Design of novel granulopoietic proteins by topological rescaffolding.

Computational protein design is rapidly becoming more powerful, and improving the accuracy of computational methods would greatly streamline protein engineering by eliminating the need for empirical optimization in the laboratory. In this work, we set out to design novel granulopoietic agents using a rescaffolding strategy with the goal of achieving simpler and more stable proteins. All of the 4 experimentally tested designs were folded, monomeric, and stable, while the 2 determined structures agreed with the design models within less than 2.5 Å. Despite the lack of significant topological or sequence similarity to their natural granulopoietic counterpart, 2 designs bound to the granulocyte colony-stimulating factor (G-CSF) receptor and exhibited potent, but delayed, in vitro proliferative activity in a G-CSF-dependent cell line. Interestingly, the designs also induced proliferation and differentiation of primary human hematopoietic stem cells into mature granulocytes, highlighting the utility of our approach to develop highly active therapeutic leads purely based on computational design.

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

The 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.

A trimeric lipoprotein assists in trimeric autotransporter biogenesis in enterobacteria.

Trimeric autotransporter adhesins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. TAAs form fibrous, adhesive structures on the bacterial cell surface. Their N-terminal extracellular domains are exported through a C-terminal membrane pore; the insertion of the pore domain into the bacterial outer membrane follows the rules of ß-barrel transmembrane protein biogenesis and is dependent on the essential Bam complex. We have recently described the full fiber structure of SadA, a TAA of unknown function in Salmonella and other enterobacteria. In this work, we describe the structure and function of SadB, a small inner membrane lipoprotein. The sadB gene is located in an operon with sadA; orthologous operons are only found in enterobacteria, whereas other TAAs are not typically associated with lipoproteins. Strikingly, SadB is also a trimer, and its co-expression with SadA has a direct influence on SadA structural integrity. This is the first report of a specific export factor of a TAA, suggesting that at least in some cases TAA autotransport is assisted by additional periplasmic proteins.

A domain dictionary of trimeric autotransporter adhesins.

Trimeric 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.

Complete fiber structures of complex trimeric autotransporter adhesins conserved in enterobacteria.

Trimeric autotransporter adhesins (TAAs) are modular, highly repetitive surface proteins that mediate adhesion to host cells in a broad range of Gram-negative pathogens. Although their sizes may differ by more than one order of magnitude, they all follow the same basic head-stalk-anchor architecture, where the head mediates adhesion and autoagglutination, the stalk projects the head from the bacterial surface, and the anchor provides the export function and attaches the adhesin to the bacterial outer membrane after export is complete. In complex adhesins, head and stalk domains may alternate several times before the anchor is reached. Despite extensive sequence divergence, the structures of TAA domains are highly constrained, due to the tight interleaving of their constituent polypeptide chains. We have therefore taken a "domain dictionary" approach to characterize representatives for each domain type by X-ray crystallography and use these structures to reconstruct complete TAA fibers. With SadA from Salmonella enterica, EhaG from enteropathogenic Escherichia coli (EHEC), and UpaG from uropathogenic E. coli (UPEC), we present three representative structures of a complex adhesin that occur in a conserved genomic context in Enterobacteria and is essential in the infection process of uropathogenic E. coli. Our work proves the applicability of the dictionary approach to understanding the structure of a class of proteins that are otherwise poorly tractable by high-resolution methods and provides a basis for the rapid and detailed annotation of newly identified TAAs.

Thalidomide mimics uridine binding to an aromatic cage in cereblon.

Thalidomide 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.

This 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.

Histones and histone variant families in prokaryotes.

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a superfamily of histones, termed α3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones.

The design of functional proteins using tensorized energy calculations.

In protein design, the energy associated with a huge number of sequence-conformer perturbations has to be routinely estimated. Hence, enhancing the throughput and accuracy of these energy calculations can profoundly improve design success rates and enable tackling more complex design problems. In this work, we explore the possibility of tensorizing the energy calculations and apply them in a protein design framework. We use this framework to design enhanced proteins with anti-cancer and radio-tracing functions. Particularly, we designed multispecific binders against ligands of the epidermal growth factor receptor (EGFR), where the tested design could inhibit EGFR activity in vitro and in vivo. We also used this method to design high-affinity Cu2+ binders that were stable in serum and could be readily loaded with copper-64 radionuclide. The resulting molecules show superior functional properties for their respective applications and demonstrate the generalizable potential of the described protein design approach.

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

[This corrects the article DOI: 10.1021/acsmedchemlett.0c00440.].

A topological refactoring design strategy yields highly stable granulopoietic proteins.

