A single methylene distinguishes Q from N. We find that this distinction profoundly alters one prominent activity of Q/N-rich proteins, prion formation, and influences another, toxicity. Changing Ns to Qs decreased prion formation and increased the accumulation of non-amyloid aggregates. These were toxic in the yeast cytoplasm and even more toxic when ectopically applied to cell lines of neuronal origin. In contrast, changing Qs to Ns enhanced prion formation and reduced toxicity. These observations were surprising, as the notion that Qs and Ns are equivalent for prion formation is pervasive (Si et al., 2003
; Ross et al., 2005b
; Decker et al., 2007
; Patel et al., 2009
; Salazar et al., 2010
). Further, algorithms for identifying amyloidogenic sequences – TANGO (Fernandez-Escamilla et al., 2004
) and Zyggregator (Tartaglia and Vendruscolo, 2008
) – do not predict clear differences in the effects of our Q and N replacements (Figure S7
), and do not predict amyloid formation by Q/N-rich PrDs (Alberti et al., 2009
) or the PrD variants we analyzed here (Figure S7
). Hopefully, the training sets created by our variants will lead to improved sequence-based predictions of amyloid formation.
Seeking a mechanistic explanation for our biological results, we asked how Qs and Ns affected various steps in both spontaneous amyloid assembly and templated assembly. The residues affected the specificity of templated and strongly altered the intrinsic efficiencies with which both the monomers and oligomers converted from the soluble disordered state to the amyloid. Molecular simulations suggest a possible rationale: the shorter N side chain enhances hydrogen bonding to the polypeptide backbone, increasing the formation of turns and β-sheets. We propose that this distinction is amplified as multiple monomers come together, allowing N-rich molecules to more effectively form ordered self-assemblies. Moreover, Ns reduced nonspecific interactions, which inhibit polymerization through off-pathway aggregation.
The disparity between our Q and N variants is likely the culmination of small contributions from many residues in the sequence, but local, contextual effects might also matter. For example, Ns (but not Qs) form hydrogen-bonded spines, or “asparagine-ladders”, in β-helix proteins (Jenkins and Pickersgill, 2001
; Lenore Cowen, personal communication). The β-helix is a model structure for functional amyloids (Shewmaker et al., 2009
) and fungal prions (Krishnan and Lindquist, 2005
; Wasmer et al., 2008
; Tessier and Lindquist, 2009
; Dong et al., 2010
). Moreover, Q stretches of sufficient length can overcome their intrinsically lower amyloid propensities, as occurs when a short segment of Sup35’s PrD, containing 13 Qs and 9 Ns, is replaced with a stretch of 62 Qs (Osherovich et al., 2004
Recently, it was reported that many Q/N-rich proteins form coiled coils which might govern their aggregation (Fiumara et al., 2010
). We note that Ns have a much lower coiled coil propensity than Qs – 0.25 vs. 0.99 using the Coils algorithm (Lupas et al., 1991
) and 0.29 vs. 0.90 using Paircoil2 (McDonnell et al., 2006
). A testable, unifying explanation for both our data and that of Fiumara arises: might a too-strong propensity to form coiled coils inhibit conversion to amyloid and favor the formation of toxic aggregates?
Other functions of Q/N rich proteins derive from the opposite conformational extreme they populate – disorder. Our Q and N variants are predicted to be highly disordered (9 of 10 disorder prediction web-servers reviewed in He et al., 2009
). Yet, IDRs are typically enriched for Qs and depleted of Ns (Radivojac et al., 2007
). Q-richness may be integral to the functions of dynamic protein assemblies: transcriptional regulatory complexes, RNA processing bodies and endocytic complexes (Xiao and Jeang, 1998
; Titz et al., 2006
; Decker et al., 2007
; Meriin et al., 2007
; Buchan et al., 2008
; Fuxreiter et al., 2008
; Alberti et al., 2009
). The conformational heterogeneity of Q-rich polypeptides might expedite the assembly and remodeling of such complexes, and grant freedom to explore new binding partners, accelerating the functional diversification of network hubs and the evolution of novel circuitries.
These desirable properties come at a price, however. Conformational disorder is a burden for protein homeostasis, in part due to mass action-driven interaction promiscuity (Vavouri et al., 2009
). This liability may drive the tightly regulated expression of proteins with IDRs in general and of “Q/N-rich” proteins in particular (Gsponer et al., 2008
). Our computational and experimental analyses indicate that Qs, specifically, increase the propensity for toxic interactions by disordered proteins, which, in turn, may contribute to the pathology of Q-rich proteins in disease. Although more comparisons will be needed between proteins expressed in, and applied to, a variety of cell types and compartments, the toxicity of non-amyloid species seems to be related to multi-factorial effects on intracellular protein-protein interaction promiscuity and extracellular membrane permeabilizing activities. Both intracellularly and extracellularly, amyloid formation reduced toxicity, consistent with previous suggestions for protective roles for amyloids (Takahashi et al., 2008
; Truant et al., 2008
; Treusch et al., 2009
). The genetic tractability of yeast prions provides a tool for investigating this very difficult problem.
The conformational transitions and protein::protein interactions of IDRs govern diverse biological processes, from regulatory networks to protein-misfolding diseases to protein-based inheritance. Further elucidating the conformational preferences of disordered proteins will be key to understanding their central roles in both normal biology and disease.