional structure of the leech protein Saratin in solution and identified its collagen binding site by NMR titration experiments. The NMR structure of Saratin consists of one alpha-helix and a five-stranded beta-sheet arranged in the topology betabetaalphabetabetabeta. The C-terminal region, of about 20 amino acids in length, adopts no regular structure. NMR titration experiments with collagen peptides show that the collagen interaction of Saratin takes place in a kind of notch that is formed by the end of the alpha-helix and the beta-sheet. NMR data-driven docking experiments to collagen model peptides were used to elucidate the putative binding mode of Saratin and collagen. Mainly, parts of the first and the end of the fifth beta-strand, the loop connecting the alpha-helix and the third beta-strand, and a short part of the loop connecting the fourth and fifth beta-strand participate in binding.

Kinetics for Drug Discovery: an industry-driven effort to target drug residence time

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership.

Scale-Up of Antibody Purification

Recent developments of highly specific biotherapeutics are mainly based on immunoaffinity mechanisms. After ups and downs of monoclonal antibodies (mAb) these molecules are now gaining respect again. Among the “Top Twenty” biopharmaceuticals the number of antibodies and related products is increasing. In the pipeline of new biological entities most of the molecules either are murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, or human monoclonal antibodies. Examples of approved monoclonal antibodies which have already a multi-million dollar market volume are the chimeric monoclonal antibodies ReoPro and Remicade (Johnson & Johnson/Centocor), Simulect (Novartis), and IDEC’s Zevlin, the human or humanized antibodies Humira (Abbott), Herceptin (Genentech/Roche), Zenapax (Roche/PDL), Mylotarg (Celltech/Wyeth), Campath (Ilex/Schering), Xanelim (Genentech/Xoma) and Avastin (Genentech) or the murine derived antibodies Bexxar (Corixa/GSK), Rituxan (IDEC/Genentech) and Orthoclone (Johnson & Johnson).

Letter to the Editor: Sequential assignment and secondary structure of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen

Collagen plays an important structural role in the extra-cellular matrix of tissues. Under normal circumstances it is not exposed to flowing blood. Upon injury of the vessel wall, collagen is exposed to flowing blood and its constituents leading to activation and release of a variety of pro-aggregatory and mitotic factors propagating aggregation and thrombosis. The adhesion of platelets to the injured arterial wall is mediated by von Willebrand factor (vWF), which binds to collagen (for review see Sixma et al., 1997). As a consequence the collagen-bound vWF then gathers platelets, leading in the end to platelet activation (for review see Sadler, 1998). Thus vWF could act as a bridge between collagen and platelets and is a prerequisite for platelet adhesion. This process in itself may only be temporary, however, requiring additional, direct interactions between collagen and other receptors on the platelet surface in order to facilitate permanent platelet adhesion, activation and aggregation (Sixma et al., 1997). Such collagen receptors on platelets are known to include, but may not be limited to GP VI, GP Ia/IIa (alpha2beta1), to a lesser extent GP IV (CD36) (Moroi and Jung, 1997) and perhaps p65 (Chiang et al., 1997). In addition, vWF and fibrinogen facilitate cross-linking and further activation of platelets via GP IIb/IIIa receptor binding (Kulkarni et al., 2000), providing stability and strength for the developing thrombus.

Ligand Desolvation Steers On-Rate and Impacts Drug Residence Time of Heat Shock Protein 90 (Hsp90) Inhibitors.

Residence time and more recently the association rate constant kon are increasingly acknowledged as important parameters for in vivo efficacy and safety of drugs. However, their broader consideration in drug development is limited by a lack of knowledge of how to optimize these parameters. In this study on a set of 176 heat shock protein 90 inhibitors, structure-kinetic relationships, X-ray crystallography, and molecular dynamics simulations were combined to retrieve a concrete scheme of how to rationally slow down on-rates. We discovered that an increased ligand desolvation barrier by introducing polar substituents resulted in a significant kon decrease. The slowdown was accomplished by introducing polar moieties to those parts of the ligand that point toward a hydrophobic cavity. We validated this scheme by increasing polarity of three Hsp90 inhibitors and observed a 9-, 13-, and 45-fold slowdown of on-rates and a 9-fold prolongation in residence time. This prolongation was driven by transition state destabilization rather than ground state stabilization.

Ligand‐Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free—Towards a Conformational Activity Screening for Drug Discovery

Abstract Investigation of protein–ligand interactions is crucial during early drug‐discovery processes. ATR‐FTIR spectroscopy can detect label‐free protein–ligand interactions with high spatiotemporal resolution. Here we immobilized, as an example, the heat shock protein HSP90 on an ATR crystal. This protein is an important molecular target for drugs against several diseases including cancer. With our novel approach we investigated a ligand‐induced secondary structural change. Two specific binding modes of 19 drug‐like compounds were analyzed. Different binding modes can lead to different efficacy and specificity of different drugs. In addition, the k obs values of ligand dissociation were obtained. The results were validated by X‐ray crystallography for the structural change and by SPR experiments for the dissociation kinetics, but our method yields all data in a single and simple experiment.