Mutations in human prion-like domains: pathogenic but not always amyloidogenic

Figures & data

Figure 1. Domain organization of hnRNPs that have been structurally characterized using cryo-EM. The RNA recognition motifs (RRMs), low complexity domains (LCDs) and the nuclear localization signals (PY-NLS and NLS) are colored in orange, light blue and dark blue, respectively.

Schematic representation of five different hnRNPs, including hnRNPA1, hnRNPA2, hnRNPDL-2, TDP-43 and FUS proteins. The proteins are represented as horizontal bars segmented into different coloured regions, which correspond to their RNA recognition motifs (RRMs), low complexity domains (LCD) and nuclear localization signals (NLS).

Figure 2. Impact of hnRNPA1 disease-associated mutations on fibril stability. (a) domain organization of hnRNPA1. Residues covered by the amyloid core in the cryo-EM hnRNPA1 fibril structure (PDB 7BX7) [17] are indicated with a brown bar. (b-g) stabilization energy maps of the amyloid fibrils formed by the WT protein (b) and their corresponding disease-associated mutants D262V (c), D262N (d), N267S (e), P288A (f) and P288S (g). Note that amyloid fibril structures are colored by energy, being red and blue the stabilizing and destabilizing residues, respectively. In (a-g) the nature of the disease-associated mutations is indicated with green (stabilizing) or red (destabilizing) filled circles.

Schematic representation of hnRNPA1 domains, indicating the residues of the amyloid core with a brown bar and the disease-associated mutations with green or red-filled circles depending on their stabilizing or destabilizing effect. Figures 2(b-g). Stabilization energy maps of the amyloid fibrils formed by the WT and mutant hnRNPA1 proteins, showing stabilizing and destabilizing residues coloured in red and blue, respectively. Next to the mutated residues on the energy maps, green or red filled circles represent the stabilizing or destabilizing nature of the mutations.

Figure 3. Impact of hnRNPA2 disease-associated mutations on fibril stability. (a) domain organization of hnRNPA2. Residues covered by the amyloid core in the cryo-EM hnRNPA2 fibril structure (PDB 6WQK) [18] are indicated with a brown bar. (b-d) stabilization energy maps of the amyloid fibrils formed by the WT (b) and their corresponding disease-associated mutants D290V (c) and P298L (d). The structures are colored according to the energy values, as described in figure 2. In (a-d), the nature of the disease-associated mutations is indicated with green (stabilizing) or red (destabilizing) filled circles.

Schematic representation of hnRNPA2 domains, indicating the residues of the amyloid core with a brown bar and the disease-associated mutations with green or red-filled circles depending on their stabilizing or destabilizing effect. Figures 3(b-d). Stabilization energy maps of the amyloid fibrils formed by the WT and mutant hnRNPA2 proteins, showing stabilizing and destabilizing residues coloured in red and blue, respectively. Next to the mutated residues on the energy maps, green or red filled circles represent the stabilizing or destabilizing nature of the mutations.

Table 1. Calculated standard free energy of stabilization of disease-associated mutations in hnRNPs.

	Mutation	Related disease	ΔΔG (kcal/mol)*	References
hnRNPA1 (PDB 7BX7) [17]	D262V	ALS, MSP	−24,19	[17,29,30]
	D262N	ALS, MSP	−11,32	[17,29,30]
	P288S	ALS, MSP	34,08	[17,29]
	P288A	ALS, MSP	15,01	[17,29]
	N267S	ALS, MSP	−5,25	[17,29]
hnRNPA2 (PDB 6WQK) [18]	D290V	MSP, ALS, Paget’s disease of bone	−2,77	[4,18,31]
	P298L	Paget’s disease of bone	12,07	[31,32]
hnRNPDL-2 (PDB 7ZIR) [25]	D259N	LGMD D3	−4,63	[10,25]
	D259H	LGMD D3	4,89	[10,25]
TDP-43 (PDB 7PY2) [27]	A315E	ALS	3,25	[33–35]
	A315T	ALS	4,75	[34–36]
	M337V	ALS	−5,29	[33,35,37]
	Q331K	ALS	14,48	[33,35,37]
TDP-43 (PDB 8CG3) [28]	A315E	ALS	4,83	[33–35]
	A315T	ALS	14,21	[34–36]
	M337V	ALS	31,86	[33,35,37]
	Q331K	ALS	22,93	[33,35,37]
FUS (PDB 7VQQ) [24]	P106L	FTLD	17,72	[38]
	S115N	ALS	−7,58	[39]

*Standard free energy of stabilization (ΔΔG in kcal/mol) upon mutation calculated using Rosetta [42] and the indicated cryo-EM structures. The mutations were classified as stabilizing or destabilizing, considering a threshold of ΔΔG <-1 kcal/mol for the first and ΔΔG >1 kcal/mol for the latest. Calculations were performed as described elsewhere [25].

