Post on 14-Jul-2020
Article
The Structure of the ZMYN
D8/Drebrin ComplexSuggests a Cytoplasmic Sequestering Mechanism ofZMYND8 by DrebrinGraphical Abstract
Highlights
d ZMYND8 PHD-BROMO-PWWP tandem specifically binds to
non-histone protein Drebrin
d The ZMYND8 PHD-BROMO-PWWP tandem/Drebrin ADF-H
domain complex structure is solved
d Drebrin binding blocks histone marker access to ZMYND8
d Drebrin can sequester ZMYND8 in cytoplasm and thus
regulate its nuclear activity
Yao et al., 2017, Structure 25, 1657–1666November 7, 2017 ª 2017 Elsevier Ltd.http://dx.doi.org/10.1016/j.str.2017.08.014
Authors
NingningYao, JianchaoLi, HaiyangLiu,
Jun Wan, Wei Liu, Mingjie Zhang
Correspondenceliuw@ust.hk (W.L.),mzhang@ust.hk (M.Z.)
In Brief
The crystal structure of ZMYND8 PHD-
BROMO-PWWP tandem in complex
Drebrin ADF-H domain solved by Yao
et al. reveals how ZMYND8 can
specifically bind to non-histone proteins
and how the histone marker reader might
be sequestered in cytoplasm by
specifically binding to an actin binding
protein.
Data Resources
5Y1Z
Structure
Article
The Structure of the ZMYND8/DrebrinComplex Suggests a CytoplasmicSequestering Mechanism of ZMYND8 by DrebrinNingning Yao,1,4 Jianchao Li,2,4 Haiyang Liu,1,2 Jun Wan,1,2 Wei Liu,1,2,* and Mingjie Zhang1,2,3,5,*1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong
University of Science and Technology Medical Center, Shenzhen 518036, China2Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Kowloon,Hong Kong3Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and
Technology, Clear Water Bay, Kowloon, Hong Kong4These authors contributed equally5Lead Contact
*Correspondence: liuw@ust.hk (W.L.), mzhang@ust.hk (M.Z.)
http://dx.doi.org/10.1016/j.str.2017.08.014
SUMMARY
Malfunctions of the actin binding protein Drebrinhave been implicated in various human diseasessuch as Alzheimer’s disease, cognitive impairments,cancer, and digestive disorders, though with poorlyunderstood mechanisms. The ADF-H domain of Dre-brin does not contain actin binding and depolymeriz-ing activity. Instead, it binds to a histone markerreader, ZMYND8. Here we present the high-resolu-tion crystal structure of Drebrin ADF-H in complexwith the ZMYND8 PHD-BROMO-PWWP tandem,elucidating the mechanistic basis governing thehighly specific interaction of the two proteins. Thestructure reveals that the ZMYND8 PHD-BROMO-PWWP tandem forms a structural supramodule thatis necessary for binding to Drebrin ADF-H. DrebrinADF-H competes with modified histone for bindingto ZMYND8. Binding of Drebrin can shuttle ZMYND8from nucleus to cytoplasm in living cells. Takentogether, our study uncovers a non-actin target bind-ing mode for ADF-H domains, and suggests thatDrebrin may regulate activities of epigenetic readerZMYND8 via its cytoplasmic sequestration.
INTRODUCTION
Shuttling proteins between nuclei and cytoplasm is a general
strategy used by eukaryotic cells to control spatially the activ-
ities of proteins (Gama-Carvalho and Carmo-Fonseca, 2001;
Xu and Massague, 2004). Neurons are no exceptions. In fact
the extremely polarized morphology of neurons often requires
more coordinated protein distributions between different com-
partments of cells including nuclei and cytoplasm. For example,
stimulations of a neuron via its synapses can induce long-lasting
transcriptional event alterations in its nuclei. Such long-range
communication between synapses and nuclei may involve direct
Stru
shuttling of proteins between these two compartments (Ch’ng
et al., 2012; Deisseroth et al., 2003; Ivanova et al., 2015; Panayo-
tis et al., 2015). As with other cell types, neurons frequently shut-
tle proteins between nucleus and cytoplasm in response to
different stimuli. For example, some highly conserved RNA bind-
ing proteins (RBPs), which are specifically expressed in the ner-
vous system, could be shuttled between nucleus and cytoplasm
in the presence of nucleo-cytoplasmic shuttling signals and play
important roles in neurite differentiation (Colombrita et al., 2013;
Kasashima et al., 1999). Similar nuclear/cytoplasm translocation
phenomena are also known in the case of the Suppressor of
fused (Sufu), a negative regulator of the Hedgehog signal trans-
duction pathway, and histone deacetylase 7 (HDAC7), a negative
gene expression regulator and the chromatin-associated high-
mobility group proteins (Li et al., 2004; Paces-Fessy et al.,
2004; Pedersen et al., 2010). Such nuclear/cytoplasm shuttling
of these proteins are often regulated by either post-translational
modification or protein-protein interactions.
Zinc-finger MYND-type-containing 8 (ZMYND8), also known
as PRKCBP1, RACK-7, and Spikar, was first identified as a pro-
tein kinase C binding partner (Fossey et al., 2000) and was later
shown to be a transcriptional regulator (Malovannaya et al.,
2011). It contains an N-terminal nuclear localization sequence
(NLS) and a PHD-BROMO-PWWP (PBP) tandem, and a C-termi-
nal coiled-coil (CC) domain and a myeloid, Nervy, and DEAF-1
(MYND) domain. Its domain organization is similar to that of
ZMYND11 (also known as BS69), which uses the N-terminal
BROMO-PWWP tandem to recognize histone H3.3K36me3
(Guo et al., 2014; Wen et al., 2014). Both ZMYND8 and
ZMYND11 were reported to be tumor suppressors (Ansieau
and Sergeant, 2003).
