Plancqueel, S. | bioRxiv Science

bioRxiv2024

The monomer/dimer switch modulates the activity of plant adenosine kinase

HighlightThe switch from active monomers to inactive dimers in plant ADKs impacts overall enzyme activity and represents a novel negative feedback-loop mechanism to maintain steady levels of adenosine and AMP. Adenosine undergoes ATP-dependent phosphorylation catalyzed by adenosine kinase (ADK). In plants, ADK also phosphorylates cytokinin ribosides, transport forms of the hormone. Here, we investigated the substrate preferences, oligomeric states and structures of ADKs from moss (Physcomitrella patens) and maize (Zea mays) alongside metabolomic and phenotypic analyses. We showed that dexamethasone-inducible ZmADK overexpressor lines in Arabidopsis can benefit from a higher number of lateral roots and larger root areas under nitrogen starvation. We discovered that maize and moss enzymes can form dimers upon increasing protein concentration, setting them apart from the monomeric human and protozoal ADKs. Structural and kinetic analyses revealed a catalytically inactive unique dimer. Within the dimer, both active sites are mutually blocked. The activity of moss ADKs, exhibiting a higher propensity to dimerize, was tenfold lower compared to maize ADKs. Two monomeric structures in a ternary complex highlight the characteristic transition from an open to a closed state upon substrate binding. This suggests that the oligomeric state switch can modulate the activity of moss ADKs and likely other plant ADKs. Moreover, dimer association represents a novel negative feedback mechanism, helping to maintain steady levels of adenosine and AMP.

plant biology↗

bioRxiv2023

Functional and structural insights into the interaction and multi-step activation of bacterial ExoY nucleotidyl cyclases toxins by profilin-bound G-actin

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs are actin-activated NC toxins. Here, we investigate the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) rather than filamentous (F-actin) actin. Vn-ExoY binds with only modest affinity to free or profilin-bound G-actin, but can capture the G-actin:profilin complex for its own activation by preventing the spontaneous or VASP- or formin-mediated assembly of G-actin:profilin at the barbed ends of F-actin in vitro. This may prolong the lifetime of the cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. A series of high-resolution crystal structures of nucleotide-free, 3-deoxy-ATP- or 3-deoxy-CTP-bound Vn-ExoY, activated by free or profilin-bound G-actin-ATP/-ADP show that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of all NC toxins. Substrate binding promotes a large, previously-unidentified, closure of their NBP. This confines catalytically important residues of the NC toxins and metal cofactors around the substrate and promotes the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. Residues that play an important role in both the purinyl and pyrimidinyl cyclase activity of NC toxins are validated in Vn-ExoY and the distantly-related ExoY from Pseudomonas aeruginosa that interact with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation reaction of nucleotides of different sizes. These structural insights into the dynamics of the actin-binding interface of actin-activated ExoYs and the multi-step activation of all NC toxins open up new perspectives for identifying ways to specifically inhibit these bacterial NC enzymes. Author SummaryExoY toxins belong to a family of bacterial nucleotidyl cyclases (NCs) that are injected into eukaryotic cells and bind to specific host cofactors to trigger their toxic, potent NC enzymatic activity. They alter host cell signalling by overproducing purine and pyrimidine cyclic nucleotides, which act as canonical and non-canonical intracellular messengers, respectively. The molecular and mechanistic details underlying the activation and catalytic specificities of NC toxins are only partially understood. Here, we investigate ExoY-type members that are unable to interact with actin filaments for their activation. We show in vitro that such ExoYs capture the actin:profilin complex for activation by disrupting its association with the most dynamic ends of actin filaments. We have captured several structural snapshots along the Vn-ExoY activation pathway by G-actin or G-actin-profilin without or with purine or pyrimidine nucleotide analogues. Our structural data reveal unprecedented mechanistic details of how the active site of all NC toxins is sequentially remodelled by cofactor and substrate binding, how they can accommodate nucleotides of different sizes as substrates, and elucidate important features of their catalytic reaction. These structural insights into the multi-step activation of NC toxins provide new perspectives for identifying ways to specifically inhibit this class of NC enzymes.

biophysics↗