Crystal structure of a dynamin GTPase domain

1

Crystal structure of a dynamin GTPase domain

Hartmut H. Niemann, Menno L. W. Knetsch, Anna Scherer, Dietmar J. Manstein & F.

Jon Kull

Max-Planck-Institute for Medical Research, Department of Biophysics, Jahnstrasse 29,

69120 Heidelberg, Germany

Dynamins form a family of large GTPases1 involved in processes including

endocytosis, vesicle trafficking and maintenance of mitochondrial morphology2.

Whether they work as signaling GTPases or function mechanochemically during

membrane fission is still a matter of debate3-5. Dynamins are multidomain proteins

sharing an amino-terminal GTPase domain, a central region, and a GTPase

effector domain (GED), which stimulates the GTPase activity2. The GTPase

domain is the most highly conserved region among dynamins from different

subfamilies and species 6. Here we report the 2.3 Å crystal structure of the

nucleotide-free GTPase domain of Dictyostelium discoideum dynamin A7. The

structure consists of a globular domain containing the G-protein core fold and

additional structural elements. The common six-stranded ββββ-sheet is extended to

eight strands by a topologically unique insertion that distinguishes dynamins from

other subfamilies of GTP binding proteins.

The dynamin A GTPase domain (residues 2-316; 60% sequence identity to

human dynamin 1) was expressed and crystallized as a fusion with the motor domain of

myosin (manuscript in preparation). The structure was determined by molecular

replacement using the myosin motor domain as search model (Table 1). The current

model contains residues 2-306. Three surface loops are disordered. The GTPase domain

consists of an eight-stranded β-sheet with six parallel and two antiparallel strands

2

surrounded by nine helices (Fig. 1). The G-domain core motif, a six-stranded β-sheet

with one antiparallel strand surrounded by five α-helices8, is present within our structure

and closely resembles that found in Ras9. Corresponding secondary-structure elements

of dynamin A and Ras, which share 10% sequence identity, can be superposed with a

root mean square deviation of 1.46 Å for the 76 common Cα atoms.

The beta-sheet is extended beyond β2 by strands β2A and β2B, which are linked

by helix αB (Fig. 1). Strands β2 and β3, which are connected by a short β-turn in most

other G-proteins are linked by a 55 amino acid insertion in dynamin A (Fig. 2). The start

of this unique insertion coincides with the only intron in the Dictyostelium dymA gene

coding for dynamin A. The Drosophila shibire10 and the C. elegans dyn-111 genes also

have splice sites close to the start or end of the insertion. The connection between β2

and β2A is the most diverse region within the dynamin GTPase domain. In dynamin A it

is 9 residues longer than in dynamin 1 (Fig. 2) and forms a long protruding loop (Fig. 1).

In the Saccharomyces cerevisiae dynamin homologues Vps1p12 and Dnm1p13 the

connection is more than 30 amino acids longer than that of dynamin A and two more β-

strands are predicted in this region. This variability, coupled with its extended

conformation, suggests that the loop connecting β2 and β2A is a site for interaction with

other proteins.

Another feature of the dynamin GTPase domain are the additional N-terminal

helix αA and the extension of the C-terminal helix α5, which contact each other. Helices

αA and αB pack against the β-sheet from opposite sides. Helix αA forms hydrogen

bonds with polar side chains of the β-sheet, which are solvent-exposed in Ras. ARFs are

Ras-like GTP-binding proteins also containing an N-terminal α-helix. However, helix

αA in dynamin A occupies a different position than the N-terminal helix of ARF, which

packs against the other side of the β-sheet 14. Helix αA is almost perpendicular to the C-

terminal helix α5 in dynamin A, whereas these helices are parallel in ARF. Helix α5 is

3

considerably longer in dynamin than in other G-proteins, forming a continuous helix,

kinked at Pro 296. The part of helix α5 C-terminal to Pro 296 makes hydrophobic

contacts only to the N-terminal helix αA and the loop connecting αA and β1, suggesting

that they form a stable structural unit. Surface potential analysis shows that αA and the

C-terminal end of α5 form a hydrophobic groove. In our structure this groove is

occupied by hydrophobic residues from the C-terminus of myosin and the linker

between the fusion-partners. In full-length dynamin this groove is probably occupied by

residues from the C-terminal region of the molecule or residues from another dynamin

molecule in the oligomeric complex.

