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 β -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 βstrands 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 Cterminal 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 Gproteins 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
32
GSQSSGKS39
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 2765) 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% PEG8000, 50 mM Tris, pH 8.5, 200 mM KCl, 5 mM MgCl2, 10% Glucose, 2% Methylpropane-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% PEG8000 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 polyAla. 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:
[email protected]).
9
Figure 1. Structure and topology of the GTPase domain of dynamin A. The Gprotein 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 Molscript
29
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 βsheets 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
Resolution (Å)
Native data
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 (%)
Rfree (%)
c
21.0
d
26.1
2
Average B factor dynamin only (Å )
2
Average B factor overall (Å )
51
31
a
Values in parentheses correspond to the highest resolution shell.
b
th
Rsym = ∑h∑i│I(h)-Ii(h)│ / ∑h∑iIi(h), where Ii(h) and I(h) are the i and mean measurements of the
intensity of reflection h.
c
Rwork = ∑h│Fo - Fc│/ ∑hFo, where Fo and Fc are the observed and calculated structure factor
amplitudes of reflection h.
d
Rfree is the same as Rwork, but calculated on the ~7% of the data excluded from refinement.
13
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