Studies of ZMYND8 have so far been mainly focused on
its roles in nucleus, where it can act as reader for post-transla-
tionally modified histones, form complex with lysine-specific
demethylase 5C (KDM5C) in regulating active enhancers, co-
localize with nucleosome remodeling and histone deacetylase
(NuRD) complex in DNA damage response, bind to REST core-
pressor 2 (RCOR2) and thus regulate neuronal differentiation,
and interact with lysine-specific demethylase 5D (KDM5D,
also known as JARID1D) to suppress metastasis-linked gene
cture 25, 1657–1666, November 7, 2017 ª 2017 Elsevier Ltd. 1657
Figure 1. Biochemical Characterizations of ZMYND8/Drebrin Interaction
(A) Domain organizations of ZMYND8 and Drebrin.
(B) Analytical gel filtration-based assay showing that ZMYND8 PBP tandem and Drebrin ADF-H domain can interact with each other in vitro.
(C) ITC-based assay showing that Drebrin ADF-H domain can bind to ZMYND8 PBP tandem with a KD of �4.3 mM.
(D) ITC results showing that including the Drebrin CC-Hel domain and ZMYND8 N-terminal NLS did not further enhance the binding.
expression (Adhikary et al., 2016; Gong et al., 2015; Li et al.,
2016; Savitsky et al., 2016; Shen et al., 2016; Spruijt et al.,
2016; Zeng et al., 2010). Recently, ZMYND8 was shown to
interact with a neuronal actin binding protein known as develop-
mentally regulated brain protein (Drebrin) (Yamazaki et al., 2014).
It was shown that the localization of ZMYND8 to the dendritic
spines depends on the binding of Drebrin, suggesting a role of
Drebrin in regulating the distribution of ZMYND8 between nu-
cleus and the synapses.
Drebrin was first discovered as the developmentally regulated
brain protein (Shirao et al., 1988). The expression levels of
different isoforms of Drebrin (Drebrin A and E) were shown to
be developmentally regulated: Drebrin E is prominently ex-
pressed during early neuronal development and Drebrin A is ex-
pressed in more mature neurons (Imamura et al., 1992; Shirao
and Obata, 1985). Drebrin is highly expressed in neuron and re-
ported to regulate actin cytoskeleton in dendritic spines (Lin and
Koleske, 2010; Sekino et al., 2007; Shirao and Gonzalez-Billault,
2013). The expression level of Drebrin is tightly controlled. Over-
expression of Drebrin can cause elongation of the spines without
affecting spine density, while knockout or knockdown of Drebrin
will lead to reduction of the density of dendritic spine and
decrease in the PSD-95 clustering in post-synaptic densities (Ta-
kahashi et al., 2003). Downregulation of the expression level of
Drebrin has also been linked to several human genetic diseases,
such as Alzheimer’s disease, Down syndrome, and mild cogni-
tive impairments (Counts et al., 2012; Harigaya et al., 1996;
Shim and Lubec, 2002). In addition to neurons, Drebrin is also ex-
1658 Structure 25, 1657–1666, November 7, 2017
pressed in other tissues including lung, intestine, and liver. Either
overexpression or underexpression of Drebrin has been impli-
cated in various human diseases including cancer and microvilli
inclusion disease, possibly due to impairments of cell polarity in
tissues caused by altered levels of Drebrin (Bazellieres et al.,
2012; Dart et al., 2017; Dun and Chilton, 2010; Lin et al., 2014;
Peitsch et al., 2005). It is tempting to hypothesize that, in addition
to directly binding to actin filaments, Drebrin may also control
transcriptional activities of ZMYND8 by modulating its nuclear
localization in neurons as well as other cell types, thereby indi-
rectly exerting diverse regulatory roles in various tissues.
Both Drebrin E and Drebrin A contain an N-terminal highly con-
served actin-depolymerizing factor homology (ADF-H) domain
(Figure 1A), which loses both actin-depolymerizing activity and
actin binding ability (Grintsevich et al., 2010; Poukkula et al.,
2011). Instead, they use their central putative CC domain and he-
lical (Hel) domain to interact with and bundle actin filaments
(Grintsevich et al., 2010; Worth et al., 2013). The lacking of actin
binding of the Drebrin ADF-H domain raises a natural question:
what is the function of this evolutionarily conserved ADF-H
domain? The recent discovery that the Drebrin ADF-H domain
can bind to ZMYND8 suggests that it may function as a general
protein binding module instead of an actin binding domain.
In this study, we elucidated the molecular basis governing
the unexpected ZMYND8 PBP/Drebrin ADF-H interaction. The
structure of the ZMYND8 PBP/Drebrin ADF-H complex revealed
that the interaction between ZMYND8 and Drebrin is highly
specific. No binding could be detected between ZMYND11
Figure 2. Overall Structure of the ZMYND8
PBP/Drebrin ADF-H Complex
(A) Ribbon representations of the ZMYND8 PBP/
Drebrin ADF-H complex crystal structure. The
Drebrin ADF-H domain is colored in green. The
PHD, BROMO, Zinc Finger, PWWP, andC-terminal
extension are colored in pink, violet, magenta,
hotpink, and purple, respectively (as shown in
Figure 1A). The zinc ions are shown as gray
spheres. This color coding applies to all figures
except when otherwise indicated.
(B) Comparison of the ZMYND8 PBP structure (in
magenta) in the ZMYND8 PBP/Drebrin ADF-H
complex with the recently reported ZMYND8 PBP
apo-form structures (PDB: 4COS in light blue;
PDB: 5B73 in light yellow). The histone binding
sites for PHD, BROMO, and PWWP domains are
indicated with cyan, marine, and blue ellipses,
respectively.
(C) Superposition of the BROMO-PWWP tandems
of ZMYND8 (magenta) and ZMYND11 (PDB: 4N4H
in yellow) showing that the overlapping binding site
of Drebrin ADF-H (green) to ZMYND8 and
H3.3K36me3 (orange) to ZMYND11.
