Droplet and fibril formation of the functional amyloid Orb2.

The functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low-complexity sequences similar to many RNA-binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility, and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. Using fluorescence microscopy, we show that soluble Orb2 can indeed phase separate into protein droplets. However, fluorescence recovery after photobleaching (FRAP) data shows that these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. Orb2 fragments that lack the C-terminal RNA-binding domain (RBD) form fibrils out of these droplets. Solid-state NMR shows that these fibrils have well-ordered static domains in addition to the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.

Structural Model of the Proline-Rich Domain of Huntingtin Exon-1 Fibrils.

Huntington's disease is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine domain that increases the propensity of huntingtin exon-1 to form cross-ß fibrils. Although the polyglutamine domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of huntingtin exon-1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800-ns long molecular dynamics simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired electron paramagnetic resonance double electron-electron resonance distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline (polyP) II helical conformation. The two polyP regions within the PRD stay in a polyP II helix for most of the simulation, whereas occasional kinks in the proline-rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD.

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

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 ß-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.

Dynamic domains of amyloid fibrils can be site-specifically assigned with proton detected 3D NMR spectroscopy.

Several amyloid fibrils have cores framed by highly dynamic, intrinsically disordered, domains that can play important roles for function and toxicity. To study these domains in detail using solid-state NMR spectroscopy, site-specific resonance assignments are required. Although the rapid dynamics of these domains lead to considerable averaging of orientation-dependent NMR interactions and thereby line-narrowing, the proton linewidths observed in these samples is far larger than what is regularly observed in solution. Here, we show that it is nevertheless possible to record 3D HNCO, HNCA, and HNcoCA spectra on these intrinsically disordered domains and to obtain site-specific assignments.