ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism

Abstract

A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering. Monomer loss kinetic studies, combined with measurements of native state protein–protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.

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

“…As such, while binding affinities for TPP in sites III, V, and VI could not be determined, we suspect similar strengths of interaction with the protein for TPP and ATP, where the predominant mode of interaction occurs through phosphate groups. This is also evident from overlaying the precipitation boundaries (Figure A,B), protein–protein interaction (Figure C,D), and ζ-potential profiles (Figure E,F) for ATP versus TPP and from the almost identical effectiveness of ATP and TPP at preventing the aggregation of globular proteins under thermal stress. , …”

“…For natively folded proteins, molecular simulations indicate that binding to loop regions leads to a reduction in their flexibility. Because these groups are often the most susceptible to unfolding upon heating, it has been hypothesized that ATP binding not only increases the protein’s thermal stability but the unfolded regions are less likely to associate due to preferential interactions with ATP clusters. , On the other hand, we showed that ATP or TPP binds to negatively charged proteins, leading to an overcharging effect and an increase in colloidal stability, which slows down aggregate growth rates . In contrast, for positively charged proteins like lysozyme and the IDP histatin-5, low concentrations of TPP have the opposite effect of causing protein precipitation. , Similarly, ATP has been shown to increase fibril formation for a series of basic IDPs, as well as an insulin fragment conjugated to octalysine .…”

“…This is also evident from overlaying the precipitation boundaries (Figure 1A,B), protein−protein interaction (Figure 1C,D), and ζ-potential profiles (Figure 1E,F) for ATP versus TPP and from the almost identical effectiveness of ATP and TPP at preventing the aggregation of globular proteins under thermal stress. 27,28 Our results indicate the nature of ATP binding to folded protein surfaces is markedly different from binding to IDPs and IDRs; for folded protein surfaces, the TPP moiety determines the non-specific interaction, and only for the specific binding site on lysozyme (site I) is there a difference between ATP and TPP affinities. On the other hand, there are more significant differences between TPP and ATP binding to IDPs and IDRs with the adenosine ring increasing the specificity and affinity of these interactions.…”

“…Consistent with this hypothesis, ATP is effective at suppressing aggregation upon thermal stress for lysozyme, ovalbumin, BSA, and ribonuclease A, but not α-chymotrypinogen (α-Cgn), which is the only protein of the group with a low arginine content. 18,27,28 ■ CONCLUSIONS In summary, we performed a comprehensive structural study of ATP and TPP interaction with lysozyme combining crystallography, NMR, and light scattering. Here, we have identified many more non-specific binding sites of ATP with millimolar affinities than previously observed in NMR studies of non-specific interactions of ATP with model proteins 32 and arginine-rich crystallins.…”

“…32,33 On the other hand, we showed that ATP or TPP binds to negatively charged proteins, leading to an overcharging effect and an increase in colloidal stability, which slows down aggregate growth rates. 28 In contrast, for positively charged proteins like lysozyme and the IDP histatin-5, low concentrations of TPP have the opposite effect of causing protein precipitation. 29,34 Similarly, ATP has been shown to increase fibril formation for a series of basic IDPs, 35 as well as an insulin fragment conjugated to octalysine.…”