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In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M−U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed.

In major histocompatibility complex (MHC) class I molecules, monomorphic β 2 -microglobulin (β 2 m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. β 2 M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed β 2 m and to understand the evolution of the interfaces between β 2 m and different HC. Using human β 2 m and the HLA–B*27:09 complex as a model system, a comparison of free and HC-bound β 2 m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of β 2 m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved β 2 m–HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that β 2 m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.

Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein–protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined prolinederived modules (ProMs) to succeed in developing a nanomolar inhibitor (K d = 120 nM,MW= 734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein–protein interaction involved in actin filament processing and cell migration.

Significance Protein–protein interactions mediated by proline-rich motifs are involved in regulation of many important signaling cascades. Protein domains specialized in recognition of these motifs expose a flat and relatively rigid binding site that preferentially interacts with sequences adopting a left-handed polyproline helix II. Here, we present a toolkit of new chemical entities that enables rational construction of selective small-molecule inhibitors for these protein domains. As proof of principle, we developed a selective, cell-permeable inhibitor of Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains of the Ena/VASP protein family. Invasive breast-cancer cells treated with our EVH1 inhibitor showed strongly reduced cell invasion. Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.

β 2 -Microglobulin (β 2 m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of β 2 m in various MHC molecules as shown by X-ray crystallography, β 2 m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether β 2 m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. First we show that human β 2 m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled β 2 m bound to the HLA-B*27:09 HC to examine the β 2 m-HC interface. We then proceed to compare the resonances of β 2 m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with four distinct peptides for which structural information is already available. We find that only the resonances at the β 2 m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables β 2 m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

In major histocompatibility complex (MHC) class I molecules, monomorphic [beta]^sub 2^-microglobulin ([beta]^sub 2^m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. [beta]^sub 2^M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed [beta]^sub 2^m and to understand the evolution of the interfaces between [beta]^sub 2^m and different HC. Using human [beta]^sub 2^m and the HLAâ[euro]\"B*27:09 complex as a model system, a comparison of free and HC-bound [beta]^sub 2^m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of [beta]^sub 2^m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved [beta]^sub 2^mâ[euro]\"HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that [beta]^sub 2^m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.[PUBLICATION ABSTRACT]

In major histocompatibility complex (MHC) class I molecules, monomorphic beta sub(2)-microglobulin ( beta sub(2)m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. beta sub(2)M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed beta sub(2)m and to understand the evolution of the interfaces between beta sub(2)m and different HC. Using human beta sub(2)m and the HLA-B*27:09 complex as a model system, a comparison of free and HC-bound beta sub(2)m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of beta sub(2)m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved beta sub(2)m-HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that beta sub(2)m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.