ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils

Abstract

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction.

Figure 1. Design of the stringent starvation protein B (SspB)-fusion protein.

(a) Overall structure of the SspB protein. The tail residues from 112 to 165, shown in dots, were not modelled because of their flexibility. The distance between Asp111 and Asp111′ is approximately 40 Å. The N-terminal hexa-histidine tag is shown as a red block. (b) Domain architecture of the SspB-CCD fusion protein. The N-terminal hexa-histidine tag, full-length SspB (residues 1 to 165), and target coiled-coil domain (CCD) are coloured red, green, and blue, respectively. (c) Schematic model of a dimer of SspB fused with a parallel CCD. The N- and C-termini of the CCs are indicated. (d) Schematic model of a possible arrangement of a tetramer of SspB fused with an antiparallel CCD. The N- and C-termini of the CCs are indicated. (e) A possible arrangement of higher-order oligomers of SspB fused with an antiparallel CCD and protein aggregation during concentration of the sample (left: soluble control protein; right: aggregation of higher-order oligomers).

Figure 2. Structures of parallel coiled-coil domains (CCDs).

(a) GCN4 from Saccharomyces cerevisiae. (b) APC, adenomatous polyposis coli protein from Home sapiens. (c) Atg16, autophagy protein 16 from S. cerevisiae. (d) SCOC, short coiled-coil protein from H. sapiens. (e) LRRFIP1, leucine-rich repeat flightless-interacting protein 1 from H. sapiens. (f) Ndel1, nuclear distribution protein nudE-like 1 from Rattus norvegicus. (g) TPM1, tropomyosin α-1 chain from Gallus gallus. (h) ROCK1, Rho-associated coiled-coil containing protein kinase 1 from H. sapiens. The length of each CCD and the Cα distance between the two N-terminal residues are provided.

Figure 3. Structures of antiparallel coiled-coil domains (CCDs).

(a) MDV1, mitochondrial division protein 1. (b) Mfn1, Mitofusin-1. (c) LMNA, prelamin-A/C. (d) BECN1, beclin-1. (e) TRIM25, E3 ubiquitin/ISG15 ligase tripartite motif-containing protein 25. (f) TRIM5, tripartite motif-containing protein 5. The length of each CCD is provided.

Figure 4. ACCORD results for parallel coiled-coils (CCs).

Size-exclusion chromatography with multi-angle light scattering data from (a) SspB-GCN4, (b) SspB-APC, (c) SspB-Atg16, (d) SspB-SCOC, (e) SspB-LRRFIP1, (f) SspB-Ndel1, (g) SspB-TPM1, and (h) SspB-ROCK1 fusion proteins. Insets show the results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the chromatography fractions. (i) Schematic model of the SspB-ROCK1 dimer. All parallel CCDs fused with the SspB protein behaved as dimers in solution.

Figure 5. ACCORD results for antiparallel coiled-coils.

Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) data from (a) stringent starvation protein B (SspB)-Mfn1, (b) SspB-MDV1, (c) SspB-TRIM25, and (d) SspB-BECN1 fusion proteins. Insets shows the results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the chromatography fractions. SEC-MALS analysis was not possible for (e) SspB-LMNA and (f) SspB-TRIM5 because of their significant precipitation. (g) Schematic model of the SspB-BECN1 tetramer. Antiparallel CCDs fused with the SspB protein behaved as tetramers or heavy aggregates in solution.

Figure 6. Validation of ACCORD results.

(a) Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) data showed that SspB-NDP52 is a dimer in solution, suggesting that the coiled-coil domain (CCD) of NDP52 is oriented in a parallel manner. Inset shows the results of SDS-PAGE of the chromatography fractions. (b) Ab initio envelope using SAXS data superimposed onto two MBP molecules (PDB ID: 3DM0) and a parallel CCD of Atg16 (PDB ID: 3A7O), which might have similar CC lengths based on the numbers of residues. (c) Molecular envelope of MBP-MDV1. The high-resolution crystal structure of MBP-MDV1 (Supplementary Fig. 2) was fitted into the low resolution molecular envelope generated from SAXS data.