HCM-Associated MuRF1 Variants Compromise Ubiquitylation and Are Predicted to Alter Protein Structure

Figures

• 

  Figure 1. Schematic representation of the MuRF1 [muscle RING (Really Interesting New Gene)-finger protein-1] domain composition and the distribution of HCM (hypertrophic cardiomyopathy)-associated variants. MuRF1 comprises six distinct domains: Really Interesting New Gene (RING) (C23-R79; red), MuRF family-specific motif (MFC) (H80-P113; yellow), B-box type 2 (B-box2) (G117-S161; blue), Coiled-coil (CC) (D214-G271; green), C-terminal subgroup One Signature-box (COS-box) (G272-E328; pink), and Acidic tail (AT) (E329-Q353; orange). Grey regions denote intrinsically disordered domains. The truncated variants are highlighted in red. This figure was generated using IBS 2.0 (https://ibs.renlab.org/#/server, accessed on 1 June–31 July 2024).

• 

  Figure 2. In vitro auto-monoubiquitylation analysis of 25 MuRF1 variants highlights functional impairments. Each reaction included MBP-MuRF1-WT (Maltose-Binding Protein-MuRF1 wild type) or the variant His-UBE1 (Histidine-UBE1) and free ubiquitin and was incubated for one hour with and without His-UBE2W. MBP-MuRF1-WT with His-UBE2W served as a positive control, while reactions without His-UBE2W acted as negative controls for each sample. The experiments with WT MBP-MuRF1, both with and without His-UBE2W, were repeated for each membrane. Ubiquitylation levels were evaluated through immunoblotting using anti-MBP and anti-FK2 (anti-ubiquitylated protein) antibodies. Coomassie blue staining was used to detect His-UBE1, His-UBE2W, and free ubiquitin in the reactions. (A) Anti-MBP immunoblot results for MuRF1 variants that map on the RING domain. (B) Anti-MBP immunoblot results for MuRF1 variants that map on the MFC and B-box2 domains. (C) Anti-MBP immunoblot results for MuRF1 variants that map on the Coiled-coil and COS-box domains. (D) Anti-MBP immunoblot results of unstructured and truncated MuRF1 variants. The results of anti-FK2 and Coomassie blue staining are shown in the Supplementary…

• 

  Figure 3. In vitro His-Titin (A168-A170) monoubiquitylation analysis demonstrates that all MuRF1 variants impair MuRF1 function. The assay included wild-type MBP-MuRF1 or the variants His-Titin (A168-A170) and His-UBE1 and free ubiquitin, which were incubated for one hour in the presence and absence of His-UBE2W. wild-type MBP-MuRF1 with His-UBE2W served as a positive control, while the reactions devoid of His-UBE2W acted as negative controls for each sample. The experiments with WT MBP-MuRF1, both with and without His-UBE2W, were repeated for each membrane. Ubiquitylation levels were assessed through immunoblotting using anti-His and anti-FK2 (anti-ubiquitylated protein). Coomassie blue staining was used to detect His-UBE1, His-UBE2W, and free ubiquitin within the reaction. (A) Anti-6x Histidine immunoblot results of MuRF1 missense variants that map on the RING domain. (B) Anti-6x Histidine immunoblot results of MuRF1 missense variants that map on the MFC and B-box2 domains. (C) Anti-6x Histidine immunoblot results of MuRF1 missense variants that map on the Coiled-coil and COS-box domains. (D) Anti-6x Histidine immunoblot results of unstructured and truncated MuRF1 variants. The results…

• 

  Figure 4. Assessing the impact of MuRF1 variants on the MuRF1-RING-MFC domain structure: Variants C23Y, C39G, and C75Y are predicted to impair zinc binding, whereas L37V, F73S, and R86C are predicted to compromise structural integrity. Ribbon representations illustrate models of the MuRF1-RING-MFC structure and its variants. (A) C23 coordinates the first zinc ion (top panel). The introduction of the C23Y variant is predicted to disrupt zinc binding (lower panel), (B) C39 coordinates the first zinc ion (top panel). The introduction of the C39G variant is anticipated to impair zinc binding (lower panel). (C) C75 coordinates the second zinc ion (top panel). The introduction of the C75Y variant is predicted to disrupt zinc binding (lower panel). (D) L37 mediates non-polar contacts with L43 and F73 (top panel). The introduction of the L37V variant is predicted to mediate fewer non-polar contacts with neighbouring residues (lower panel). (E) F73 mediates extensive networks of non-polar contacts with L37 and V82 (top panel). The introduction of the F73S variant is predicted to mediate fewer contacts in this region (lower panel). (F) R86 mediates cation–π interactions with H87 (top panel). The…

• 

  Figure 5. Assessing the impact of MuRF1 variants on the MuRF1-B-box2, CC, and COS-box domain structures showed that C145Y (B-box2) disrupted zinc binding, while I130M (B-box2), T232M (CC), and L319P (COS-box) weakened the core region. (A) Assessing the impact of the C145Y variant on the MuRF1-B-box2 domain structure. Ribbon representation of the MuRF1-B-box2 domain, highlighting that C75 coordinates the zinc ion (grey sphere). The black dashed line represents zinc binding (top panel). The introduction of the C145Y variant is predicted to disrupt zinc binding (lower panel). (B) Evaluating the effect of the I130M mutation on the MuRF1-B-box2 domain structure. Ribbon representation of the MuRF1-B-box2 domain, illustrating that I130 mediates non-polar contacts with P140 and L159 (top panel). The introduction of the I130M variant is predicted to decrease the number of non-polar contacts (lower panel). (C) Probing the effect of the T232M variant on the MuRF1-CC domain structure. Ribbon representation of the MuRF1-CC domain model, showcasing that T232 mediates a hydrogen bond with Q235. The black dashed line represents the hydrogen bond (top panel). The introduction of the T232M variant is…