Identification and Structural Characterization of the N-terminal Amyloid Core of Orb2 isoform A

Silvia A Cervantes,Ansgar B Siemer,Thalia H Bajakian,Alexander S Falk,Maria A Soria,Rachel J Service,Ralf Langen

doi:10.1038/srep38265

Abstract

Orb2 is a functional amyloid that plays a key role in Drosophila long-term memory formation. Orb2 has two isoforms that differ in their N-termini. The N-terminus of the A isoform (Orb2A) that precedes its Q-rich prion-like domain has been shown to be important for Orb2 aggregation and long-term memory. However, besides the fact that it forms fibrillar aggregates, structural information of Orb2 is largely absent. To understand the importance of the N-terminus of Orb2A and its relation to the fibril core, we recorded solid-state NMR and EPR data on fibrils formed by the first 88 residues of Orb2A (Orb2A88). These data show that the N-terminus of Orb2A not only promotes the formation of fibrils, but also forms the fibril core of Orb2A88. This fibril core has an in-register parallel β-sheet structure and does not include the Q-rich, prion-like domain of Orb2. The Q-rich domain is part of the unstructured region, which becomes increasingly dynamic towards the C-terminus.

Highlights

There are two Orb[2] isoforms, Orb2A and Orb2B
We present the first detailed structural study of fibrils formed by Orb2A88 to address two specific questions: (1) What is the role of the N-terminal domain of Orb2A in amyloid formation? (2) What is the exact location of the amyloid core of Orb2A88? We find that the N-terminal domain of Orb2A can form amyloid by itself and that the Q-rich domain is less important for amyloid formation than originally thought
We found that Orb2A88 forms fibrils under a variety of different conditions and our NMR spectra of Orb2A88 fibrils showed no batch dependence nor did they depend on whether they were prepared quiescent at 4 °C or agitated at 25 °C, in the presence and absence of glycerol, MgCl2, and 100–200 mM NaCl

Summary

Introduction

There are two Orb[2] isoforms, Orb2A and Orb2B. Both isoforms share a C-terminus containing domains common to all CPEBs: two RNA recognition motifs (RRMs) and a zinc finger[10]. Orb2A, and in particular its Q-rich and N-terminal domains, is essential for the formation of amyloid-like aggregates by the isoform Ob2B and for LTM. Orb2A functions without its RNA binding domain (RBD), whereas Orb2B is still able to induce LTM in the absence of its Q-rich domain[11] Taken together, these data suggest that the N-terminus of Orb2A is essential for Orb2B fibril formation and function. We find that the N-terminal domain of Orb2A can form amyloid by itself and that the Q-rich domain is less important for amyloid formation than originally thought These results give a direct structural explanation for why Orb2A can aggregate more readily than Orb2B

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Results

Conclusion