Evolution of acyl-ACP thioesterases and β-ketoacyl-ACP synthases revealed by protein–protein interactions

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein–protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners, whereas KSs showed promiscuous behavior across bacterial, plant, and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic cross-linking, and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.     

Alternative modulation of protein–protein interactions by small molecules

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Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

REDOR-based experiments with simultaneous ¹H–¹³C and ¹H?¹⁵N dipolar dephasing are explored for investigating intermolecular protein–protein interfaces in complexes formed by a U–¹³C,¹⁵N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U–¹³C,¹⁵N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by ¹H–¹³C or ¹H–¹⁵N cross polarization, permitting to establish the residues of the U–¹³C,¹⁵N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into ¹³C–¹³C correlation experiments. We established the validity of this approach on U–¹³C,¹⁵N-histidine and on a structurally characterized complex of dynactin’s U–¹³C,¹⁵N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.     

Scoring functions for protein–protein interactions

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Trimethylsilyl tag for probing protein–ligand interactions by NMR

Protein–ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D ¹H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B–NS3 protease and the human prolyl isomerase FK506 binding protein.     

Comparative mapping of host–pathogen protein–protein interactions

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Peptide-based covalent inhibitors of protein–protein interactions

Protein–protein interactions (PPI) are involved in all cellular processes and many represent attractive therapeutic targets. However, the frequently rather flat and large interaction areas render the identification of small molecular PPI inhibitors very challenging. As an alternative, peptide interaction motifs derived from a PPI interface can serve as starting points for the development of inhibitors. However, certain proteins remain challenging targets when applying inhibitors with a competitive mode of action. For that reason, peptide-based ligands with an irreversible binding mode have gained attention in recent years. This review summarizes examples of covalent inhibitors that employ peptidic binders and have been tested in a biological context.     

Molecular glues to stabilise protein–protein interactions

Targeting protein–protein interactions (PPIs) has become a common approach to tackle various diseases whose pathobiology is driven by their mis-regulation in important signalling pathways. Modulating PPIs has tremendous untapped therapeutic potential and different approaches can be used to modulate PPIs. Initially, therapeutic effects were mostly sought by inhibiting PPIs. However, by gaining insight in the mode of action of certain therapeutic compounds, it became clear that stabilising (i.e. enhancing) PPIs can also be useful. The latter strategy is recently gaining a lot of attention, as stabilising physiologic, or even inducing novel interactions of a target protein with E3 ubiquitin ligases forms the basis of the targeted protein degradation (TPD) approach. An emerging additional example for drug discovery based on PPI stabilisation are the 14-3-3 proteins, a family of regulatory proteins, which engages in many protein–protein interactions, some of which might become therapeutical targets.     

More than protein? Bee–flower interactions and effects of disturbance regimes revealed by rare pollen in bee nests

Bees and their host flower populations were studied by identifying pollen to species or genus, from trap nests where bees were reared. Rare plant species in bee diets, and disturbance regimes, have not previously been researched and are emphasized here. Two focal bee groups with one species each (Megachilidae and Apidae) were studied in a 500,000-ha tropical reserve in the Yucatán Peninsula nine complete years. The number of rare or major pollen species in nests had no statistical correlation; thus, rare pollen analysis complements study of major brood provisions. We found most nests (87 % Megachile zaptlana, 93 % Centris analis) contained rare pollen; only 12 % of the 438 nests contained major pollen alone. Rare pollen sometimes indicated an energy source rather than a scarce protein resource. Trichome nectar of Cydista, along with Ipomoea and Caesalpinia, were nectar sources. Malpighiaceae, despite lacking nectar, often provided the complete Centris diet. Considering rare pollen, only Centris responded to drought, or competition from immigrant honeybees. Neither bee responded to hurricanes. Drought years coincided with low bee populations; Centris nests contained more rare species then. After feral Africanized honeybees colonized, Centris had more major species and fewer rare. Some herbarium vouchers from the study area contained exotic pollen, demonstrating in situ floral contamination and ecological generalization by bees, but this rarely occurred in plants found among the bee diets. Megachile and Centris responded differently to competition and resource scarcity, and plausibly evolved under different disturbance regimes, yet appeared well adapted to hurricane disturbance.     

Probing membrane protein–lipid interactions

Structure determination of membrane proteins has highlighted the many roles played by lipids in influencing overall protein architecture. It is now widely accepted that lipids surrounding membrane proteins play crucial roles by modulating their conformational, structural, and functional properties. Capturing often transient lipid interactions and defining their chemical identity, however, remains challenging. Recent advances in mass spectrometry have resolved questions concerning lipid interactions by providing the molecular composition of intact complexes in association with lipids. Together with other biophysical tools, a picture is emerging of the dynamic nature of lipid-mediated interactions and their effects on conformation, interactions, and signaling.