(D) Structural details of the overlapping binding
of Drebrin and histone on PWWP domain. D1:
ZMYND8/Drebrin. D2: ZMYND11/H3.3K36me3.
D3: superposition of ZMYND8/Drebrin and
ZMYND11/H3.3K36me3. Critical residues are
shown as sticks.
andDrebrin or between ZMYND8 andDrebrin-like. The ZMYND8
PBP/Drebrin ADF-H complex structure further suggests that
Drebrin binding and histone binding are mutually exclusive in
the ZMYND8 PBP tandem. Our structure also explains why the
Drebrin ADF-H domain fails to bind actin filaments. Finally, we
demonstrated using a heterologous cell culture assay that nu-
clear localized ZMYND8 can be effectively shuttled out to the
cytoplasm by co-expression of wild-type Drebrin but not by
ZMYND8 binding-defective mutants designed on the basis of
the complex structure.
RESULTS
ZMYND8 Can Bind to Drebrin ADF-H Domain with ItsPHD-BROMO-PWWP TandemA previous study has shown that the interaction between
ZMYND8 and Drebrin is mediated by the PBP tandem and
ADF-H domain (Yamazaki et al., 2014) (Figure 1A). Here we first
purified the recombinant ZMYND8 PBP tandem (amino acids
Structu
[aa] 103–426) and the Drebrin ADF-H
domain (aa 1–135) to validate and quantify
the interaction. On an analytical gel filtra-
tion column, a 1:1 mixture of ZMYND8
PBP and Drebrin ADF-H was eluted at a
volume clearly smaller than each of the
two individual proteins alone (Figure 1B),
suggesting that the two proteins in the
mixture form a stable complex. An
isothermal titration calorimetry (ITC) assay
was used to quantify the interaction, and
the titration experiment showed that the two proteins bind to
each other with a 1:1 stoichiometry and with a dissociation con-
stant (KD) of�4.3 mM (Figure 1C). Extending ZMYND8 to include
the conserved N-terminal NLS sequence (aa 1–426) and extend-
ing Drebrin ADF-H to include the C-terminal CC-Hel region (aa
1–309) did not alter the binding affinity between the two proteins
(Figures 1C and 1D), indicating that ZMYND8 PBP and Drebrin
ADF-H are the minimal regions responsible for the two proteins
to interact with each other.
Overall Structure of ZMYND8 PHD-BROMO-PWWP/Drebrin ADF-H ComplexTo understand the molecular basis of the interaction, we set out
to determine the crystal structure of the ZMYND8 PBP/Drebrin
ADF-H complex. After numerous trials, we obtained several
well-diffracted (to 2.65-A resolution) crystals by fusing the Dre-
brin ADF-H domain at the C terminus of ZMYND8 PBP with a
22-residue flexible linker (Figure 2A and Table 1). Consistent
with the recently solved crystal structures in its apo form (Li
re 25, 1657–1666, November 7, 2017 1659
Table 1. X-Ray Crystallographic Data Collection and Model
Refinement
Data Collection
Datasets ZMYND8 PBP/Drebrin ADF-H
Space group P212121
Wavelength (A) 0.9780
Unit cell parameters (A) a = 54.18, b = 138.10, c = 161.16
a = b = g = 90�
Resolution range (A) 50–2.65 (2.70–2.65)
No. of unique reflections 34,299 (1,638)
Redundancy 6.5 (6.3)
I/s 25.9 (2.5)
Completeness (%) 98.9 (94.5)
Rmergea (%) 9.2 (95.1)
CC1/2 (last-resolution shell)b 0.758
Structure Refinement
Resolution (A) 15–2.65 (2.77–2.65)
Rcrystc/Rfree
d(%) 17.81/23.37 (27.97/33.62)
RMSD bonds (A)/angles (�) 0.009/0.941
Average B factor (A2)e 58.1
No. of atoms
Protein atoms 6,702
Water 48
Ligands 42
No. of reflections
Working set 32,354 (2,717)
Test set 1,648 (149)
Ramachandran plot regionse
Favored (%) 97.6
Allowed (%) 2.4
Outliers (%) 0
Numbers in parentheses represent the value for the highest-resolu-
tion shell.aRmerge = S jIi � <I>j/SIi, where Ii is the intensity of measured reflection
and <I> is the mean intensity of all symmetry-related reflections.bCC1/2 was defined by Karplus and Diederichs (2012).cRcryst = S jjFcalcj � jFobsjj/SFobs, where Fobs and Fcalc are observed and
calculated structure factors.dRfree = ST jjFcalcj � jFobsjj/SFobs, where T is a test dataset of about 5% of
the total unique reflections randomly chosen and set aside prior to
refinement.eB factors and Ramachandran plot statistics are calculated using Mol-
Probity (Chen et al., 2010).
et al., 2016; Savitsky et al., 2016), the ZMYND8 PBP tandem
consists of an N-terminal PHD domain bound with two Zn2+
ions, a BROMO domain and a PWWP domain separated by a
zinc finger. The four domains of the ZMYND8 PBP tandem
interact with each other sequentially and intimately to form an in-
tegral structural supramodule. The binding of Drebrin ADF-H
domain does not induce obvious conformational changes of
the PBP supramodule (root-mean-square deviation [RMSD]
with the apo structures PDB: 4COS and PDB: 5B73 of 0.55 A
and 0.58 A, respectively; Figure 2B). The Drebrin ADF-H domain
fits snugly into the groove formed by the BROMO, zinc-finger,
and PWWP domains of the ZMYND8 PBP supramodule, and
1660 Structure 25, 1657–1666, November 7, 2017
makes contacts with both the BROMO domain and the PWWP
domain. The structure suggests that formation of the ZMYND8
PBP supramodule is essential for its binding to Drebrin. The
structure of the ZMYND8 PBP supramodule solved here also
showed a high similarity to that of the ZMYND11 BROMO-
PWWP tandem (RMSD of 1.21 A, Figure 2C).
