Crystal structure of the FYCO1 RUN domain suggests possible interfaces with small GTPases

Abstract

FYCO1 is a multidomain adaptor protein that plays an important role in autophagy by mediating the kinesin-dependent microtubule plus-end-directed transport of autophagosomes. FYCO1 contains a RUN domain, which is hypothesized to function as a specific effector for members of the Ras superfamily of small GTPases, but its role has not been well characterized and its interaction partner(s) have not been identified. Here, the crystal structure of the FYCO1 RUN domain was determined at 1.3 Å resolution. The overall structure of the FYCO1 RUN domain was similar to those of previously reported RUN domains. Detailed structural comparisons with other RUN domains and docking studies suggested a possible interaction interface of the FYCO1 RUN domain with small GTPases of the Ras superfamily.

Keywords: FYCO1; RUN domain; X-ray crystallography; autophagy; small GTPase binding.

Figure 1

Domain structure and amino-acid sequence of FYCO1. (a) Schematic representation of the domain structure of human FYCO1. The RUN (RPIP8, UNC-14 and NESCA) domain, coiled-coil region, FYVE (Fab1, YOTB/ZK632.12, Vac1 and EEA1) domain, LIR (LC3-interacting region) motif and GOLD (Golgi dynamics) domain are shown in green, blue, orange, purple and yellow, respectively. (b) Sequence alignment of FYCO1 RUN domains from mammals. The secondary-structural elements are indicated above the alignments. Nonconserved residues are highlighted in blue.

Figure 2

Crystal structure of the FYCO1 RUN domain. (a) Overall structure of the FYCO1 RUN domain. The N- and C-termini and the structural elements are labelled. (b) Electron-density map of the phenylalanine residues in the native crystal. The 2F _o − F _c difference electron-density map is contoured at the 1.5σ level with a blue mesh. The hole in the aromatic ring is clearly visible in the density map. (c) Electron-density map of water molecules. The 2F _o − F _c difference electron-density map is contoured at the 1.0σ level with a blue mesh. (d) Electron density of the mercury-derivatized crystal at 1.6 Å resolution. The 2F _o − F _c difference electron-density map is contoured at the 1.5σ level with a blue mesh. The anomalous difference Fourier map is contoured at the 5.0σ level with a red mesh.

Figure 3

Sequence and structural comparisons of RUN domains. (a) Sequence alignment of RUN domains. Secondary-structural elements are indicated above the alignments. The conserved residues are highlighted in red. The residues of Rab6IP1 that interact with Rab6 and those of RUSC2 that interact with Rab35 are highlighted in yellow and purple, respectively. (b) Superposition of RUN domains. The RUN domains of FYCO1, RPIPx (Kukimoto-Niino et al., 2006 ▸), Rab6IP1 (Recacha et al., 2009 ▸), NESCA (Sun et al., 2012 ▸) and RUSC2 (Lin et al., 2019 ▸) are shown in green, blue, yellow, red and purple, respectively. (c) Structural differences among the five RUN domains. Secondary-structural elements are indicated.

Figure 4

Interaction surface of the RUN–small GTPase complex. (a) Crystal structures of the Rab6IP1–Rab6 and RUSC2–Rab35 complexes (Recacha et al., 2009; Lin et al., 2019 ▸). Rab6IP1, Rab6, RUSC2 and Rab35 are shown in yellow, red, purple and orange, respectively. (b) Electrostatic surface potentials of the five RUN domains. The electrostatic surface potential is coloured from −8 kT e⁻¹ (red) to +8 kT e⁻¹ (blue).

Figure 5

Docking models of the FYCO1 RUN domain and small GTPases. The docking models were calculated by ZDOCK (Pierce et al., 2011 ▸). The FYCO1 RUN domain is shown as a ribbon model in green. The top five models of small GTPases from each calculation are shown as tube models in red, orange, light green, cyan and blue. The PDB entries used as input models for small GTPases are shown under the models.