Expanding the versatility of natural and de novo designed coiled coils and helical bundles

Natural helical bundles (HBs) constitute a ubiquitous class of protein folds built of two or more longitudinally arranged α-helices. They adopt topologies that include symmetric, highly regular assemblies all the way to asymmetric, loosely packed domains. The diverse functional spectrum of HBs ranges from structural scaffolds to complex and dynamic effectors as molecular motors, signaling and sensing molecules, enzymes, and molecular switches. Symmetric HBs, particularly coiled coils, offer simple model systems providing an ideal entry point for protein folding and design studies. Herein, we review recent progress unveiling new structural features and functional mechanisms in natural HBs and cover staggering advances in the de novo design of HBs, giving rise to exotic structures and the creation of novel functions

Structural diversity of coiled coils in protein fibers of the bacterial cell envelope

he cell envelope of bacteria shows great diversity in architecture and composition, to a large extent due to its proteome. Proteins localized to the cell envelope, whether integrally embedded in the membrane, membrane-anchored, or peripherally associated as part of a macromolecular complex, often form elongated fibers, in which coiled coils represent a prominent structural element. These coiled-coil segments show a surprising degree of structural variability, despite being shaped by a small number of simple biophysical rules, foremost being their geometry of interaction referred to as 'knobs-into-holes'. Here we will review this diversity, particularly as it has emerged over the last decade

Sweet and Blind Spots in E3 Ligase Ligand Space Revealed by a Thermophoresis-Based Assay

Repurposing E3 ubiquitin ligases for targeted protein degradation via customized molecular glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable molecular glues and PROTACs is our fragmentary understanding of E3 ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of cereblon (CRBN)

A domain dictionary of trimeric autotransporter adhesins

AbstractTrimeric autotransporter adhesins (TAAs) are modular, highly repetitive outer membrane proteins that mediate adhesion to external surfaces in many Gram-negative bacteria. In recent years, several TAAs have been investigated in considerable detail, also at the structural level. However, in their vast majority, putative TAAs in prokaryotic genomes remain poorly annotated, due to their sequence diversity and changeable domain architecture. In order to achieve an automated annotation of these proteins that is both detailed and accurate we have taken a domain dictionary approach, in which we identify recurrent domains by sequence comparisons, produce bioinformatic descriptors for each domain type, and connect these to structural information where available. We implemented this approach in a web-based platform, daTAA, in 2008 and demonstrated its applicability by reconstructing the complete fiber structure of a TAA conserved in enterobacteria. Here we review current knowledge on the domain structure of TAAs

A FRET-Based Assay for the Identification and Characterization of Cereblon Ligands

Cereblon serves as an ubiquitin ligase substrate receptor that can be tuned toward different target proteins by various cereblon-binding agents. This offers one of the most promising avenues for targeted protein degradation in cancer therapy, but cereblon binding can also mediate teratogenic effects. We present an effective assay that is suited for high-throughput screening of compound libraries for off-target cereblon interactions but also can guide lead optimization and rational design of novel cereblon effector molecules

Structural dynamics of the cereblon ligand binding domain.

Cereblon, a primary target of thalidomide and its derivatives, has been characterized structurally from both bacteria and animals. Especially well studied is the thalidomide binding domain, CULT, which shows an invariable structure across different organisms and in complex with different ligands. Here, based on a series of crystal structures of a bacterial representative, we reveal the conformational flexibility and structural dynamics of this domain. In particular, we follow the unfolding of large fractions of the domain upon release of thalidomide in the crystalline state. Our results imply that a third of the domain, including the thalidomide binding pocket, only folds upon ligand binding. We further characterize the structural effect of the C-terminal truncation resulting from the mental-retardation linked R419X nonsense mutation in vitro and offer a mechanistic hypothesis for its irresponsiveness to thalidomide. At 1.2Å resolution, our data provide a view of thalidomide binding at atomic resolution

Thalidomide mimics uridine binding to an aromatic cage in cereblon

AbstractThalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity

Your personalized protein structure: Andrei N. Lupas fused to GCN4 adaptors

AbstractThis work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date