Detailed Structural Analysis Shows that the Interactionbetween ZMYND8 PHD-BROMO-PWWP and DrebrinADF-H Is Highly SpecificADF-H binds to two separate sites of the ZMYND8 PBP tandem,
one on the PWWP domain and the other on the BROMO domain
(Figure 3A). The interface involving the PWWP domain is mainly
mediated by the aA of Drebrin ADF-H. A salt bridge formed by
Arg10D (the superscript ‘‘D’’ represents Drebrin) and Asp332Z
(‘‘Z’’ denotes ZMYND8) is critical for the interaction (Figure 3B),
as charge-reversemutations of either of the two residues individ-
ually totally disrupted the binding (Figure 3D). Hydrophobic inter-
actions by Leu11D, Leu14D, and Tyr17D and Phe308Z, Thr355Z,
and Ile358Z further anchor the aA helix of ADF-H to the
PWWP site (Figure 3B). In addition, a hydrogen bond between
Glu107D and His331Z also contributes to the binding (Figure 3B).
Mutating the His331Z to Ala abolished the binding (Figure 3D).
The binding in the second site is much less extensive and only
involves hydrophobic residues of Cys96D from Drebrin ADF-H
aC and Ile257Z, Val260Z from the ZMYND8 BROMO domain.
Substitutions of Cys96D or Val260Z to polar Gln led to a 10- to
100-fold decrease of the binding (Figure 3D).
Both the Drebrin and Drebrin-like protein (also known as
HIP-55, mAbp1, and SH3P7) are mammalian orthologs of yeast
actin binding protein 1 (Abp1), amember of the ABP family of pro-
teins (Kessels et al., 2000). They share similar domain architec-
tures in their N terminus. Especially their ADF-H domains share
more than 40% sequence identity (Figure S1A), and the crystal
structure reported here can be superimposedwell with the previ-
ously solved nuclear magnetic resonance structure of Drebrin-
like ADF-H domain (RMSD �1.16 A) (Goroncy et al., 2009). We
wondered whether ZMYND8 can also interact with the Drebrin-
like ADF-H domain. We carefully analyzed the sequences and
found that although the ADF-H domain of Drebrin-like shared
high identity with Drebrin and only a few residues involved in
ZMYND8binding are different, Drebrin-like ADF-Hhas nodetect-
able binding to ZMYND8 PBP (Figures 3D, S1B, and S1C). The
Drebrin ADF-H and ZMYND8 PBP complex structure can explain
the lack of binding of Drebrin-like ADF-H to ZMYND8 PBP. The
residue corresponding to Arg10 in Drebrin is a Gly in Drebrin-
like (Figure S2), and Arg10 is absolutely essential for Drebrin
ADF-H to bind to ZMYND8 PBP (Figures 3B and 3D). As ex-
pected, when Arg10 of Drebrin ADF-H was replaced by Gly, the
mutant protein failed to interact with ZMYND8 (Figures 3D and
S1D). In parallel, ZMYND11, a close relative of ZMYND8, cannot
bind toDrebrin either (Figures 3D, S1E, andS1F). Taken together,
these biochemical data further support that the interaction be-
tween ZMYND8 and Drebrin is highly specific.
The Drebrin ADF-H Binding and Histone Peptide Bindingon ZMYND8 PBP Tandem Are Mutually ExclusivePHD, BROMO, and PWWP domains are well known to recog-
nize histone markers (Filippakopoulos and Knapp, 2012; Qin
Figure 3. The Detailed Interactions between ZMYND8 PBP and Drebrin ADF-H
(A) The ZMYND8 PBP/Drebrin ADF-H interface can be divided into two parts as highlighted by solid and dashed boxes.
(B) Stereo view of the detailed interactions in the ADF-H/PWWP interface.
(C) Detailed interactions in the ADF-H/BROMO interface.
(D) Summary of the ITC-derived dissociation constants showing that mutations of the critical residues in either of the interfaces weakened or abolished the
binding. Also, no binding was detected between ZMYND8/Drebrin-like and ZMYND11/Drebrin. WT, wild-type.
and Min, 2014; Sanchez and Zhou, 2011). In the apo form of
the PBP supramodule, all the known histone binding sites
are accessible to their respective binders (Figure 2B). Upon
forming a complex with Drebrin ADF-H, the histone binding
site on the PWWP domain is occupied by ADF-H, indicating
that the Drebrin and histone binding on ZMYND8 PBP supra-
module may be mutually exclusive. Consistent with this anal-
ysis, the histone H3.3K36me3 binding site on the ZMYND11
PWWP domain (thus inferring also the histone peptide binding
site of ZMYND8 PWWP) overlaps with the Drebrin binding site
(Figures 2C and 2D) (Wen et al., 2014). In particular, the gua-
nidinium group of Arg10 in Drebrin and the trimethylated
amide group of K36 in H3.3 are at a near identical position
in the two structures (Figure 2D), suggesting that Drebrin
ADF-H can block histone peptide from binding to ZMYND8
PWWP. This analysis is supported by a recent finding showing
that mutations in the PWWP domain weaken the ZMYND8/his-
tone peptide binding (Savitsky et al., 2016). Considering that
the binding affinity of ZMYND8 PBP to Drebrin ADF-H is com-
parable with those of ZMYND8 PBP to histone peptides (with
KD in the micromolar range) (Li et al., 2016; Savitsky et al.,
2016), it is biochemically possible that Drebrin can compete
with histone peptides for binding to the ZMYND8 PBP
supramodule.
