Nonspecific binding of adenosine tripolyphosphate and tripolyphosphate modulates the phase behavior of lysozyme

DOI: 10.1021/jacs.2c09615

Authors: Matja Zalar (The University of Manchester) , Jordan Bye (The University of Manchester) , Robin A. Curtis (The University of Manchester)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of The American Chemical Society , VOL 2

State: Published (Approved)
Published: January 2023
Diamond Proposal Number(s): 24447

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.

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Added On: 09/01/2023 08:31

Documents:
jacs.2c09615.pdf

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)