Designed nanomolar small-molecule inhibitors of Ena/VASP EVH1 interaction impair invasion and extravasation of breast cancer cells.

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 proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] 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.

A modular toolkit to inhibit proline-rich motif-mediated protein-protein interactions.

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.

SEPT9 negatively regulates ubiquitin-dependent downregulation of EGFR.

Septins constitute a family of GTP-binding proteins that are involved in a variety of biological processes. Several isoforms have been implicated in disease, but the molecular mechanisms underlying pathogenesis are poorly understood. Here, we show that depletion of SEPT9 decreases surface levels of epidermal growth factor receptors (EGFRs) by enhancing receptor degradation. We identify a consensus motif within the SEPT9 N-terminal domain that supports its association with the adaptor protein CIN85 (also known as SH3KBP1). We further show CIN85-SEPT9 to be localized exclusively to the plasma membrane, where SEPT9 is recruited to EGF-engaged receptors in a CIN85-dependent manner. Finally, we demonstrate that SEPT9 negatively regulates EGFR degradation by preventing the association of the ubiquitin ligase Cbl with CIN85, resulting in reduced EGFR ubiquitylation. Taken together, these data provide a mechanistic explanation of how SEPT9, though acting exclusively at the plasma membrane, impairs the sorting of EGFRs into the degradative pathway.

NMR spectroscopy reveals unexpected structural variation at the protein-protein interface in MHC class I molecules.

ß2-Microglobulin (ß2m) 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 ß2m in various MHC molecules as shown by X-ray crystallography, ß2m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether ß2m 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 ß2m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled ß2m bound to the HLA-B*27:09 HC to examine the ß2m-HC interface. We then proceed to compare the resonances of ß2m 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 ß2m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables ß2m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

The clip-segment of the von Willebrand domain 1 of the BMP modulator protein Crossveinless 2 is preformed.

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.

The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNA-binding protein.

During bacterial DNA replication, DnaG primase and the χ subunit of DNA polymerase III compete for binding to single-stranded DNA-binding protein (SSB), thus facilitating the switch between priming and elongation. SSB proteins play an essential role in DNA metabolism by protecting single-stranded DNA and by mediating several important protein-protein interactions. Although an interaction of SSB with primase has been previously reported, it was unclear which domains of the two proteins are involved. This study identifies the C-terminal helicase-binding domain of DnaG primase (DnaG-C) and the highly conserved C-terminal region of SSB as interaction sites. By ConSurf analysis, it can be shown that an array of conserved amino acids on DnaG-C forms a hydrophobic pocket surrounded by basic residues, reminiscent of known SSB-binding sites on other proteins. Using protein-protein cross-linking, site-directed mutagenesis, analytical ultracentrifugation and nuclear magnetic resonance spectroscopy, we demonstrate that these conserved amino acid residues are involved in the interaction with SSB. Even though the C-terminal domain of DnaG primase also participates in the interaction with DnaB helicase, the respective binding sites on the surface of DnaG-C do not overlap, as SSB binds to the N-terminal subdomain, whereas DnaB interacts with the ultimate C-terminus.

Large-scale purification of ribosome-nascent chain complexes for biochemical and structural studies.

Here we present a method to purify large amounts of highly pure and stably arrested ribosome-nascent chain complexes (RNCs) from Escherichia coli cells. It relies on the combined use of translation-arrest sequences to generate nascent polypeptides of specified length and subsequent tag purification of the RNCs. Moreover, we adapted this method for the in vivo production of RNCs with specific isotope labeling of the nascent chains for nuclear magnetic resonance (NMR) studies. This method opens therefore possibilities for a wide range of biochemical and structural studies exploring conformations of nascent chains during the early steps of protein folding and targeting.