Comparison of ADF-H Domain Complex StructuresSuggests a Non-canonical Function of Drebrin ADF-HDomainThe ZMYND8 PBP/Drebrin ADF-H complex structure solved in
this work reveals a previously unknown function of the ADF-H
domain. Instead of binding to actin, Drebrin ADF-H binds to his-
tonemarker recognition domains. The aC helix of Drebrin ADF-H
interacts with the ZMYND8 BROMO domain (Figure 4A). Previ-
ous biochemical and structural studies have focused on how
other ADF-H domains bind to actin (Lappalainen et al., 1998;
Poukkula et al., 2011). The structures of twinfilin C-terminal
ADF-H or cofilin ADF-H domain in complex with G-actin or
F-actin showed that the long aC, situated at the cleft between
actin subdomains 1 and 3, constitutes the major actin binding
element of ADF-H (Figure 4B) (Galkin et al., 2011; Paavilainen
et al., 2008). A similar binding mode has also been observed in
the structure of GMF ADF-H bound to the actin-related protein
2 (ACTR2) of Arp2/3 complex (Luan and Nolen, 2013). Interest-
ingly, the ADF-H domain of cofilin and twinfilin is structurally
homologous to Gelsolin segment 1, and they both utilize their
respective aC to bind to the same groove between subdomains
1 and 3 of actin, although the detailed interacting residues are
slightly different (Mclaughlin et al., 1993; Paavilainen et al.,
2008). On another note, the crystal structure of cofilin ADF-H
Structure 25, 1657–1666, November 7, 2017 1661
Figure 4. aC in ADF-H Domains Is Commonly Used to Recognize Targets
(A–C) Crystal structures of Drebrin ADF-H/ZMYND8 (A, magenta), twinfilin ADF-H/Actin (B, orange), and cofilin ADF-H/LIMK kinase (C, salmon) complexes
showing that the aC (highlighted in green) is the key element for these ADF-H domains (shown in gray) to interact with their respective targets. Residues critical in
binding are shown as blue sticks.
(D) Sequence alignments of the commonly used aC from all human ADF-H domains. The residues critical for actin binding are highlighted in red boxes and the
residue critical for ZMYND8 binding in Drebrin is highlighted in a magenta box.
domain in complex with its regulator LIMK1 kinase domain has
recently been reported (Hamill et al., 2016). LIMK1 is known to
phosphorylate cofilin and inhibit cofilin’s actin binding and depo-
lymerizing capacity. Interestingly, the aC of cofilin ADF-H is also
chiefly responsible for binding to the LIMK1 kinase domain
(Figure 4C).
Sequence alignments of the aC from all identified human
ADF-H domains showed that several critical residues (two posi-
tively charged residues Arg/Lys at position 2, Arg/Lys at position
11, and the two hydrophobic residues Val/Leu/Ile at position 1
and Met at position 5) responsible for actin binding are con-
served in cofilin or twinfilin but not in Drebrin or Drebrin-like (Fig-
ure 4D). This is consistent with the extremely low actin binding
affinity of Drebrin and Drebrin-like ADF-H domains (Grintsevich
et al., 2010; Kessels et al., 2000; Poukkula et al., 2011). The
Drebrin ADF-H domain instead has gained a new function in
binding to the histone marker reader ZMYND8. The Cys at the
1662 Structure 25, 1657–1666, November 7, 2017
fifth position of aC of Drebrin is used for hydrophobic interaction
with the ZMYND8 BROMO domain (Figure 4D). The correspond-
ing residue in cofilin is Met5, which is responsible for both actin
and LIMK1 binding. The corresponding residue at this position in
Drebrin-like is aGly (Figure 4D), andwe have shown that Drebrin-
like was not able to bind to ZMYND8. The structural and amino
acid sequence analyses suggest that the diverse amino acid se-
quences of the structurally invariant aC helix of ADF-H domains
may allow these ADF-H domains to bind to proteins with very
different functions.
Drebrin Binding-Induced ZMYND8 CytoplasmLocalizationThe Drebrin-dependent synaptic localization of ZMYND8 in
neurons (Yamazaki et al., 2014) suggested that Drebrin can
sequester ZMYND8 at synapses and prevent it from entering
the nucleus to perform its nuclear function as a transcriptional
Figure 5. ZMYND8/Drebrin Interaction Is
Essential for the Cytoplasmic Localization
of ZMYND8
(A) Representative fluorescent images of HEK293T
cells expressing Myc-ZMYND8 alone or co-ex-
pressing Myc-ZMYND8. A1: Myc-ZMYND8 wild-
type (WT) alone; A2: Myc-ZMYND8 WT and HA-
Drebrin WT; A3: Myc-ZMYND8 and HA-Drebrin
R10G; A4: Myc-ZMYND8 H331A and HA-Drebrin
WT. The nuclei were shown by DAPI (blue) staining.
(B) Quantification of percentages of cells with
ZMYND8 in nuclei in different groups of experi-
ments; 300–800 cells were quantified in each
group of experiments. Values are expressed in
means ± SD and analyzed using one-way ANOVA
with Tukey’s multiple comparison test by Graph-
Pad Prism 6. ns, not significant; ****p < 0.0001.
(C) Schematic cartoon showing the proposed
function of ZMYND8/Drebrin complex. C1: the
synaptic localization of ZMYND8 by binding to actin
cytoskeletal associating protein Drebrin. C2: the
epigenetic function of ZMYND8 by binding to his-
tone markers.
regulator. We used HEK293T heterologous cells to test this hy-
pothesis. When overexpressed alone, ZMYND8 was predomi-
nantly localized in the nucleus as shown by its co-localization
with DAPI (Figures 5A1 and 5B). Co-expression of hemagglutinin
(HA)-Drebrin with Myc-ZMYND8 led to effective redistribution of
ZMYND8 from nucleus to cytoplasm (Figure 5A2). Importantly,
specific mutations either on Drebrin (R10G) or ZMYND8
(H331A), both of which disrupt the formation of the Drebrin/
ZMYND8 complex, were defective in Drebrin-induced cyto-
plasm localization of ZMYND8, indicating that the redistribution
of ZMYND8 from nucleus to cytoplasm is Drebrin binding depen-
dent (Figures 5A3, 5A4, and 5B).
