Ure2p of S. cerevisiae
is a 354-residue protein, consisting of a 245-residue C-terminal domain (CTD) with a globular fold, structurally homologous to glutathione S-transferase1,2
, and a 110-residue, Asn- and Gln-rich N-terminal segment that is natively unfolded3-5
. Under typical experimental conditions, the CTD dimerizes through multiple noncovalent interactions, principally hydrophobic in nature, on its flat face1,2,6
. The normal biological function of Ure2p is to regulate genes involved in catabolism of certain nitrogen sources by binding to the transcription factor Gln3p. [URE3], a non-Mendelian genetic element of S. cerevisiae
described in 1971 by Lacroute7
, was shown in 1994 to be an autoinactivating infectious form of Ure2p8
, making Ure2p the first non-mammalian prion protein to be identified. Aggregation of Ure2p into amyloid fibrils proved to be the basis of [URE3]9,10
, as is likely the case for the mammalian prion diseases11-14
. [URE3] is undetectable in wild yeast strains15
, implying that [URE3] is most likely a prion “disease”, not an advantageous or functional condition.
Polypeptides from the N-terminal segment (e.g.
, and Ure2p10-3910,16,17
, with Ure2pn-m
representing residues n to m of Ure2p) form amyloid fibrils in vitro
, protease treatment of full-length Ure2p amyloid yields only N-terminal fragments18
, [URE3] propagates stably in cells that express only the N-terminal segment (but not in cells that express only the CTD)19
, and protease-treated Ure2p amyloid induces [URE3] when transfected into S. cerevisiae20
. Overexpression of N-terminal polypeptides (e.g.
) within S. cerevisiae
also induces [URE3]9
. Therefore, the N-terminal segment (more specifically, the Asn- and Gln-rich portion thereof, residues 1-89) is the “prion domain” (PD) of Ure2p. Solid state nuclear magnetic resonance (NMR) measurements on Ure2p1-8916
fibrils indicate that the cross-β structure within these fibrils, identified by electron diffraction21
, has an in-register parallel organization, as also observed in full-length β-amyloid fibrils22
, amylin fibrils23
, and other yeast prion fibrils24-27
. In-register, parallel β-sheet formation is apparently favored by intermolecular interactions among Gln and/or Asn sidechains, which are automatically aligned by the parallel structure so as to permit “polar zipper” interactions28-31
regardless of the order or spacing of Gln and Asn residues within the amino acid sequence17,25
Recently, Loquet et al.
have reported the first solid state NMR studies of full-length Ure2p fibrils, prepared with uniform 15
N and 13
. Two-dimensional (2D) solid state NMR spectra of these fibrils clearly show strong, sharp signals attributable to the CTD, nearly identical to the signals observed in 2D spectra of microcrystals formed by the CTD alone. Thus, it appears that the CTD retains its globular fold in full-length Ure2p fibrils. This observation is consistent with experiments by Baxa et al.
, which showed that fibrils formed by fusions of the Ure2p PD with several globular partners (including barnase, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein) retain the activity of the partner protein33
and that the calorimetric signature of the CTD unfolding is identical in soluble and fibrillized Ure2p3
, as well as experiments by Bai et al.
, which showed that Ure2p retains glutathione peroxidase activity in the amyloid state34
. An important implication of the solid state NMR data of Loquet et al.
is that the CTD is not highly mobile in full-length Ure2p fibrils32
, ruling out the possibility that CTD monomers are tethered to the fibril core by a disordered linker segment that is sufficiently long to permit large and rapid reorientational motions of the CTD.
Loquet et al.
suggest that their results may support a model in which the core of biologically relevant Ure2p fibrils is constructed from CTD dimers, with the N-terminal segment located on the periphery (see Fig. S9
of ref. 32
). Such a model is inconsistent with previous work described above, especially the infectivity of fibrils that contain only the PD, the induction of [URE3] by overexpression of the PD, and the stable propagation of [URE3] in cells that express only the PD9,19,20
. An alternative interpretation of the solid state NMR results of Loquet et al.
is that the PD forms a rigid, cross-β core that is contained within a helical shell of CTD units, which are immobilized by a combination of covalent linkage at their N-termini to the PD core, noncovalent CTD-CTD dimerization, and contacts among CTD dimers. Below, we present solid state NMR data to support this alternative interpretation. Data below also provide further evidence that the structural basis for distinct, self-propagating strains or variants of [URE3] lies in the PD core.