Abstract Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.
Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B -factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.
The creation of complex neuronal networks relies on ligand-receptorReceptors interactions that mediate attraction or repulsion towards specific targets. Roundabouts comprise a family of single-pass transmembrane receptorsReceptors facilitating this process upon interaction with the soluble extracellular ligand SlitSlit proteinProtein family emanating from the midline. Due to the complexity and flexible nature of RoboRobo receptorsReceptors , their overall structureStructure has remained elusive until now. Recent structural studies of the RoboRobo 1 and RoboRobo 2 ectodomains have provided the basis for a better understanding of their signallingSignalling mechanism. These structuresStructure reveal how RoboRobo receptorsReceptors adopt an auto-inhibited conformation on the cell surface that can be further stabilised by cis and/or trans oligmerisation arrays. Upon SlitSlit -N binding RoboRobo receptorsReceptors must undergo a conformational change for Ig4 mediated dimerisation and signaling, probably via endocytosis. Furthermore, it's become clear that RoboRobo receptorsReceptors do not only act alone, but as large and more complex cell surface receptorReceptors assemblies to manifest directional and growth effects in a concerted fashion. These context dependent assemblies provide a mechanism to fine tune attractive and repulsive signals in a combinatorial manner required during neuronal development. While a mechanistic understanding of SlitSlit mediated RoboRobo signaling has advanced significantly further structural studies on larger assemblies are required for the design of new experiments to elucidate their role in cell surface receptorReceptors complexes. These will be necessary to understand the role of SlitSlit -Robo Robo signaling in neurogenesis, angiogenesis, organ development and cancerCancer progression. In this chapter, we provide a review of the current knowledge in the field with a particular focus on the Roundabout receptorReceptors family.
Pathogenic species from the Mycobacterium genus are responsible for a number of adverse health conditions in humans and animals that threaten health security and the economy worldwide. Mycobacteria have up to five specialized secretion systems (ESX-1 to ESX-5) that transport virulence factors across their complex cell envelope to facilitate manipulation of their environment. In pathogenic species, these virulence factors influence the immune system's response and are responsible for membrane disruption and contributing to cell death. While structural details of these secretion systems have been recently described, gaps still remain in the structural understanding of the secretion mechanisms of most substrates. Here, we describe the crystal structure of Mycobacterium tuberculosis ESX-1 secretion-associated substrate EspB bound to its chaperone EspK. We found that EspB interacts with the C-terminal domain of EspK through its helical tip. Furthermore, cryogenic electron microscopy, size exclusion chromatography analysis, and small-angle X-ray scattering experiments show that EspK keeps EspB in its secretion-competent monomeric form and prevents its oligomerization. The structure presented in this study suggests an additional secretion mechanism in ESX-1, analogous to the chaperoning of proline-glutamate (PE)-proline-proline-glutamate (PPE) proteins by EspG, where EspK facilitates the secretion of EspB in Mycobacterium species.
New therapeutic strategies targeting influenza are actively sought due to limitations in current drugs available. Host-directed therapy is an emerging concept to target host functions involved in pathogen life cycles and/or pathogenesis, rather than pathogen components themselves. From this perspective, we focused on an essential host partner of influenza viruses, the RED–SMU1 splicing complex. Here, we identified two synthetic molecules targeting an α-helix/groove interface essential for RED–SMU1 complex assembly. We solved the structure of the SMU1 N-terminal domain in complex with RED or bound to one of the molecules identified to disrupt this complex. We show that these compounds inhibiting RED–SMU1 interaction also decrease endogenous RED-SMU1 levels and inhibit viral mRNA splicing and viral multiplication, while preserving cell viability. Overall, our data demonstrate the potential of RED-SMU1 destabilizing molecules as an antiviral therapy that could be active against a wide range of influenza viruses and be less prone to drug resistance.
