Activation of an anti-bacterial toxin by the biosynthetic enzyme CysK: mechanism of binding, interaction specificity and competition with cysteine synthase

Abstract

Contact-dependent growth inhibition (CDI) is a wide-spread mechanism of inter-bacterial competition. CDI+ bacteria deliver CdiA-CT toxins into neighboring bacteria and produce specific immunity proteins that protect against self-intoxication. The CdiA-CT toxin from uropathogenic Escherichia coli 536 is a latent tRNase that is only active when bound to the cysteine biosynthetic enzyme CysK. Remarkably, the CysK:CdiA-CT binding interaction mimics the ‘cysteine synthase’ complex of CysK:CysE. The C-terminal tails of CysE and CdiA-CT each insert into the CysK active-site cleft to anchor the respective complexes. The dissociation constant for CysK:CdiA-CT (K d ~ 11 nM) is comparable to that of the E. coli cysteine synthase complex (K d ~ 6 nM), and both complexes bind through a two-step mechanism with a slow isomerization phase after the initial encounter. However, the second-order rate constant for CysK:CdiA-CT binding is two orders of magnitude slower than that of the cysteine synthase complex, suggesting that CysE should outcompete the toxin for CysK occupancy. However, we find that CdiA-CT can effectively displace CysE from pre-formed cysteine synthase complexes, enabling toxin activation even in the presence of excess competing CysE. This adventitious binding, coupled with the very slow rate of CysK:CdiA-CT dissociation, ensures robust nuclease activity in target bacteria.

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action

“…The authors, in addition to the remarkable finding of a moonlighting function for CysK, proposed that the toxin forms a complex with CysK using the same structural motif used by CysE (i.e., insertion of the C-terminus in CysK active site). Further structural [70] and functional [24] studies confirmed the original finding and shed light on the details of complex formation and toxin activation. CysK/CdiA-CT complex is formed by two toxin monomers that bind to one CysK dimer with the C-terminal carboxylate engaging to the same binding site that is occupied by the substrate of the enzyme (Figure 2A).…”

“…Cysteine metabolism (i.e., de novo biosynthesis and degradation) [17], is intimately connected with many bacterial functions relevant to infection, such as resistance to oxidative stress and resistance to antibiotics [18,19,20,21,22], biofilm formation [23], and toxin activation [24,25]. For this reason, this metabolic pathway has received much attention in the last ten years as a potential target for the development of antibiotics or antibiotic enhancers [17,26,27,28,29,30,31,32,33,34,35,36,37,38].…”

Refining the structure−activity relationships of 2-phenylcyclopropane carboxylic acids as inhibitors of O-acetylserine sulfhydrylase isoforms

“…This hampers the accurate determination of the inhibition mechanism due to limitations to the range of inhibitor concentration that can be explored. However, since partial inhibition is a typical feature of allosteric inhibitors, we used a method developed by our group 24 to assess if compound 25 is able to displace a low affinity active site binder, 1-ethylcyclopropane-1,2-dicarboxylic acid, that forms a highly fluorescent complex with OASS. Compound 25 is unable to displace the compound up to an equimolar concentration of about 200 μM.…”

“…When the K d was lower than the concentration of the protein used in the assay, the dependence of the fluoresecnce emission intensity on ligand concentration was fitted to a quadratic equation that describes tight binding 23 . The fluorescence competitive binding assay, described elsewhere 24 , was used to evaluate whether compounds bind at the active site of the enzyme or to an allosteric site. The assay is based on the formation of a low-affinity, highly fluorescent complex between OASS and 1-ethylcyclopropane-1,2-dicarboxylic acid.…”

Insights into multifaceted activities of CysK for therapeutic interventions

“…The presence of an immunity protein known as CdiI is responsible for the phenomenon (Aoki et al 2011). CdiI forms a complex with CdiA-CT to neutralize its tRNase activity but apparently this complex dissociates rapidly (Benoni et al 2017;Kaundal et al 2016). A study by Kaundal et al categorically demonstrates the role of CysK in stabilizing the binary complex CdiI/CdiA-CT with ~ 130 fold decrease in dissociation rate therefore, proving CysK to be a modulator of CdiA-CT/CdiI activity (Kaundal et al 2016;Johnson et al 2016).…”