Structure and mechanism of a cytosine transport protein

CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB also to be important in the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence in the organism. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4Å resolution in complex with cytosine. We show that the protein carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 make specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.

The Impact of Retinal Configuration on the Protein–Chromophore Interactions in Bistable Jumping Spider Rhodopsin-1

Bistable rhodopsins have two stable forms that can be interconverted by light. Due to their ability to act as photoswitches, these proteins are considered as ideal candidates for applications such as optogenetics. In this work, we analyze a recently crystalized bistable rhodopsin, namely the jumping spider rhodopsin-1 (JSR1). This rhodopsin exhibits identical absorption maxima for the parent and the photoproduct form, which impedes its broad application. We performed hybrid QM/MM simulations to study three isomers of the retinal chromophore: the 9-cis, 11-cis and all-trans configurations. The main aim was to gain insight into the specific interactions of each isomer and their impact on the absorption maximum in JSR1. The absorption spectra were computed using sampled snapshots from QM/MM molecular dynamics trajectories and compared to their experimental counterparts. The chromophore–protein interactions were analyzed by visualizing the electrostatic potential of the protein and projecting it onto the chromophore. It was found that the distance between a nearby tyrosine (Y126) residue plays a larger role in the predicted absorption maximum than the primary counterion (E194). Geometric differences between the isomers were also noted, including a structural change in the polyene chain of the chromophore, as well as changes in the nearby hydrogen bonding network.

Comprehensive Profiling of Mammalian Tribbles Interactomes Implicates TRIB3 in Gene Repression

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles’ interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

Structure and dynamics of pathway activation by the Toll immunoreceptor from the viral mosquito vector Aedes aegypti

A. aegypti has evolved to become an efficient vector for arboviruses such as Dengue but the mechanisms of host-pathogen immune tolerance are unknown. Toll receptors and Spaetzle (Spz) ligands have undergone duplication raising the possibility of neofunctionalization and mutualism to develop between arboviruses and mosquitoes. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A-Spz1C complexes that display transient but specific interactions. Binding of the first ligand alters receptor-receptor interactions and promotes asymmetric contacts in the vicinity of the Z-loop in Toll5A. This conformation then restricts binding of a second ligand, while temporarily bridging the C-termini that promote signalling. Increased receptor concentrations promote inactivating head-to-head receptor assemblies. Furthermore, the transcriptional signature of Spz1C differs from other Spz cytokines in the control of genes involved in innate immunity, lipid metabolism and tissue regeneration. Given the remarkable DENV-induced expression patterns of these proteins, our data rationalises how Spz1C upregulation might promote antimicrobial defence in the midgut, and Toll5A upregulation, viral tolerance in the salivary glands.