Get5 carboxyl-terminal domain is a novel dimerization motif that tethers an extended Get4/Get5 complex

Abstract

Tail-anchored trans-membrane proteins are targeted to membranes post-translationally. The proteins Get4 and Get5 form an obligate complex that catalyzes the transfer of tail-anchored proteins destined to the endoplasmic reticulum from Sgt2 to the cytosolic targeting factor Get3. Get5 forms a homodimer mediated by its carboxyl domain. We show here that a conserved motif exists within the carboxyl domain. A high resolution crystal structure and solution NMR structures of this motif reveal a novel and stable helical dimerization domain. We additionally determined a solution NMR structure of a divergent fungal homolog, and comparison of these structures allows annotation of specific stabilizing interactions. Using solution x-ray scattering and the structures of all folded domains, we present a model of the full-length Get4/Get5 complex.

FIGURE 1.

Crystal structure of Get5-C. A, schematic of the domain organization of Get5 and sequence alignment of the carboxyl domain in fungi. Lines indicate flexible regions. Sequences are: S. cere, S. cerevisiae; A. goss, Ashbya gossypii; S. scle, Sclerotina sclerotiorum; A. fumi, A. fumigatus. Residues that mediate intermolecular contacts in both Get5 and AfGet5 dimers are highlighted in red, and residues specific to Get5 or AfGet5 are highlighted in blue or green, respectively. B, dimerization interface of a monomer of Get5-C with σ_a-weighted 2|F_o| −|F_c| electron density contoured at 1.5σ. C, asymmetric unit of Get5-C. One monomer is color ramped from amino (blue) to carboxyl (red) terminus. The 2-fold axis is indicated with a dotted line. Side chains that make intermolecular contacts are shown (left).

FIGURE 2.

Solution structures of Get5-C and AfGet5-C. A and B, ensembles of the 10 lowest energy solution structures of the ordered regions of Get5-C (A) and AfGet5-C (B). C, ribbon diagram of AfGet5-C with side chains making intermolecular contacts. AfGet5-C is rotated from the orientation in the top of B as indicated by the sphere in the corner. Inset, view of the environment around Val-192.

FIGURE 3.

Get5-C is a stable dimer. Thermal melting curves of Get5-C (solid line) and AfGet5-C (dashed line) measured by CD spectroscopy.

FIGURE 4.

SAXS of Get4/Get5. A, pair-distance distribution function of the Get4/Get5 heterotetramer derived from SAXS. B, averaged ab initio reconstruction of Get4/Get5 shown as a gray surface in two orientations. C, rigid body models generated by independent simulated annealing calculations using known structures against SAXS data. In each model, the two copies of Get5 are colored magenta and orange, and the two copies of Get4 are colored cyan and blue. The three top models are shown ranked according to χ² fit to the experimental SAXS curve.

FIGURE 5.

Model of the Get4/Get5/Sgt2 sorting complex and its interaction with Get3. Schemes are drawn to scale, with the exception of chaperones bound to Sgt2. The arrow indicates the path of the TA protein from chaperones to Sgt2 to Get3, which is held by Get4.