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Crystal structure of ATP-bound Get3–Get4–Get5 complex reveals regulation of Get3 by Get4

Nature Structural & Molecular Biology volume 21, pages 437–442 (2014)Cite this article

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Abstract

Correct localization of membrane proteins is essential to all cells. Chaperone cascades coordinate the capture and handover of substrate proteins from the ribosomes to the target membranes, yet the mechanistic and structural details of these processes remain unclear. Here we investigate the conserved GET pathway, in which the Get4–Get5 complex mediates the handover of tail-anchor (TA) substrates from the cochaperone Sgt2 to the Get3 ATPase, the central targeting factor. We present a crystal structure of a yeast Get3–Get4–Get5 complex in an ATP-bound state and show how Get4 primes Get3 by promoting the optimal configuration for substrate capture. Structure-guided biochemical analyses demonstrate that Get4-mediated regulation of ATP hydrolysis by Get3 is essential to efficient TA-protein targeting. Analogous regulation of other chaperones or targeting factors could provide a general mechanism for ensuring effective substrate capture during protein biogenesis.

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Figure 1: Get4–5 prefers ATP-bound state of Get3.
Figure 2: 5.4-Å crystal structure of an ATP-bound Get3–Get4–5 complex.
Figure 3: Get3-Get4 binding interfaces.
Figure 4: Get4–5 regulates Get3 ATPase activity.
Figure 5: A working model for Get4–5.

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Acknowledgements

We are grateful to V. Denic (Harvard University) for providing the Δget3 Δget5 yeast strain. We thank G. Card, A. Gonzalez and M. Soltis for help with data collection at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2. We are grateful to G. Moore and B. Moore for support of the Molecular Observatory at the California Institute of Technology. Operations at the SSRL are supported by the US Department of Energy and US National Institutes of Health (NIH). This work was supported by career awards from the David and Lucile Packard Foundation and the Henry Dreyfus Foundation (S.S.), US National Science Foundation Graduate Research Fellowship DGE-1144469 (M.E.R.), NIH training grant 5T32GM007616-33 (H.B.G. and M.R.) and NIH research grant R01GM097572 (W.M.C. Jr.).

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  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA

    Harry B Gristick, Meera Rao, Justin W Chartron, Michael E Rome, Shu-ou Shan & William M Clemons Jr

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  1. Harry B Gristick
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Contributions

H.B.G. performed all experiments except those noted. M.R. performed Get4 inhibition studies, and M.E.R. performed translocation experiments, both supervised by S.S. J.W.C. initiated the project and aided in refinement. S.S. contributed to experimental design and interpretation. W.M.C. Jr. conceived and supervised the project. H.B.G. and W.M.C. Jr. wrote the manuscript. All authors discussed the results and implications and commented on the manuscript.

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Correspondence to William M Clemons Jr.

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Integrated supplementary information

Supplementary Figure 1 Representative ITC isotherms for nucleotide-dependent complex formation.

(a) Representative measurements for Get3 in various nucleotide states. Raw data are shown in the top panel of each trial represented as the power input into the sample cell over time. Integrated data are shown in the bottom panels in terms of the total energy required for equilibration as a function of the molar ratio of Get4-5N:Get3. The solid line represents the best-fit model used to calculate affinities. The affinities indicated for each nucleotide state are the average from at least three experiments. In all experiments Get4-5N was titrated into the sample cell containing Get3. (b) Representative measurements for Get3 mutants from the Anchoring interface, top, or the Regulatory interface, bottom. The indicated affinity for each mutant represents the value obtained from a single experiment carried out in the presence of ATP. (c) Representative measurements for Get4 mutants from the various interfaces as in (b).

Supplementary Figure 2 Surface properties of Get3 and Get4.

(a) Left, Surface representation of closed Get3 dimer (gray) from (b) showing Get4 binding site (blue), Get1 cytoplasmic domain binding site (yellow) and the overlap (green). Right, Similar surface representation highlighting the Get4 binding site (blue), Get2 cytoplasmic domain binding site (red) and the overlap (brown). (b) Left, orientation of Get3 used in subsequent panels to highlight the binding interfaces. Monomers are colored as in Figure 2. For surface representations one monomer is outlined in black and dashed lines (blue) highlight the interaction surface of Get4 on Get3. Center, accessible surface color ramped based on electrostatic potential from -12 kT/e (red) to +12 kT/e (blue). Get5N colored gray. Right, accessible surface color ramped based on conservation from ≤50% (white) to ≥90% (purple). Get5N colored gray. Coloring is based on a ClustalW 34 alignment using the following sequences: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus fumigatus, Candida albicans, Pichia pastoris, Nematostella vectensis, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus laevis, Mus musculus, Homo sapiens, Arabidopsis thaliana, Neurospora crassa, Anolis carolinensis. (c) Surface properties of Get4 as in (b), except Get4 is color-ramped from N-terminus (blue) to C-terminus (red).

Supplementary Figure 3 Stereo views of the Get3D-Get4–5N interface.

(a) The Get3D-Get4 interface. Coloring is the same as in Figure 3a. Get3 Lys69 and Get4 Asp74 are highlighted by open arrowheads. (b) 2FO-FC Electron density contoured at 1.5σ highlighting the Get3D-Get4 interface.

Supplementary Figure 4 TA targeting assay.

(a) Representative autoradiograph images used to calculate Get3 translocation efficiency corresponding to Figure 4b for wildtype (Top) and K69D (Bottom). (b) Comparison of Get3 translocation efficiencies in Δget3 and Δget3/Δmdy2 lysates. (c) Representative autoradiograph images used to calculate Get3 translocation efficiencies for Δget3/Δmdy2 lysate in (b).

Supplementary Figure 5 Tetramers of Get3.

(a) Tetramers of Get3 seen in various crystal forms with Get3 dimers similar to Figure 5a,b. Each is highlighted with the archaeal Get3 (Methanocaldococcus jannaschii) from PDB ID 3UG6, the Get2-Get3 complex from PDB ID 3SJD, and the Get3D-Get4-5N crystal structure. (b) Stereo view of the 2Fo-Fc density contoured at 1.5σ showing the loops involved in formation of the Get3D-Get4-5N complex tetramer. (c) Comparison of the Archaeal MjGet3 dimer aligned to one dimer of the Get3D-Get4-5N complex dimer.

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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 64948 kb)

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Gristick, H., Rao, M., Chartron, J. et al. Crystal structure of ATP-bound Get3–Get4–Get5 complex reveals regulation of Get3 by Get4. Nat Struct Mol Biol 21, 437–442 (2014). https://doi.org/10.1038/nsmb.2813

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