DISCUSSION
In the current study, by detailed biochemical and structural char-
acterizations, we demonstrate that ZMYND8 PBP tandem can
specifically interact with the Drebrin ADF-H domain. We also
show that ZMYND8 can relocalize from nucleus to cytoplasm
upon binding to Drebrin in living cells. Drebrin is an important
actin-regulating protein in synapses. Although ZMYND8 is well
known for its epigenetic function in the nucleus, the discovery
Structu
of the ZMYND8/Drebrin interaction sug-
gests that the nuclear level of ZMYND8
may be regulated by Drebrin-mediated
binding in neuronal synapses (Figure 5C).
Considering that the expression levels of
Drebrin vary at different brain develop-
mental stages (Shirao and Obata, 1985),
the nuclear distribution of ZMYND8 might
also be developmentally regulated via
binding to Drebrin. We wish to emphasize
that the current biochemical, structural,
and heterologous living cell studies,
together with an earlier study showing
Drebrin-dependent synaptic localization
of ZMYND8 in cultured neurons (Yamazaki et al., 2014), suggest
a possible Drebrin-mediated nuclear/cytoplasm shuttling mech-
anism of ZMYND8 in neurons. When Drebrin expression level is
high, this cytoskeleton-associated actin binding protein can act
as a cytoplasmic anchor to sequester ZMYND8 in the cytosol by
the interaction described in this work.Without such sequestering
(due to either lowered expression level of Drebrin or regulated
dissociation of the Drebrin/ZMYND8 complex), ZMYND8 can
enter the nucleus via its NLS-mediated transportation to perform
its epigenetic function. Further studies are required to verify
whether such a shuttling mechanism is indeed adopted in neu-
rons. Nevertheless, the structural information obtained in this
study will be valuable for guiding future functional studies of
the ZMYND8/Drebrin interaction in nervous systems.
The ZMYND8 PBP/Drebrin ADF-H complex presented here
has also revealed a previously unrecognized role of the ADF-H
domain. ADF-H domains were originally identified as actin-
severing modules to promote filamentous actin depolymeriza-
tion (Lappalainen et al., 1998). ADF-H domain-containing pro-
teins can be grouped into five classes. However, members of
only two classes (cofilin and twinfilin) have been unequivocally
shown to contain actin binding and severing activities (Poukkula
re 25, 1657–1666, November 7, 2017 1663
et al., 2011). The GMF class does not bind to actin but can asso-
ciate with an actin-related protein (ACTR2) in the Arp2/3 complex
(Luan and Nolen, 2013). The fact that Coactosin or the Drebrin
classes can only very weakly interact with F-actin suggests
that the ADF-H domain may have functions other than binding
to and severing actin filaments. In the Drebrin ADF-H case, res-
idues critical for actin binding in the aChelix aremissing. Instead,
this commonly used actin binding region becomes the ZMYND8
BROMO domain binding site. Together with a second site that
binds to the PWWP domain of ZMYND8, the interaction between
Drebrin ADF-H and ZMYND8 PBP is highly specific and with a
quite high affinity (Figure 3).
PHD, BROMO, and PWWP, the three well-characterized do-
mains involved in epigenetics regulation, are well known as
readers of epigenetically modified histones. In this study, we
elucidated the molecular mechanism of the interaction between
the ZMYND8 PBP tandem and the synaptic cytoskeletal protein
Drebrin. To the best of our knowledge, this is the first structural
study elucidating how histone marker readers might also bind
to non-histone targets. This finding expands our knowledge of
how histone marker readers may be regulated by binding
proteins outside the cell nucleus. Recently, several BROMO
domain-containing proteins have been reported to have both nu-
cleus and cytoplasm localizations (Filippakopoulos and Knapp,
2012). Similar cytoplasm localization of histone recognition do-
mains containing proteins, e.g., bromodomain-containing pro-
tein 7 (BRD7), bromodomain and PHD finger-containing protein
1 (BRPF1), and PHD finger protein 20-like protein 1 (PHF20L1),
are also found in the Human Protein Atlas database (http://
www.proteinatlas.org/). Although it is not clear how the nu-
clear/cytoplasm localizations of these proteins are determined,
our study of the Drebrin/ZMYND8 interaction provides a clue
to how histone maker readers may also play roles in cellular
localizations via binding to proteins outside the nucleus.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
166
B HEK293T Cells Culture
d METHOD DETAILS
B Cloning and Protein Purification
B The Size Exclusion Chromatography Coupled with
Multiple Angle Light Scattering Assay
B The Isothermal Titration Calorimetry Assay
B Crystallography
B Cell Imaging and Data Analysis
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
B Data Resources
SUPPLEMENTAL INFORMATION
Supplemental Information includes two figures and can be found with this
article online at http://dx.doi.org/10.1016/j.str.2017.08.014.
4 Structure 25, 1657–1666, November 7, 2017
AUTHOR CONTRIBUTIONS
J.L., W.L., J.W., and M.Z. designed the experiments. N.Y. and H.L. performed
biochemistry and cell biology experiments; J.L. is responsible for the structural
biology experiments. All authors analyzed the results. J.L., W.L., and M.Z.
wrote the manuscript. M.Z. coordinated the research.
ACKNOWLEDGMENTS
We thank the Shanghai Synchrotron Radiation Facility (SSRF) BL17U1
and BL19U1 for X-ray beam times. This work was supported by grants
from the National Basic Research Program of China (2014CB910204)
and National Key R&D Program (2016YFA0501903) from the Minister of
Science and Technology of China, Natural Science Foundation of
Guangdong Province (2016A030312016), Shenzhen Basic Research grant
(JCYJ20160229153100269), and RGC of Hong Kong (16103614; 16149516;
and AoE-M09-12) to M.Z., and grants from the National Natural Science Foun-
dation of China (No. 31400647 and No. 31670765) and Shenzhen Basic
Research grants (JCYJ20160427185712266 and JCYJ20170411090807530)
to W.L. M.Z. is a Kerry Holdings Professor in Science and a Senior Fellow of
IAS at HKUST.
