Toni Kühl

Structurally Diverse μ‐Conotoxin PIIIA Isomers Block Sodium Channel NaV1.4

Certain VGSC subtypes (NaV1.3, 1.7, 1.8, and 1.9) are expressed in the peripheral nervous system and mediate the transmission of signals leading to the sensation of different kinds of pain, such as nociception (NaV1.8), acute inflammatory (NaV1.7), and neuropathic (NaV1.3) pain. [2] Therefore, VGSCs are potential targets for novel analgesics, ideally those with strong channel specificity. Among sodium channel antagonists, m- and mO-conotoxins from the venoms of marine cone snails have attracted considerable attention because of their analgesic potency. [2a, 3] m-Conotoxins are 14to 26-mer peptides with six cysteine residues (Supporting Information, Table S1). [4] They inhibit muscle and/or neuronal VGSCs by occluding the ion channel pore. [5] A specific cysteine framework, that is, CCXnCXnCXnCC, confers conformational restriction to their three-dimensional structure upon formation of three disulfide bonds. It is generally accepted that the native fold of the toxins carries the disulfide connectivities Cys1–Cys4, Cys2–Cys5, and Cys3–Cys6 (numbered in the order of occurrence in the amino acid sequence). [3, 5b] However, three-dimensional structures are available only for a limited subset of m-conotoxins, that is, PIIIA, [6]

Analysis of Fe(III) Heme Binding to Cysteine-Containing Heme-Regulatory Motifs in Proteins

Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420-430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420-430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV-vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CP-peptide-heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzymes activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.

Lack of beta-arrestin signaling in the absence of active G proteins

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.Arrestins terminate signaling from GPCRs, but several lines of evidence suggest that they are also able to transduce signals independently of G proteins. Here, the authors systematically ablate G proteins in cell lines, and show that arrestins are unable to act as genuine signal initiators.

Regulatory FeII/III Heme: The Reconstruction of a Molecule's Biography†

More than 20 years of research on heme as a temporary effector molecule of proteins have revealed its widespread impact on virtually all primary functions in the human organism. As our understanding of this influence is still growing, a comprehensive overview of compiled data will give fresh impetus for creativity and developing new strategies in heme‐related research. From known data concerning heme‐regulated proteins and their involvement in the development of diseases, we provide concise information of FeII/III heme as a regulator and the availability of “regulatory heme”. The latter is dependent on the balance between free and bound FeII/III heme, here termed “hemeostasis”. Imbalance of this system can lead to the development of diseases that were not always attributed to this small molecule. Diseases such as cancer or Alzheimers disease highlight the reawakened interest in heme, whose function was previously believed to be completely understood.

Determination of Hemin‐Binding Characteristics of Proteins by a Combinatorial Peptide Library Approach

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron‐ion‐containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate‐covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)4(C/H/Y)(X)4 to characterize peptide ligands with considerable hemin binding capacities. Some of the library‐selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His‐based (≈39 %) and Tyr‐based (≈40 %) sequences over Cys‐based ones (≈21 %) was detected. The binding affinities for the library‐selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme‐binding domain.

Synthesis and functional characterization of tridegin and its analogues: inhibitors and substrates of factor XIIIa.

Tridegin, a 66‐mer peptide isolated from the leech Haementeria ghilianii, is a potent inhibitor of the coagulation factor XIIIa. This paper describes the chemical synthesis of tridegin by two different strategies—solid‐phase assembly and native chemical ligation—both followed by oxidation in solution phase. Tridegin and truncated analogues were examined for their activity and revealed a particular importance of the C‐terminal region of the parent peptide. Based on these studies a minimal sequence required for factor XIIIa inhibition could be identified. Our data revealed that the glutamine residue at position 52 (Q52) of tridegin most likely binds to the active site of factor XIIIa and was therefore suggested to react with the enzyme. The function of the N‐terminal region is also discussed, as the isolated C‐terminal segment of tridegin lost its inhibitory activity rapidly in the presence of factor XIIIa, whereas this was not the case for the full‐length inhibitor.

Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM)

The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine-Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV-Visible (UV-Vis) spectroscopy.

Role of the Chemical Environment beyond the Coordination Site: Structural Insight into Fe(III) Protoporphyrin Binding to Cysteine-Based Heme-Regulatory Protein Motifs.

The importance of heme as a transient regulatory molecule has become a major focus in biochemical research. However, detailed information about the molecular basis of transient heme–protein interactions is still missing. We report an in‐depth structural analysis of FeIII heme–peptide complexes by a combination of UV/Vis, resonance Raman, and 2D‐NMR spectroscopic methods. The experiments reveal insights both into the coordination to the central iron ion and into the spatial arrangement of the amino acid sequences interacting with protoporphyrin IX. Cysteine‐based peptides display different heme‐binding behavior as a result of the existence of ordered, partially ordered, and disordered conformations in the heme‐unbound state. Thus, the heme‐binding mode is clearly the consequence of the nature and flexibility of the residues surrounding the iron ion coordinating cysteine. Our analysis reveals scenarios for transient binding of heme to heme‐regulatory motifs in proteins and demonstrates that a thorough structural analysis is required to unravel how heme alters the structure and function of a particular protein.

Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli

BACKGROUND The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzymes heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 μM. CONCLUSIONS It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.

Modulation of SHP-1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high-affinity binding partner for protein tyrosine phosphatase SHP-1 and was recently used as lead structure to analyze the recognition requirements for the enzymes N-SH2 domain. Here, we focused on a set of peptides comprising C-terminally extended linear and conformationally constrained side chain-bridged cyclic N-SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP-1 through binding to both, the N-SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE--NH(2), displayed favorable binding affinity, but reduced ability to stimulate SHP-1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP-1. In summary, results presented in this study suggest that inhibitory N-SH2 ligands of SHP-1 may be obtained by designing bivalent compounds that associate with the N-SH2 domain and simultaneously occupy a specific binding site on the PTP domain.