Nonspecific Binding of Adenosine Tripolyphosphate and Tripolyphosphate Modulates the Phase Behavior of Lysozyme

Abstract

Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein−ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme−ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein−protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.

Liquid-Droplet-Mediated ATP-Triggered Amyloidogenic Pathway of Insulin-Derived Chimeric Peptides: Unraveling the Microscopic and Molecular Processes

“…Because such flanking sequences also occur in vivo , mechanistic insights into their role in the amyloidogenic context are essential for the understanding of pathogenesis of amyloid-associated diseases. The use of ATP is also motivated by the fact that ATP serves not only as an energy source to drive biochemical reactions but also, owing to its high intracellular concentration, affects protein stability, solubility, and aggregation propensity. − The attraction with peptides can be due to the adenosin part for neutral side chains and is due to the triphosphate group for positively charged side chains as studied here. Our expectation was that for sufficiently long poly-K chains, the tendency to form liquid droplets should compete with the canonical nucleation/elongation pathway of amyloid fibril formation.…”

Adenosine Triphosphate Mediates Phase Separation of Disordered Basic Proteins by Bridging Intermolecular Interaction Networks

“…Biphasic effects of ATP on the phase separation of the disordered C-terminal region of CAPRIN1 and the condensation of the folded basic protein lysozyme were also reported. 28,29 This effect was recapitulated by coarse-grained molecular dynamics (MD) simulations. 30 The interactions of ATP with proteins have been probed by various techniques.…”

“…33 For lysozyme, 13 C HSQC identified six ATP binding sites, all of which contain arginine but only one contains lysine, suggesting a strong preference of ATP for arginine over lysine. 29 Three of these sites were captured by a lysozyme-ATP crystal structure. Interestingly, in the crystal lattice, the ATP molecules bridge between lysozyme molecules.…”

“…Likewise, Dang et al showed that ATP up to 1.5 mM promoted the phase separation of the TDP-43 prion-like domain but the promotional effect diminishes with a further increase in ATP concentration, reminiscent of the biphasic regulatory effects of RNA. Biphasic effects of ATP on the phase separation of the disordered C-terminal region of CAPRIN1 and the condensation of the folded basic protein lysozyme were also reported. , This effect was recapitulated by coarse-grained molecular dynamics (MD) simulations …”

Protein–macrocycle polymorphism: crystal form IV of the Ralstonia solanacearum lectin–sulfonato-calix[8]arene complex

“…Despite differences in the crystallization mechanisms, either ammonium sulfate (salting-out) or polyethylene glycol (depletion attraction) can act as the precipitant. Chicken egg-white lysozyme ($14 kDa), which is possibly the best crystallographically characterized protein, with $1000 entries in the Protein Data Bank (PDB), crystallizes in at least six space groups (most frequently in P4 3 2 1 2), with some evidence that anion binding can select the space group (Lanza et al, 2019;Plaza-Garrido et al, 2018;Vaney et al, 2001;Zalar et al, 2023). Co-crystals of the polyanionic sulfonato-calix [4]arene with lysozyme or methylated lysozyme reveal the polyanion to play key roles in the crystal packing (McGovern et al, 2014(McGovern et al, , 2015.…”