Two other prominent insertions are present in dynamin A when compared to

Ras. Firstly, the loop connecting β3 and α2 is 13 residues longer. Although several of

the additional residues extend helix α2 N-terminally, making it longer than in Ras, most

of these residues are disordered in our structure. This is not surprising as in other G-

proteins α2 and the preceding loop have been shown to move between different

nucleotide states and are often flexible in the absence of γ-phosphate. A second insertion

is located between β6 and α5. It contains helix αC, which does not pack against either

side of the β-sheet, but rather runs perpendicular to the strands.

Despite of the absence of nucleotide, three of the four consensus elements15

responsible for nucleotide binding (G1 to G4) are well resolved. The G4 motif

(consensus sequence N/TKxD) is responsible for base binding and specificity and

adopts a conformation similar to that found in GTPases bound to nucleotide (Fig. 3). In

dynamin A the corresponding sequence is 207TKLD210. The backbone and side chains of

Thr 207 and Lys 208 are very close to the conformation found in nucleotide bound Ras

or Rac. The side chain of Thr 207 makes a hydrogen bond to the carbonyl of Ser 36 in

the P-loop, as seen in Rac1 16, which shares the sequence TKLD. In most other GTPases

an Asn occupies the position of this Thr and forms a hydrogen bond to the base. In the

4

Rac1 structure with bound nucleotide, no hydrogen bonding occurs between the Thr side

chain and the base. Therefore it seems likely in dynamin the base will not interact with

Thr 207. Asp 210 needs to move inwards about 2 Å, in order to hydrogen bond to the

base in a way similar to Ras or Rac. The G5-motif involved in base binding in many

small GTPases (145SAK147 in Ras) is absent in dynamin A, where 237INR239 occupy

roughly equivalent positions. It seems likely that the residues between β6 and αC

including the short helix αC' could rearrange upon nucleotide binding, in order to

facilitate more favorable interactions with the base, as seen in the human guanylate

binding protein 1 (hGBP1)17,18. The G1 motif (GxxxxGKS/T) is responsible for binding

of the phosphates (often called the P-loop). In dynamin the motif 32GSQSSGKS39

adopts an unusual conformation. The carbonyl of Gln 34 is flipped when compared to

GTPases bound to nucleotide. A similar peptide-flip is seen in nucleotide free hGBP118.

Lys 38 hydrogen bonds to the side chain of Asp 138. This residue is part of the G3 motif

(see below) and the interaction of the P-loop Lys with elements from or close to G3

seems to be a general way to stabilize P-loops having neither nucleotide nor a phosphate

or sulphate ion bound. In the nucleotide free complex of EF-Tu with its exchange factor

EF-Ts, the Lys binds the side chain of the corresponding Asp19. In the Ras-SOS

complex20 the Lys interacts with a Glu following G3, and in nucleotide free hGBP1, it

binds the carbonyl of a Thr that is equivalent to Leu 139 in dynamin18. The G3 (DxxG)

and G2 (a conserved Thr) motifs, are involved in the coordination of the γ-phosphate

and the Mg2+-ion and move significantly between the GDP- and the GTP-bound form of

Ras 9,21 and EF-Tu22,23. Thr 59 (G2) is not well resolved in our structure. Gly 141 from

G3 adopts a conformation that is closer to that of the corresponding Gly 60 of Ras-GTP

than that of Ras-GDP. G2 and G3 are part of structural elements often referred to as

switch I (G2) and switch II (G3).

Several mutations have been described in the dynamin GTPase domain that

impair function. Two temperature sensitive mutations were found in the Drosophila

5

shibire gene10. In the Shits1 allele, the Gly corresponding to Gly 275 at the start of helix

α5 is mutated to Asp. This Gly has backbone angles forbidden for non-Gly residues.