Received: April 11, 2017
Revised: July 27, 2017
Accepted: August 28, 2017
Published: September 28, 2017
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monoclonal anti-HA Santa Cruz Biotechnology Cat#sc7392; RRID: AB_627809
Rabbit polyclonal anti-Myc Proteintech Cat#16286-1-AP; RRID: AB_11182162
Chemicals, Peptides, and Recombinant Proteins
Dulbecco’s Modified Eagle Medium (DMEM) Thermo Fisher Scientific Cat#11995065
fetal bovine serum (FBS) Thermo Fisher Scientific Cat#16000044
penicillin-streptomycin Thermo Fisher Scientific Cat#15140122
Recombinant protein: Human Drebrin ADF-H (aa:
1-135, ref# NP_004386.2)
This paper N/A
Recombinant protein: Human Drebrin ADF-H-CC-
Hel (aa: 1-309, ref# NP_004386.2)
This paper N/A
Recombinant protein: Human ZMYND8 NLS-PBP
(aa: 1-426, ref# NP_898868.1)
This paper N/A
Recombinant protein: Human ZMYND8 PBP (aa:
103-426, ref# NP_898868.1)
This paper N/A
Recombinant protein: Mouse Drebrin-like ADF-H
(aa: 1-138, ref# NP_001139780.1)
This paper N/A
Recombinant protein: Mouse ZMYND11 Bromo-
PWWP (aa: 155-367, ref# NP_001334401.1)
This paper N/A
Recombinant protein: ZMYND8 PBP-linker-
Drebrin ADF-H (ZMYND8 Q103-S426-
GSGSGENLYFQGGSGGSGGSGS-Drebrin
M1-N135)
This paper N/A
Critical Commercial Assays
ViaFect� Transfection Reagent Promega Cat#E4981
Deposited Data
Crystal structure of ZMYND8 PBP/Drebrin ADF-H This paper PDB: 5Y1Z
Experimental Models: Cell Lines
Human: HEK293T cells ATCC CRL-3216
Experimental Models: Organisms/Strains
Escherichia coli: Rosetta (DE3) Novagen Cat#70954
Escherichia coli: BL21 (DE3) Invitrogen Cat#C600003
Recombinant DNA
Plasmid: HA-Drebrin This paper N/A
Plasmid: Myc-ZMYND8 This paper N/A
Plasmid: S142A Drebrin-YFP Addgene Cat#58335
Plasmid: GFP-ZMYND8 Addgene Cat#65401
Plasmid: pET32M.3C Human Drebrin ADF-H This paper N/A
Plasmid: pET32M.3C Human Drebrin ADF-H-
CC-Hel
This paper N/A
Plasmid: pET32M.3C Human ZMYND8 NLS-PBP This paper N/A
Plasmid: pET32M.3C Human ZMYND8 PBP This paper N/A
Plasmid: pET32M.3C Mouse Drebrin-like ADF-H This paper N/A
Plasmid: pET32M.3C Mouse ZMYND11
Bromo-PWWP
This paper N/A
Plasmid: pET32M.3C ZMYND8 PBP-linker-Drebrin
ADF-H
This paper N/A
(Continued on next page)
Structure 25, 1657–1666.e1–e3, November 7, 2017 e1
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Software and Algorithms
Origin7.0 OriginLab http://www.originlab.com/
GraphPad Prism GraphPad Software Inc. http://www.graphpad.com/scientific-software/
prism/
HKL2000 (Otwinowski and Minor, 1997) http://www.hkl-xray.com/
PHASER (Mccoy et al., 2007) http://www.phaser.cimr.cam.ac.uk/
PHENIX (Adams et al., 2010) http://www.phenix-online.org/
Coot (Emsley et al., 2010) http://www2.mrc-lmb.cam.ac.uk/Personal/
pemsley/coot/
PyMOL DeLano Scientific LLC http://www.pymol.org/
ASTRA6 Wyatt http://www.wyatt.com/products/software/
astra.html
ImageJ NIH https://imagej.nih.gov/ij/
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to, and will be fulfilled by the Lead Contact Mingjie Zhang (mzhang@
ust.hk).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
HEK293T Cells CultureHEK293T cells (fromATCC) were cultured in Dulbecco’sModified EagleMedium (DMEM) supplementedwith 10% fetal bovine serum
(FBS), and 1% of penicillin-streptomycin at 37�C with 5% CO2.
METHOD DETAILS
Cloning and Protein PurificationThe coding sequences of Drebrin fragments (residues 1-135 & 1-309) were PCR amplified using the full-length human 70 kDa Drebrin
cDNA template (NCBI accession code: NM_004395.3) . The coding sequences of ZMYND8 (residues 103-426&1-426) fragments
were PCR amplified using the full-length human 130 kDa ZMYND8 cDNA template (NCBI accession code: NM_183047.3). The
coding sequences of Drebrin-like (residues 1-138, NCBI accession code: NM_001146308.1) and ZMYND11 (residues 155-367,
NCBI accession code: NM_001347472.1) were PCR amplified from a mouse cDNA library. For the fusion construct used for crystal-
lization and the complex structure determination, Drebrin 1-135 was fused to ZMYND8 103-426 C-terminus by standard two-step
PCR with a 22-residue flexible linker ‘GSGSGENLYFQGGSGGSGGSGS’ consisted of a TEV cleavage site ‘ENLYFQ’ flanking by
Glycine and Serine residues. All point mutations were created by the standard PCR-based mutagenesis method and confirmed
by DNA sequencing. All of these coding sequences were cloned into a modified pET32a vector for protein expression. All Drebrin
or Drebrin-like proteins were expressed in Escherichia coli BL21 (DE3), while all ZMYND8 and ZMYND11 proteins were expressed
in Escherichia coli Rosetta (DE3) with additional ZnCl2 (final concentration in medium: 200 mM). The N-terminal thioredoxin-His6-
tagged proteins were purified with a Ni2+ Sepharose� 6 Fast Flow column and followed by a Superdex-200 prep grade size-exclu-
sion chromatography. The thioredoxin tag was cut by 3C protease and the proteins were further purified by another Superdex-200
prep grade size-exclusion chromatography.
