The EMBO Journal Vol.18 No.11 pp.2923–2929, 1999
The crystal structure of the Physarum polycephalum
actin–fragmin kinase: an atypical protein kinase with
a specialized substrate-binding domain
Stefan Steinbacher1, Peter Hof,
Ludwig Eichinger2, Michael Schleicher2,
Jan Gettemans3, Joe ¨l Vandekerckhove3,
Robert Huber and Jo ¨rg Benz4
Abteilung Strukturforschung, Max-Planck-Institut fu ¨r Biochemie,
Ludwig-Maximilians-Universita ¨t, 80336 Mu ¨nchen, Germany and
3Flanders Interuniversity Institute of Biotechnology,
University of Gent, 9000 Gent, Belgium
4Present address: Department fu ¨r Chemie und Biochemie,
Universita ¨t Bern, 3012 Bern, Switzerland
Coordinated temporal and spatial regulation of the
actin cytoskeleton is essential for diverse cellular pro-
cesses such as cell division, cell motility and the forma-
tion and maintenance of specialized structures in
differentiated cells. In plasmodia of Physarum poly-
cephalum, the F-actin capping activity of the actin–
fragmin complex is regulated by the phosphorylation
of actin. This is mediated by a novel type of protein
kinase with no sequence homology to eukaryotic-type
protein kinases. Here we present the crystal structure
of the catalytic domain of the first cloned actin kinase
in complex with AMP at 2.9 Å resolution. The three-
dimensional fold reveals a catalytic module of ~160
residues, in common with the eukaryotic protein kinase
superfamily, which harbours the nucleotide binding
site and the catalytic apparatus in an inter-lobe cleft.
Several kinases that share this catalytic module differ
in the overall architecture of their substrate recognition
domain. The actin–fragmin kinase has acquired a
unique flat substrate recognition domain which is
supposed to confer stringent substrate specificity.
Keywords: actin phosphorylation/crystal structure/
cytoskeleton reorganization/fragmin/protein kinase
Protein phosphorylation plays a key role in regulating the
dynamic rearrangements of the cytoskeleton starting with
upstream signalling cascades (Eby et al., 1998) resulting
in phosphorylation of downstream effectors which directly
interact with actin or tubulin. For example, the signalling
pathway of Rac-mediated stimulus-induced actin reorgan-
ization results in phosphorylation of cofilin by LIM-kinase
(Arber et al., 1998) which abolishes cofilin’s actin binding
and depolymerization activities (Arber et al., 1998; Yang
et al., 1998). Phosphorylation of actin itself has been
observed repeatedly. Treatment of mammalian cells with
epidermal growth factor induces rapid phosphorylation of
actin in the cortical skeleton (van Delft et al., 1995).
© European Molecular Biology Organization
Stage-dependent phosphorylation of actin at Tyr53 in
Dictyostelium discoideum (Howard et al., 1993; Jungbluth
et al., 1995) is associated with morphological alterations
and reorganization of the actin cytoskeleton.
Plasmodial fragmin from the slime mould Physarum
polycephalum is a member of the gelsolin family which
has been implicated in cellular processes that require rapid
actin cytoskeleton reorganization, and interferes with the
growth of F-actin by severing actin filaments and capping
their barbed ends. The EGTA-resistant 1:1 complex
between actin and fragmin has been identified as the sole
in vivo target for a specific protein kinase (actin–fragmin
kinase, AFK) that phosphorylates actin mainly at Thr203
and to a minor extent at Thr202 in the actin–fragmin
complex but not in G-actin (Gettemans et al., 1992;
De Corte et al., 1996). The phosphorylation sites are
located at the minor contact site for DNase I (Kabsch
et al., 1990) and at one of the proposed actin–actin contact
sites along the long-pitch helix of F-actin (Holmes et al.,
1990). The F-actin nucleating activity of actin–fragmin is
abolished upon phosphorylation in vitro and its capping
activity becomes Ca2?-dependent. The latter observ-
ation was corroborated by microinjection of the (un)phos-
phorylated actin–fragmin complex in mammalian cells
(Constantin et al., 1998). These data indicate that actin
polymerization in Physarum can be controlled by actin
phosphorylation in a Ca2?-dependent manner.
Biochemical studies and cloning of the AFK resulted
in the identification of an 80 kDa protein representing a
novel type of protein kinase (Eichinger et al., 1996). Two
domains of ~35 kDa are linked by a sequence stretch of
50amino acidsrich inproline andserineresidues. Whereas
the C-terminal part harbours six so-called ‘kelch’-repeats,
indicating a six-bladed propeller structure (Bork and
Doolittle, 1994), the N-terminal part consists of a cata-
lytically active protein kinase domain (cAFK) which,
most notably, does not show any sequence similarities to
eukaryotic protein kinases and lacks all signature motifs
that characterize this super-family (Hanks and Hunter,
1995; Eichinger et al., 1996). Here we report on the
crystal structure of cAFK that was solved to gain insight
structural relationships to typical protein kinases.
Results and discussion
Recombinant cAFK was crystallized from high salt con-
ditions in the presence of AMP. Other nucleotides or
nucleotide analogues did not give crystals suitable for
structure determination. The structure was solved by single
isomorphous replacement and two-fold non-crystallo-
graphic symmetry averaging. The structure has been
refined to a crystallographic R-factor of 19.9% and Rfree
Crystal structure of actin–fragmin kinase
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Received March 9, 1999; revised and accepted April 7, 1999