Protein therapeutics frequently face major challenges, including complicated production, instability, poor solubility, and aggregation. De novo protein design can readily address these challenges. Here, we demonstrate the utility of a topological refactoring strategy to design novel granulopoietic proteins starting from the granulocyte-colony stimulating factor (G-CSF) structure. We change a protein fold by rearranging the sequence and optimising it towards the new fold. Testing four designs, we obtain two that possess nanomolar activity, the most active of which is highly thermostable and protease-resistant, and matches its designed structure to atomic accuracy. While the designs possess starkly different sequence and structure from the native G-CSF, they show specific activity in differentiating primary human haematopoietic stem cells into mature neutrophils. The designs also show significant and specific activity in vivo. Our topological refactoring approach is largely independent of sequence or structural context, and is therefore applicable to a wide range of protein targets.

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.

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).

The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase.

The reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer-coupled IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array. Here we report that parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative E. coli strain showed increased sensitivity toward hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of (15)N-labeled Par10 in the presence of the AhpC(115-132) peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10-AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline cis and trans isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as a substrate for Par10 enzymatic action. On the basis of these data, we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress.

De-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide Derivatives.

Targeted protein degradation via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clinical settings, in which the substrate specificity of CRBN is altered via the binding of small-molecule effectors. To date, such effectors are derived from thalidomide and confer a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular approach to design novel and minimalistic CRBN effectors. In this approach, we took advantage of the binding modes of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallography. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chemical moiety that imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to degrade the neo-substrate IKZF3 in the cell culture.

Characterization of MCU-Binding Proteins MCUR1 and CCDC90B - Representatives of a Protein Family Conserved in Prokaryotes and Eukaryotic Organelles.

Membrane-bound coiled-coil proteins are important mediators of signaling, fusion, and scaffolding. Here, we delineate a heterogeneous group of trimeric membrane-anchored proteins in prokaryotes and eukaryotic organelles with a characteristic head-neck-stalk-anchor architecture, in which a membrane-anchored coiled-coil stalk projects an N-terminal head domain via a ß-layer neck. Based on sequence analysis, we identify different types of head domains and determine crystal structures of two representatives, the archaeal protein Kcr-0859 and the human CCDC90B, which possesses the most widespread head type. Using mitochondrial calcium uniporter regulator 1 (MCUR1), the functionally characterized paralog of CCDC90B, we study the role of individual domains, and find that the head interacts directly with the mitochondrial calcium uniporter (MCU) and is destabilized upon Ca2+ binding. Our data provide structural details of a class of membrane-bound coiled-coil proteins and identify the conserved head domain of the most widespread type as a mediator of their function.

A new expression system for protein crystallization using trimeric coiled-coil adaptors.

We repeatedly experienced difficulties in obtaining pure protein of a defined oligomeric state when expressing domains that consist partially or entirely of coiled coils. We therefore modified an established expression vector, pASK-IBA, to generate N- and C-terminal fusions of the cloned domain in heptad register with the GCN4 leucine zipper. GCN4 is a well-characterized coiled coil, for which stable dimeric, trimeric and tetrameric forms exist. To test this expression system, we produced a series of constructs derived from the trimeric autotransporter adhesin STM3691 of Salmonella (SadA), which has a highly repetitive structure punctuated by coiled-coil regions. The constructs begin and end with predicted coiled-coil segments of SadA, each fused in the correct heptad register to the trimeric form of GCN4, GCN4pII. All constructs were expressed at high levels, trimerized either natively or after refolding from inclusion bodies, and yielded crystals that diffracted to high resolution. Thus, fusion to GCN4pII allows for the efficient expression and crystallization of proteins containing trimeric coiled coils. The structure of short constructs can be solved conveniently by molecular replacement using the known GCN4 structure as a search model. The system can be adapted for constructs with dimeric or tetrameric coiled coils, using the corresponding GCN4 variants.

Chemical Ligand Space of Cereblon.

The protein cereblon serves as a substrate receptor of a ubiquitin ligase complex that can be tuned toward different target proteins by cereblon-binding agents. This approach to targeted protein degradation is exploited in different clinical settings and has sparked the development of a growing number of thalidomide derivatives. Here, we probe the chemical space of cereblon binding beyond such derivatives and work out a simple set of chemical requirements, delineating the metaclass of cereblon effectors. We report co-crystal structures for a diverse set of compounds, including commonly used pharmaceuticals, but also find that already minimalistic cereblon-binding moieties might exert teratogenic effects in zebrafish. Our results may guide the design of a post-thalidomide generation of therapeutic cereblon effectors and provide a framework for the circumvention of unintended cereblon binding by negative design for future pharmaceuticals.

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.

α/ß coiled coils.

Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short ß-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/ß coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold.