Functional characterization of the ATPase-like activity displayed by a catalytic amyloid

https://doi.org/10.1016/j.bbagen.2020.129729Get rights and content

Highlights

  • Peptide SDIDVFI self-assembles into a catalytic amyloid with ATPase-like activity.

  • Hydrolysis of phosphoanhydride bonds is achieved with different ribonucleotides.

  • One aspartate residue is sufficient to exert hydrolytic activity.

  • Secondary amyloid-substrate interactions may modulate activity.

Abstract

Background

Amyloids are highly ordered polypeptide aggregates stabilized by a beta-sheet structural core. Though classically associated to pathology, reports on novel functional roles of these proteins have increasingly emerged in the past decade. Moreover, the recent discovery that amyloids formed with rationally designed small peptides can exhibit catalytic reactivity has opened up new opportunities in both biology and biotechnology. The observed activities typically require the binding of divalent metals, giving rise to active metal-amyloid complexes.

Methods

Peptide (SDIDVFI) was aggregated in vitro. The structure of the self-assembled species was analyzed using fluorescence, transmission electron microscopy, circular dichroism and computational modeling. A kinetic characterization of the emerging catalytic activity was performed.

Results

The peptide self-assembled into canonical amyloids that exhibited catalytic activity towards hydrolysis of the phosphoanhydride bonds of adenosine triphosphate (ATP), partially mimicking an ATPase-like enzyme. Both amyloid formation and activity are shown to depend on manganese (Mn2+) binding. The activity was not restricted to ATP but also affected all other ribonucleotides (GTP, CTP and UTP). Peptides carrying a single aspartate exhibited a similar activity.

Conclusions

The phosphoanhydride bonds appear as the main specificity target of the Mn2+-amyloid complex. A single aspartate per peptide is sufficient to enable the hydrolytic activity.

General significance

Catalytic amyloids are shown for the first time to catalyze the hydrolysis of all four ribonucleotides. Our results should contribute towards understanding the biological implications of amyloid-mediated reactivity as well as in the design of future catalytic amyloids for biotechnological applications.

Introduction

Protein and peptides are known to undergo conformational transitions under certain circumstances into highly ordered intermolecular aggregates known as amyloids [1]. The formation of amyloid structures can occur both in vivo and in vitro. The formation of amyloids in vivo has been classically associated to the onset and progression of different pathologies such as Alzheimer's, Parkinson's and Creutzfeldt–Jakob diseases, among many others [2,3]. The common element on these maladies is the progressive accumulation of amyloid structures in different regions of the brain. Although the precise mechanisms of toxicity for these diseases are not completely understood, there are several pathways of toxicity that have been shown to be relatively general and directly associated to these structures and their formation process [4,5].

The classical association of amyloids with pathology has been greatly contrasted in the last two decades with the increasing reports of newly discovered amyloids that function as part of the normal physiology of organisms [6,7]. These “functional amyloids” appear to be ubiquitous in nature, since their presence has been described not only in mammals but also in yeast and bacteria [7,8]. Such widespread occurrence seems to correlate with the seemingly universality of the amyloid fold [9].

Amyloids are very stable conformations that exhibit convergent structural features such as the presence of a central beta-sheet core stabilized by intermolecular contacts [10]. These contacts include hydrogen bonds and hydrophobic interactions that stabilize the overall conformation. The aggregates typically form elongated fibrillar structures that grow indefinitely and perpendicular to the beta-sheet axis [11]. Such structural arrangement allows for several residue groups to be exposed to the solvent in a highly ordered and repetitive fashion. Such regularity can in principle allow the emergence of surface reactivity when certain reactive groups are strategically placed in the amyloid sequence, giving rise to catalytically active amyloids that partially mimic the active sites of certain enzymes [12]. A proof of this concept was reported several years ago with the development of the first catalytic amyloid [13]. Alternation of histidines with hydrophobic and polar residues in 7-mer peptides was shown to yield self-assembled peptide amyloids exhibiting an esterase-like activity towards the chemical substrate analog 4-nitrophenyl acetate when zinc ions (Zn2+) were present in the mixture. The amyloid structure resembled the active site of carbonic anhydrase, which have its catalytic Zn2+-bound histidines located in a beta-sheet motif. Follow-up works showed that modulating the sequence of these peptides results in catalytic amyloids with different degrees of esterase-like activity, evidencing a direct correlation between the activity and both the exposed residue groups and the amyloid structure [14].

Our group recently developed a different type of catalytic amyloid based on the negatively charged active sites of nucleotidyltransferases [15]. The self-assembled peptide (NADFDGDQMAVHV) contained the conserved sequence of the active site of multi-subunit RNA polymerases, which is also located within a beta-sheet motif. As in the histidine-based catalytic amyloids, our peptide contained alternating charged residues as the active groups. However, the peptide exhibited hydrolytic activity towards the phosphoanhydride bonds of ATP. This ATPase-like activity was strictly dependent on manganese ions (Mn2+). Considering that ATP is a ubiquitous and fundamental molecule in biological systems, we were interested in studying essential enzymatic aspects such as substrate specificity but the size and complexity of the peptide sequence along with the lack of tools to model its assembled amyloid state made such analysis difficult. In this work, we report the development of a catalytic amyloid displaying an ATPase-like activity based on a peptide carrying the sequence (SDIDVFI) of the active site of a DNA polymerase. The simple and smaller sequence of this peptide enabled us to perform for the first time a detailed characterization of the amyloid-based ATPase-like activity in terms of substrate specificity as well as the sequence-dependence. We expect our results to give new insights on the activity of catalytic amyloids and also to pave the way towards the future rational design of novel catalytic amyloids with desired activities.

Section snippets

Chemicals

All reagents were purchased of the highest quality available from Sigma-Aldrich (St. Louis, MO, USA). Peptides were chemically synthesized at >98% purity by Genscript (NJ, USA) with acetylated N-terminal and amidated C-terminal.

Peptide sample preparation

All peptides were dissolved in 10 mM sodium hydroxide to a concentration of 4 mM for the preparation of concentrated stocks. For the working solutions, the peptides from freshly prepared stocks were diluted to 500 μM in solutions containing 50 mM Tris-HCl pH 8.0 and

Self-assembly of SDIDVFI into amyloids

In a previous work, we explored the self-assembly into amyloids of several negatively charged 7-mer peptides that alternated carboxylate groups in their sequence and therefore had the potential to convey a similar ATPase-like activity to that observed with our previous catalytic amyloid formed by peptide NADFDGDQMAVHV, by partially mimicking the active sites of nucleotydiltransferases [19]. All those peptides exhibited a metal-dependent transition to the amyloid state at neutral and basic pHs

Amyloid-mediated activity of peptide SDIDVFI

Most catalytic amyloids described so far are known to convey their activities through formation of active metal-amyloid complexes [23]. Acceleration rate is believed to proceed through a combination of surface-based effects and the presence of regularly arranged catalytic residues partially mimicking the active sites of enzymes [13,24,25]. Peptide SDIDVFI exhibited both features. The overall tridimensional location of the aspartate groups in our models is similar to that observed for similar

Conclusions

We have developed a catalytic amyloid that displays an ATPase-like activity. The small sequence size of the peptide allowed us to explore essential aspects of enzymatic activity such as substrate specificity and sequence dependence. For the first time, we show that the activity is not restricted to ATP but can also be exerted towards other types of nucleotides, including deoxyribonucleotides, placing the phosphoanhydride bond as the specific target for hydrolysis. The finding that the bond

Funding

This work was supported by research grants CONICYT PAI/CONCURSO NACIONAL INSERCIÓN EN LA ACADEMIA, CONVOCATORIA 2016 FOLIO PAI79160143, CONICYT/FONDECYT/11160554 and Proyecto DICYT código 021943DE-PAP Universidad de Santiago de Chile, USACH.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We would like to thank Tatiana Cruces Ayala for her contributions on some of the aggregation experiments.

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