Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation

Abstract

The synucleins are a family of intrinsically disordered proteins involved in various human diseases. alpha-Synuclein has been extensively characterized due to its role in Parkinson's disease where it forms intracellular aggregates, while gamma-synuclein is overexpressed in a majority of late-stage breast cancers. Despite fairly strong sequence similarity between the amyloid-forming regions of alpha- and gamma-synuclein, gamma-synuclein has only a weak propensity to form amyloid fibrils. We hypothesize that the different fibrillation tendencies of alpha- and gamma-synuclein may be related to differences in structural propensities. Here we have measured chemical shifts for gamma-synuclein and compared them to previously published shifts for alpha-synuclein. In order to facilitate direct comparison, we have implemented a simple new technique for re-referencing chemical shifts that we have found to be highly effective for both disordered and folded proteins. In addition, we have developed a new method that combines different chemical shifts into a single residue-specific secondary structure propensity (SSP) score. We observe significant differences between alpha- and gamma-synuclein secondary structure propensities. Most interestingly, gamma-synuclein has an increased alpha-helical propensity in the amyloid-forming region that is critical for alpha-synuclein fibrillation, suggesting that increased structural stability in this region may protect against gamma-synuclein aggregation. This comparison of residue-specific secondary structure propensities between intrinsically disordered homologs highlights the sensitivity of transient structure to sequence changes, which we suggest may have been exploited as an evolutionary mechanism for fast modulation of protein structure and, hence, function.

Figure 1.

Sequence alignment of α-, β-, and γ-synuclein showing the N-terminal, amyloid-forming, and C-terminal regions. Black shading highlights identical sequences among all three synucleins, while gray shading highlights identical shading among two of the synucleins.

Figure 2.

ΔδC^α − ΔδC^β secondary chemical shifts for γ-synuclein (A) and α-synuclein (B). Positive values indicate α-structure propensity and negative values indicate β-structure propensity.

Figure 3.

Distribution of referencing offsets for ¹³C^α and ¹³C^β chemical shifts calculated for 247 proteins from RefDB.

Figure 4.

Secondary structure propensity (SSP) scores for α-synuclein (dotted lines) and γ-synuclein (dashed lines) calculated using ¹³C^α and ¹³C^β chemical shifts. Positive values represent α-structure propensity and negative values represent β-structure propensity. Upper and lower lines represent chemical shifts adjusted ±0.2 ppm from the calculated referencing offset for each protein.

Figure 5.

GOR4 helix predictions for α-synuclein (dotted line) and γ-synuclein (dashed line).

Figure 6.

Sequence conservation in the synucleins as represented by normalized Shannon's entropy calculated from a multiple sequence alignment of 27 synucleins.