Figure 4. Impact of hnRNPDL-2 disease-associated mutations on fibril stability. (a) domain organization of hnRNPDL-2. The residues covered by the amyloid core in the cryo-EM hnRNPDL-2 fibril structure (PDB 7ZIR) [25] are indicated with a brown bar. (b-d) stabilization energy maps of the amyloid fibrils formed by the WT (b) and their corresponding disease-associated mutants D259N (c) and D259H (d). The structures are colored according to the energy values, as described in figure 2. In (a-d), the nature of the disease-associated mutations is indicated with green (stabilizing) or red (destabilizing) filled circles.

Schematic representation of hnRNPDL-2 domains, indicating the residues of the amyloid core with a brown bar and the disease-associated mutations with green or red-filled circles depending on their stabilizing or destabilizing effect. Figures 4(b-d). Stabilization energy maps of the amyloid fibrils formed by the WT and mutant hnRNPDL-2 proteins, showing stabilizing and destabilizing residues coloured in red and blue, respectively. Next to the mutated residues on the energy maps, green or red filled circles represent the stabilizing or destabilizing nature of the mutations.

Figure 5. Impact of TDP-43 disease-associated mutations on fibril stability. (a) domain organization of TDP-43. The residues encompassed by the amyloid core of two ex vivo fibril structures of TDP-43, derived from patients with ALS-FTLD (PDB 7PY2) [27] and type A FTLD-TDP (PDB 8CG3) [28], are indicated with brown bars. b-k) stabilization energy maps of the ALS-FTLD (b-f) and type A FTLD-TDP (g-k) amyloid fibrils. The structures of the WT (b and g) and their corresponding disease-associated mutants A315E (c and h), A315T (d and i), M337V (e and j) and Q331K (f and k) are shown. The structures are colored according to the energy values, as described in figure 2. In a-k), the nature of the disease-associated mutations is indicated with green (stabilizing) or red (destabilizing) filled circles.

Schematic representation of TDP-43 domains, indicating the residues of the amyloid cores with brown bars and the disease-associated mutations with green or red-filled circles depending on their stabilizing or destabilizing effect. Figures 5(b-k). Stabilization energy maps of the amyloid fibrils formed by the WT and mutant TDP-43 proteins derived from patients with ALS-FTLD and type A FTLD-TDP. Energy maps show stabilizing and destabilizing residues coloured in red and blue, respectively. Next to the mutated residues on the energy maps, green or red filled circles represent the stabilizing or destabilizing nature of the mutations.

Figure 6. Impact of FUS disease-associated mutations on fibril stability. (a) domain organization of FUS. The residues covered by the amyloid core in the cryo-EM FUS fibril structure (PDB 7VQQ) [24] are indicated with a brown bar. (b-d) stabilization energy maps of the amyloid fibrils formed by the WT protein (b) and their corresponding disease-associated mutants P106L (c) and S115N (d). The structures are colored according to the energy values, as described in figure 2. In a-d), the nature of the disease-associated mutations is indicated with green (stabilizing) or red (destabilizing) filled circles.

Schematic representation of FUS domains, indicating the residues of the amyloid core with a brown bar and the disease-associated mutations with green or red-filled circles depending on their stabilizing or destabilizing effect. Figures 6(b-d). Stabilization energy maps of the amyloid fibrils formed by the WT and mutant FUS proteins, showing stabilizing and destabilizing residues coloured in red and blue, respectively. Next to the mutated residues on the energy maps, green or red filled circles represent the stabilizing or destabilizing nature of the mutations.

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Data availability statement

Data sharing does not apply to this article as no new data were created or analysed in this study.