Jens Schneider-Mergener

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP‐dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non‐native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose‐bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in β‐sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

FAN, a Novel WD-Repeat Protein, Couples the p55 TNF-Receptor to Neutral Sphingomyelinase

The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor-receptor (TNF-R55) appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By combined use of the yeast interaction trap system and a peptide scanning library, the novel WD-repeat protein FAN has been identified, which specifically binds to a cytoplasmic nine amino acid binding motif of TNF-R55. This region has been previously recognized as a distinct functional domain that is both required and sufficient for the activation of neutral sphingomyelinase (N-SMase). Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, while truncated mutants of FAN produced dominant negative effects. The data suggest that FAN regulates ceramide production by N-SMase, which is a crucial step in TNF signaling.

Changing the Antigen Binding Specificity by Single Point Mutations of an Anti-p24 (HIV-1) Antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr32Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe94Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe94Ala up to the level of the e-pep affinity to the wild-type scFv.

Applications of peptide arrays prepared by the SPOT-technology.

The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.

Its substrate specificity characterizes the DnaJ co-chaperone as a scanning factor for the DnaK chaperone.

The evolutionarily conserved DnaJ proteins are essential components of Hsp70 chaperone systems. The DnaJ homologue of Escherichia coli associates with chaperone substrates and mediates their ATP hydrolysis‐dependent locking into the binding cavity of its Hsp70 partner, DnaK. To determine the substrate specificity of DnaJ proteins, we screened 1633 peptides derived from 14 protein sequences for binding to E.coli DnaJ. The binding motif of DnaJ consists of a hydrophobic core of approximately eight residues enriched for aromatic and large aliphatic hydrophobic residues and arginine. The hydrophobicity of this motif explains why DnaJ itself can prevent protein aggregation. Although this motif shows differences from DnaKs binding motif, DnaJ and DnaK share the majority of binding peptides. In contrast to DnaK, DnaJ binds peptides consisting of L‐ and D‐amino acids, and therefore is not restricted by backbone contacts. These features allow DnaJ to scan hydrophobic protein surfaces and initiate the functional cycle of the DnaK system by associating with hydrophobic exposed patches and subsequent targeting of DnaK to these or to hydrophobic patches in spatial neighbourhood.

Distribution of Binding Sequences for the Mitochondrial Import Receptors Tom20, Tom22, and Tom70 in a Presequence-carrying Preprotein and a Non-cleavable Preprotein

Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.

Substrate recognition by the AAA+ chaperone ClpB

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

Molecular Basis for the Binding Promiscuity of an Anti-p24 (HIV-1) Monoclonal Antibody

Multiple binding capabilities utilized by specific protein-to-protein interactions in molecular recognition events are being documented increasingly but remain poorly understood at the molecular level. We identified five unrelated peptides that compete with each other for binding to the paratope region of the monoclonal anti-p24 (HIV-1) antibody CB4-1 by using a synthetic positional scanning combinatorial library XXXX[B1,B2,B3,X1,X2,X3]XXXX (14 mers; 68,590 peptide mixtures in total) prepared by spot synthesis. Complete sets of substitution analogs of the five peptides revealed key interacting residues, information that led to the construction of binding supertopes derived from each peptide. These supertope sequences were identified in hundreds of heterologous proteins, and those proteins that could be obtained were shown to bind CB4-1. Implications of these findings for immune escape mechanisms and autoimmunity are discussed.

Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

ClpS is an essential component of the N-end rule pathway in Escherichia coli.

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS–ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.