The Size Exclusion Chromatography Coupled with Multiple Angle Light Scattering AssayProtein samples (typically 100 mL at a concentration of 50 mM, pre-equilibrated with column buffer) were injected into an AKTA FPLC
system with a Superose 12 10/300 GL column (GE Healthcare) using the column buffer of 50 mM Tris-HCl (pH 7.5), 1 mM DTT, and
100 mM NaCl. The chromatography system was coupled to a multi-angle light scattering system equipped with a 18 angles static
light scattering detector (Dawn, Wyatt) and a differential refractive index detector (Optilab, Wyatt). The elution profiles were analyzed
using the ASTRA 6 software (Wyatt).
The Isothermal Titration Calorimetry AssayIsothermal titration calorimetry (ITC) measurements were carried out on the MicroCal ITC200 calorimeter (Malvern) at 16�C. All pro-teins were dissolved in 50mMTris buffer containing 100mMNaCl, and 1mMDTT at pH 7.5. The protein concentrations in the syringe
e2 Structure 25, 1657–1666.e1–e3, November 7, 2017
vary from 500 mM-600 mM,while the concentrations of the binding partners in the cell vary from 50 mM-60 mM. Each titration point was
performed by injecting a 2 mL aliquot of the protein sample from the syringe into the protein sample in the cell at a time interval of 120
seconds to ensure that the titration peak returned to the baseline. The titration data were analyzed by Origin7.0 (Microcal) and fitted
by the one-site binding model.
CrystallographyCrystals of the Drebrin and ZMYND8 complex (in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT buffer) were obtained by
the sitting drop vapor diffusion method at 16�C. The crystals were grown in buffer containing 0.2 M ammonium sulfate, 0.1 M Bis-
Tris, pH 6.0 and 23%w/v polyethylene glycol 3,350 and then soaked in crystallization solution containing additional 10% v/v glycerol
for cryo-protection. Diffraction data were collected at the Shanghai Synchrotron Radiation Facility BL17U1 at 100 K. Data were pro-
cessed and scaled using HKL2000 (Otwinowski and Minor, 1997).
Structure of the ZMYND8 PBP/Drebrin ADF-H complex was solved by molecular replacement with the models of the ZMYND8
PBP apo form (PDB code: 4COS) and the Drebrin-like ADF-H domain (PDB code: 1X67) using PHASER (Mccoy et al., 2007). Further
manual model building and refinement were completed iteratively using Coot (Emsley et al., 2010) and PHENIX (Adams et al., 2010).
The final model was validated byMolProbity (Chen et al., 2010). The final refinement statistics are summarized in Table 1. All structure
figures were prepared by PyMOL (http://www.pymol.org).
Cell Imaging and Data AnalysisHEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and
1% of penicillin-streptomycin at 37�C with 5% CO2. Cells were transfected using ViaFect� Transfection Reagent (Promega) as per
manufacturer’s protocol.
All the images were acquired using a Zeiss LSM 710 laser-scanning confocal microscope, with a 633 1.4 oil objective and pinhole
setting to 1 Airy unit. Data were collected from 3 independent batches of cultures. In each batch, at least 300 fluorescence-positive
cells were counted for each group of experiments. Experimentswere conducted in a double-blinded fashion. The distribution ratios in
nucleus and cytoplasm were quantified and analyzed using GraphPad Prism 6. Data were expressed as mean ± SD. All other groups
were compared with Myc-ZMYND8 only group by one-way ANOVA with Tukey’s multiple comparison test.
QUANTIFICATION AND STATISTICAL ANALYSIS
Data of nuclear localization assays were collected from three independent batches of cultures (>300 fluorescence positive cells per
group per batch). Data were expressed as mean ± SD and analyzed using one-way ANOVAwith Tukey’s multiple comparison test by
GraphPad Prism 6; ns: not significant; ****: p<0.0001. All experiments were performed in a double-blinded fashion.
DATA AND SOFTWARE AVAILABILITY
Data ResourcesThe atomic coordinates of ZMYND8 PBP/Drebrin ADF-H complex have been deposited to the Protein Data Bank under accession
code PDB: 5Y1Z.
Structure 25, 1657–1666.e1–e3, November 7, 2017 e3
Structure, Volume 25
Supplemental Information
The Structure of the ZMYND8/Drebrin
Complex Suggests a Cytoplasmic
Sequestering Mechanism of ZMYND8 by Drebrin
Ningning Yao, Jianchao Li, Haiyang Liu, Jun Wan, Wei Liu, and Mingjie Zhang
Figure S1: Biochemical characterizations of ZMYND8/Drebrin-like and ZMYND11/Drebrin
interaction. Related to Figure 3.
(A) Domain organizations of Drebrin/Drebrin-like and ZMYND8/ZMYND11. (B) Analytical gel
filtration based assay and ITC-based assay showing no interaction between ZMYND8 PBP
tandem and Drebrin-like ADF-H domain. (D) ITC-based assay showing that Drebrin ADF-H
domain carrying R10G mutation failed to bind to ZMYND8 PBP tandem. (E&F) Analytical gel
filtration based assay and ITC-based assay showing that ZMYND11 BP tandem cannot bind to
Drebrin ADF-H domain in vitro.
Figure S2: Sequence alignment of Drebrin and Drebrin-like ADF-H domains from different
species. Related to Figure 3.
The secondary structure elements are labelled according to the Drebrin ADF-H structure. Residues
that are identical and highly similar are indicated in dark green and light green boxes, respectively.
The residues involved in ZMYND8 binding are highlighted with magenta boxes.