Overcoming challenges in structural biology with integrative approaches and nanobody-derived technologies.

A full understanding of protein structure is key to unraveling how these systems work, how mutations affect their function, and discovering new hotspots for drug discovery. Research tackling this field began with the analysis of globular proteins. In recent years, as technology has improved, research efforts have broadened their focus to include the study of multidomain proteins and the analysis of conformational variability, flexibility, and dynamic systems. Here, we have selected five recent examples that integrate complementary structural methods to provide insight into the behavior of modular, flexible, and transient contacts. We also describe the structural application of domains derived from single-chain antibodies, which are instrumental in overcoming the size limitation of cryogenic electron microscopy (cryoEM) studies. As these methods are continuously developed, they will lead to the interrogation of more complex systems, revealing how large signaling and transcriptional machines are assembled in the context of health and disease.

De Novo Engineering of Pd-Metalloproteins and Their Use as Intracellular Catalysts.

The development of transition metal-based catalytic platforms that promote bioorthogonal reactions inside living cells remains a major challenge in chemical biology. This is particularly true for palladium-based catalysts, which are very powerful in organic synthesis but perform poorly in the cellular environment, mainly due to their rapid deactivation. We now demonstrate that grafting Pd(II) complexes into engineered ß-sheets of a model WW domain results in cell-compatible palladominiproteins that effectively catalyze depropargylation reactions inside HeLa cells. The concave shape of the WW domain ß-sheet proved particularly suitable for accommodating the metal center and protecting it from rapid deactivation in the cellular environment. A thorough NMR and computational study confirmed the formation of the metal-stapled peptides and allowed us to propose a three-dimensional structure for this novel metalloprotein motif.

Structures of the germline-specific Deadhead and thioredoxin T proteins from Drosophila melanogaster reveal unique features among thioredoxins.

Thioredoxins (Trxs) are ubiquitous enzymes that regulate the redox state in cells. In Drosophila, there are two germline-specific Trxs, Deadhead (Dhd) and thioredoxin T (TrxT), that belong to the lethal(3)malignant brain tumor signature genes and to the 'survival network' of genes that mediate the cellular response to DNA damage. Dhd is a maternal protein required for early embryogenesis that promotes protamine-histone exchange in fertilized eggs and midblastula transition. TrxT is testis-specific and associates with the lampbrush loops of the Y chromosome. Here, the first structures of Dhd and TrxT are presented, unveiling new features of these two thioredoxins. Dhd has positively charged patches on its surface, in contrast to the negatively charged surfaces commonly found in most Trxs. This distinctive charge distribution helps to define initial encounter complexes with DNA/RNA that will lead to final specific interactions with cofactors to promote chromatin remodeling. TrxT contains a C-terminal extension, which is mostly unstructured and highly flexible, that wraps the conserved core through a closed conformation. It is believed that these new structures can guide future work aimed at understanding embryo development and redox homeostasis in Drosophila. Moreover, due to their restricted presence in Schizophora (a section of the true flies), these structures can help in the design of small-molecular binders to modulate native redox homeostasis, thereby providing new applications for the control of plagues that cause human diseases and/or bring about economic losses by damaging crop production.