Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein - PubMed (original) (raw)

Eva Klinglmayr et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012.

Abstract

The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis.

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Figures

Figure 1

Figure 1

Construct design and schematic representation of the Drp1 GG fusion protein. (a) Full-length Drp1 consists of an N-terminal GTPase domain (green) and a helical middle domain (blue) followed by an unstructured region (insert B or variable domain) and a C-terminal GED (pink). (b) The GG fusion protein consists of the complete GTPase domain (amino acids 1–327) fused to a C-terminal fragment of the GED (amino acids 711–736) via a (GS)4 linker.

Figure 2

Figure 2

Purification of the Drp1 GG fusion protein. (a) Size-exclusion analysis. After Ni–NTA purification, proteins were loaded onto a Superdex 75 gel-filtration column and fractions containing protein were collected. In the absence of nucleotides, the protein eluted as a monomer at a retention volume of approximately 11 ml, which corresponds to a molecular mass of approximately 40 kDa. The retention volumes of molecular-mass standards (GE Healthcare) are displayed at the top. (b) SDS–PAGE analysis. Protein fractions were loaded onto a 15% SDS–PAGE gel and visualized by Coomassie Brilliant Blue staining. Lane E, eluted protein; lane M, protein molecular-mass markers (labelled in kDa on the left). The retention volumes are indicated at the top.

Figure 3

Figure 3

The Drp1 GG fusion protein displays GTPase activity: steady-state GTPase activities of Drp1 GG fusion protein (native Drp1 GG fusion protein) in comparison to Drp1 full-length isoform 2 and enzymatically inactive K38A mutant (Drp1 GG fusion protein K38A). Data are the mean ± standard deviation from three independent measurements.

Figure 4

Figure 4

Crystal forms of the Drp1 GG fusion protein. (a) In condition A only star-shaped intergrown crystals were present which could not be reproduced. (b) Reproducible crystals from condition B were only about 50 × 40 × 10 µm in size.

Figure 5

Figure 5

Diffraction image of the Drp1 GG fusion protein: a diffraction image of the crystals produced in condition B collected on BL14.1 at BESSY II, Berlin, Germany.

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References

    1. Bereiter-Hahn, J. & Vöth, M. (1994). Microsc. Res. Tech. 27, 198–219. - PubMed
    1. Bossy, B., Petrilli, A., Klinglmayr, E., Chen, J., Lutz-Meindl, U., Knott, A. B., Masliah, E., Schwarzenbacher, R. & Bossy-Wetzel, E. (2010). J. Alzheimers Dis. 20, S513–S526. - PMC - PubMed
    1. Chang, C.-R. & Blackstone, C. (2010). Ann. N. Y. Acad. Sci. 1201, 34–39. - PMC - PubMed
    1. Chang, C.-R., Manlandro, C. M., Arnoult, D., Stadler, J., Posey, A. E., Hill, R. B. & Blackstone, C. (2010). J. Biol. Chem. 285, 32494–32503. - PMC - PubMed
    1. Chappie, J. S., Acharya, S., Leonard, M., Schmid, S. L. & Dyda, F. (2010). Nature (London), 465, 435–440. - PMC - PubMed

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