However, the presence of Asp would not be sterically blocked, and could stabilize the

helix by favorably interacting with backbone amides that are otherwise unable to form

hydrogen bonds. This interaction is predicted to become weaker with increasing

temperature. The Gly mutated to Ser in Shits2 corresponds to Gly 148 in the loop before

helix α2. This helix is part of the switch II, a region that is essential for

mechanochemical coupling and not well defined in our structure. A temperature

sensitive mutation in the C. elegans dyn-1 gene changes Pro 62 at the start of β2 to

Ser11. This residue is located in a hydrophobic environment. The change to a polar

residue might be tolerated at lower, but not at higher temperature, where hydrophobic

forces become stronger. Recently several mutations that impair the GTP-hydrolysis of

dynamin 1, its function in endocytosis and the affinity for nucleotide have been

reported4. All of the residues affected are conserved between dynamin 1 and dynamin A.

They are located in the critical switch elements and are not well resolved in our

structure, indicating high mobility in the absence of nucleotide

The hGBP1 protein has been suggested to belong to the dynamin family of large

GTPases based on biochemical and anticipated structural similarities18. Comparison of

the fold topology, however, indicates that dynamin and hGBP1 are significantly

different and represent distinct subfamilies of GTP-binding proteins. Despite these gross

structural differences, they do share several structural features, and may be

evolutionarily convergent, functional analogues. For example, the loop connecting α1

and β2 contains the switch I region and varies considerably between GTPases in terms

of length, sequence and structure. In dynamin A, the trajectory and length of this loop is

very similar to that in hGBP1, but different from other GTPases. In the triphosphate

form of hGBP1, this loop forms the so-called phosphate cap17. It seems possible that in

dynamin these residues, which are flexible in the absence of nucleotide, may have a

6

similar function. Two of the insertions found in dynamin A are present in hGBP1 as

well. In both proteins, α2 and the loop connecting β3 and α2 are longer than in Ras and

β6 does not connect to α5 directly, but goes through additional helices. Although in

hGBP1 the central β-sheet is extended to an eight-stranded sheet with the additional

strands next to β2 as in dynamin A, these β-strands are topologically different. In

hGBP1 they are formed by an N-terminal extension, while they are an insertion between

β2 and β3 in dynamin. Furthermore, the direction of the additional strands is reversed

between hGBP1 and dynamin and there is no spatial overlap between β2A of dynamin

A and β0 of hGBP1. Regarding the recognition of the guanine-base, dynamins are

related much closer to virtually any other GTPase than hGBP1.

The crystal structure described here defines the extent and fold of dynamin’s

GTPase domain, shows how the empty P-loop is stabilized and provides us with a

framework to speculate about interactions with the C-terminal GTPase effector domain

(GED). In other GTP-binding proteins, the switch II helix plays a critical role in

conformational rearrangements and interactions with effector proteins. For example, in

EF-Tu, another multidomain GTPase, large movements of the additional domains are

mediated via interactions with the switch II helix on the backside of the molecule. In our

structure, the corresponding area is empty and the switch II helix has no obvious

interaction partner. The start of the switch II helix, the loop preceding it and two

adjacent surface loops are flexible and poorly defined. Additionally, we observe a line

of solvent exposed hydrophobic residues leading from the hydrophobic groove to the

switch II region. Previous studies have shown that the GED interacts with dynamin’s

GTPase domain. Our structure is consistent with this finding and we suggest that contact

will occur via the structural elements described above. This interaction will cover the

exposed hydrophobic side chains, stabilize the flexible loops and place the effector

domain adjacent to the switch elements. Further elucidation of the specific interactions

in this molecule awaits the determination of a full-length crystal structure.

7

Methods

Protein expression, purification and crystallization. Amino acids 2-316 of dynamin

A were fused to the C-terminus of the Dictyostelium myosin II motor domain (residue 2-

765) carrying an N-terminal His-tag. The protein domains are separated by a thrombin

cleavage site, which acts as a linker. The protein was expressed in Dictyostelium and

purified essentially as described for the myosin part alone24. The fusion protein was

concentrated to 5 mg/ml in 50 mM Tris pH 8.0, 1mM MgCl2, 1mM DTT, 3% Sucrose,

for crystallization 2 mM ADP/ MgCl2 was added. Reservoir solution was 11% PEG-

8000, 50 mM Tris, pH 8.5, 200 mM KCl, 5 mM MgCl2, 10% Glucose, 2% Methyl-

propane-diol, 1 mM EGTA, 5 mM DTT. Crystals were grown by the hanging drop

vapor diffusion method at 4° C. Equal amounts of protein and reservoir solutions were

mixed and micro-seeding was used. Crystals grew within two weeks to a size of about

300 x 300 x 15 µm. Crystals were cryo-protected in reservoir solution with 12.5% PEG-

8000 and 20% glycerol and flash-frozen in liquid nitrogen.

Data collection and structure determination. A native data set was collected at Elettra

in Trieste at 100 K using a MAR image plate. Multiple rounds of crystal annealing were

necessary to increase diffraction quality. Data were processed with XDS 25. Initial

phases were obtained by molecular replacement in CNS 26 using the myosin catalytic

domain 27 as search model and data from 15 – 3.5 Å. After rigid body refinement, the

myosin converter domain (residue 686 – 761) was repositioned manually into density

using the program O 28, as it had moved relatively to the rest of the molecule. The initial

density for the dynamin GTPase was used to build six β-strands and two helices as poly-

Ala. Repetitive rounds of refinement using simulated annealing and gradient energy

minimization in CNS followed by manual rebuilding using O added more residues and

side chains as they became visible. When the R-factor had dropped to 25%, 4σ peaks in

8

the fo-fc map within hydrogen-bonding distance to an N or O atom were interpreted as

waters. More cycles of water picking followed. B-factors were refined individually. 87%

of the residues are in the most favored region of the Ramachandran plot, and no non-Gly

residues are in disallowed regions. Atomic coordinate have been deposited in the Protein Data Bank under accession code xxxx. We would like to thank M. Degano and the staff at beamline 5.2R, Elettra, Trieste, B. Klockow and E. Hofmann for helpful discussions, and K. Holmes for continuous support. H.H.N. is supported by the Boehringer Ingelheim Fonds.

Correspondence and requests for materials should be addressed to F.J.K. (e-mail:

kull@mpimf-heidelberg.mpg.de).

9

Figure 1. Structure and topology of the GTPase domain of dynamin A. The G-

protein core fold is shown in green. αA (residues 2-22) is yellow, β2A, αB, β2B

(73-129) are red, αC' and αC are orange (242-273) and the extension of α5

after the kink is blue (297-306). The asterisk marks the variable loop connecting

β2 and β2A. Missing loops are in white. a, Front view. The β-sheet is extended

beyond β2 to a total of eight strands. GDP was modeled into the nucleotide free

structure in order to highlight regions involved in nucleotide binding. b, Side

view. αA and αB pack against the sheet from different sides, while αC runs

perpendicular to the β-strands. αA and the extension of α5 make contacts at

the backside of the β-sheet. c, Topology diagram. Sheets coming out of the

paper plane are triangles with tip up, while those running into the plane are tip

down. Figures were produced using Molscript29 and Raster3d29,30

10

Figure 2. Structure-based sequence alignment of dynamin A from Dictyostelium

discoideum, human dynamin 1 and Ras. Rectangles indicate helices, arrows β-

sheets and dashed lines disordered regions. Colors of secondary structure

elements correspond to those in Fig. 1. The G-protein consensus elements are

boxed and labeled G1 to G4. Residues identical in at least two of the proteins

are boxed in black, conservative substitutions are shaded in gray.

11

Figure 3. Comparison of the nucleotide-binding site of Rac-GDP16 and

nucleotide-free dynamin A. The two molecules were aligned based on the

position of shared α-carbon atoms. After alignment, the GDP was placed into

the dynamin A nucleotide binding site in exactly the same position it occupies in

the Rac-GDP structure. Side chains are shown for the conserved residues

involved in base binding (Thr, Lys, Asp), for the Lys in the P-loop, and for the

Asp of G3. Conserved structural motifs and their adjacent β-strands are shown

in green (G1/P-loop), purple (G2/switch I), blue (G3/switch II), and yellow (G4).

Black dashed lines indicate observed hydrogen bonds and red dashed lines

indicate predicted hydrogen bonds. a, In Rac1-GDP, the Thr from the N/TKxD

motif makes a hydrogen bond to a carbonyl from the P-loop. The Asp makes

two specific hydrogen bonds with the guanine base and the Lys packs over the

base. The P-loop Lys binds to the β-phosphate and the Asp of G3 coordinates

the Mg2+ via a water (not shown). b, Thr 207 and Lys 208 in nucleotide-free

dynamin A occupy positions very similar to those of the corresponding residues

in Rac. Asp 210 would need to move in order to form the predicted hydrogen

bonds (red). Lys 38 from the P-loop binds to Asp 138 from G2.

12

Table 1. Crystallographic data statistics

Data collection and phase determination by molecular replacement method

Crystal space group P21

Unit cell parameters a = 54.45 Å b = 62.04 Å

c = 181.2 Å

α = γ = 90°, β= 94.79 °

Parameter Native data

Resolution (Å)a 15 – 2.3 (2.4 – 2.3) Wavelength (Å) 1.000 Completeness (%) 97.9 (94.6) Unique reflections 52742 Redundancy 3.7 (3.0)

I/σ 19.30 (4.98) Rsym (%)b 5.0 (21.5)

Refinement statistics

Resolution (Å) 15.0 –2.3

Reflections (work set/test set) 49050/3692

Protein atoms 8247

Ligand atoms 28

Water molecules 358

Rwork (%)c 21.0

Rfree (%)d 26.1

Average B factor dynamin only (Å2) 51

Average B factor overall (Å2) 31

aValues in parentheses correspond to the highest resolution shell.

bRsym = ∑h∑i│I(h)-Ii(h)│ / ∑h∑iIi(h), where Ii(h) and I(h) are the ith and mean measurements of the intensity of reflection h.

cRwork = ∑h│Fo - Fc│/ ∑hFo, where Fo and Fc are the observed and calculated structure factor amplitudes of reflection h.

dRfree is the same as Rwork, but calculated on the ~7% of the data excluded from refinement.

13

1. Obar, R. A., Collins, C. A., Hammarback, J. A., Shpetner, H. S. & Vallee, R. B.

Molecular cloning of the microtubule-associated mechanochemical enzyme

dynamin reveals homology with a new family of GTP-binding proteins. Nature

347, 256-61. (1990).

2. van der Bliek, A. M. Functional diversity in the dynamin family. Trends Cell

Biol. 9, 96-102. (1999).

3. Sever, S., Muhlberg, A. B. & Schmid, S. L. Impairment of dynamin's GAP

domain stimulates receptor-mediated endocytosis. Nature 398, 481-6. (1999).

4. Marks, B. et al. GTPase activity of dynamin and resulting conformation change

are essential for endocytosis. Nature 410, 231-235. (2001).

5. van der Bliek, A. M. Is dynamin a regular motor or a master regulator? Trends

Cell Biol. 9, 253-4. (1999).

6. Schmid, S. L., McNiven, M. A. & De Camilli, P. Dynamin and its partners: a

progress report. Curr. Opin. Cell. Biol. 10, 504-12. (1998).

7. Wienke, D. C., Knetsch, M. L., Neuhaus, E. M., Reedy, M. C. & Manstein, D. J.

Disruption of a dynamin homologue affects endocytosis, organelle morphology,

and cytokinesis in Dictyostelium discoideum. Mol. Biol. Cell 10, 225-43. (1999).

8. Kjeldgaard, M., Nyborg, J. & Clark, B. F. The GTP binding motif: variations on

a theme. FASEB J. 10, 1347-68. (1996).

9. Pai, E. F. et al. Refined crystal structure of the triphosphate conformation of H-

ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.

EMBO J. 9, 2351-9. (1990).

14

10. van der Bliek, A. M. & Meyerowitz, E. M. Dynamin-like protein encoded by the

Drosophila shibire gene associated with vesicular traffic. Nature 351, 411-4.

(1991).

11. Clark, S. G., Shurland, D. L., Meyerowitz, E. M., Bargmann, C. I. & van der

Bliek, A. M. A dynamin GTPase mutation causes a rapid and reversible

temperature-inducible locomotion defect in C. elegans. Proc. Natl. Acad. Sci.

USA 94, 10438-43. (1997).

12. Rothman, J. H., Raymond, C. K., Gilbert, T., O'Hara, P. J. & Stevens, T. H. A

putative GTP binding protein homologous to interferon-inducible Mx proteins

performs an essential function in yeast protein sorting. Cell 61, 1063-74. (1990).

13. Gammie, A. E., Kurihara, L. J., Vallee, R. B. & Rose, M. D. DNM1, a dynamin-

related gene, participates in endosomal trafficking in yeast. J. Cell Biol. 130,

553-66. (1995).

14. Amor, J. C., Harrison, D. H., Kahn, R. A. & Ringe, D. Structure of the human

ADP-ribosylation factor 1 complexed with GDP. Nature 372, 704-8. (1994).

15. Bourne, H. R., Sanders, D. A. & McCormick, F. The GTPase superfamily:

conserved structure and molecular mechanism. Nature 349, 117-27. (1991).

16. Scheffzek, K., Stephan, I., Jensen, O. N., Illenberger, D. & Gierschik, P. The

Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins

by RhoGDI. Nat. Struct. Biol. 7, 122-6. (2000).

17. Prakash, B., Renault, L., Praefcke, G. J., Herrmann, C. & Wittinghofer, A.

Triphosphate structure of guanylate-binding protein 1 and implications for

nucleotide binding and GTPase mechanism. EMBO J. 19, 4555-64. (2000).

15

18. Prakash, B., Praefcke, G. J., Renault, L., Wittinghofer, A. & Herrmann, C.

Structure of human guanylate-binding protein 1 representing a unique class of

GTP-binding proteins. Nature 403, 567-71. (2000).

19. Kawashima, T., Berthet-Colominas, C., Wulff, M., Cusack, S. & Leberman, R.

The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution.

Nature 379, 511-8. (1996).

20. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D. & Kuriyan, J. The structural

basis of the activation of Ras by Sos. Nature 394, 337-43. (1998).

21. Milburn, M. V. et al. Molecular switch for signal transduction: structural

differences between active and inactive forms of protooncogenic ras proteins.

Science 247, 939-45. (1990).

22. Kjeldgaard, M. & Nyborg, J. Refined structure of elongation factor EF-Tu from

Escherichia coli. J. Mol. Biol. 223, 721-42. (1992).

23. Berchtold, H. et al. Crystal structure of active elongation factor Tu reveals major

domain rearrangements. Nature 365, 126-32. (1993).

24. Manstein, D. J. & Hunt, D. M. Overexpression of myosin motor domains in

Dictyostelium: screening of transformants and purification of the affinity tagged

protein. J. Muscle Res. Cell Motil. 16, 325-32. (1995).

25. Kabsch, W. Automatic processing of rotation diffraction data from crystals of

initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795-800

(1993).

16

26. Brunger, A. T. et al. Crystallography & NMR system: A new software suite

for macromolecular structure determination. Acta Crystallogr. D 54, 905-21.

(1998).

27. Kliche, W., Fujita-Becker, S., Kollmar, M., Manstein, D. J. & Kull, F. J.

Structure of a genetically engineered molecular motor. EMBO J. 20, 40-46.

(2001).

28. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for

building protein models in electron density maps and the location of errors in

these models. Acta Crystallogr. A 2, 110-19 (1991).

29. Kraulis, P. J. MOLSCRIPT: A Program to Produce Both Detailed and Schematic

Plots of Protein Structures. J. Appl. Crystallogr. 24, 946-950 (1991).

30. Merritt, E. A. & Bacon, D. J. Raster3D: Photorealistic Molecular Graphics.

Methods Enzymol. 277, 505-524 (1997).