Báo cáo khoa học: Structural basis of p63a SAM domain mutants involved in AEC syndrome ppt

Structural basis of p63a SAM domain mutants involved in
AEC syndrome
Aruna Sathyamurthy
1
, Stefan M. V. Freund
2
, Christopher M. Johnson
2
, Mark D. Allen
2
and Mark Bycroft
2
1 MRC Centre for Protein Engineering, Cambridge, UK
2 MRC Laboratory of Molecular Biology, Cambridge, UK
Keywords
5-helix bundle; AEC syndrome; mutations;
p53; p63; p73; sterile alpha motif
Correspondence
M. D. Allen, MRC Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 0QH,
UK
Fax: +44 (0)1223 213556
Tel: +44 (0)1223 402409
E-mail:
(Received 9 July 2010, revised 11 April
2011, accepted 24 May 2011)
doi:10.1111/j.1742-4658.2011.08194.x
p63 is a member of the p53 tumour suppressor family that includes p73.
The p63 gene encodes a protein comprising an N-terminal transactivation
domain, a DNA binding domain and an oligomerization domain, but var-
ies in the organization of the C-terminus as a result of complex alternative

splicing. p63a contains a C-terminal sterile a motif (SAM) domain that is
thought to function as a protein–protein interaction domain. Several mis-
sense and heterozygous frame shift mutations, encoded within exon 13 and
14 of the p63 gene, have been identified in the p63a SAM domain in
patients suffering from ankyloblepharon–ectodermal dysplasia–clefting syn-
drome. Here we report the solution and high resolution crystal structures
of the p63a SAM domain and investigate the effect of several mutations
(L553F ⁄ V, C562G ⁄ W, G569V, Q575L and I576T) on the stability of the
domain. The possible effects of other mutations are also discussed.
Database
Coordinates are available in the Protein Data Bank database under the accession numbers
2Y9U and 2Y9T.
Introduction
p63 is a member of the p53 tumour suppressor family
that includes p73; however, p63 does not function as a
classical tumour suppressor and is rarely mutated in
human cancers. Sequence homology and initial studies
revealed that p63 could act as a DNA-sequence spe-
cific transcription factor for target genes that leads to
apoptosis or cell-cycle arrest and suggested a function
for p63 as a tumour suppressor [1,2]. Unlike p53-
knockout mice that developed spontaneous tumours,
p63-knockout mice exhibited developmental defects
implying that p63 plays an important role in mamma-
lian development [3–5]. Consistent with this, mutations
of p63 are primarily associated with developmental dis-
orders [6–14]. p63 and p73 genes share significant
sequence identity with each other and with p53 in the
N-terminal transactivation domain (TAD), the DNA
binding domain and the oligomerization domain, but

differ in having an extended C-terminal coding region
that undergoes complex alternative splicing to form six
different isoforms [1]. TAp63a, TAp63b and TAp63c
contain the N-terminal TAD, whereas the DNp63a,
DNp63b and DNp63c are transcribed from an alterna-
tive internal promoter and lack the full-length TAD.
Only splice variants a in both p63 and p73 have a five
helical bundle domain at the C-terminus called a sterile
a motif (SAM).
The p63 gene undergoes mutations in its DNA bind-
ing domain, causing ectrodactyly–ectodermal dysplasia–
cleft lip ⁄ palate (EEC) syndrome, where it is predicted to
lose its DNA binding capacity [15,16]. Several missense
Abbreviations
AEC, ankyloblepharon–ectodermal dysplasia–clefting; EEC, ectrodactyly–ectodermal dysplasia–cleft lip ⁄ palate; SAM, sterile a motif;
TAD, transactivation domain.
2680 FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS
mutations with amino acid substitutions and heterozy-
gous frame shift mutations, encoded within exon 13 of
the p63 gene, have also been identified in the p63a SAM
domain in patients suffering from ankyloblepharon–
ectodermal dysplasia–clefting (AEC) syndrome [17–19].
The AEC phenotype is similar to that of the EEC
patients and yet differs distinctly in its features.
p53, TAp63 and TAp73 can all activate p53-
response elements through their TADs that share
significant homologies. The activation of promoters for
MDM2 and p21, and the repression of Hsp70 pro-
moter, appears to depend on the isoform of p63.
TAp63 proteins with a mutated SAM domain or no

SAM domain (TAp63b and TAp63c) all showed a dis-
tinct activation of MDM2 and p21, and a decreased
repression of Hsp70 [20], compared with wild-type
TAp63a. As such, the SAM domain of p63 has been
implicated in the inhibition of p63-mediated transacti-
vation of both MDM2 and p21. This result is further
supported by mutations occurring in the p63 SAM
domain resulting in the activation of Hsp70.
Mutations can inactivate a protein either by altering
functionally important residues or by the global desta-
bilization of the overall fold. It is important to know
which mechanism is occurring in order to be able to
correctly identify functional sites in the protein. The
solution structure of p63a SAM domain
1RG6 pro-
vided an insight into how several of the AEC muta-
tions observed may cause destabilization of the
domain, and indeed two of the mutations studied by
the group could only be analysed using molecular
dynamics. We have determined high resolution crystal
and solution structures of the wild-type p63a SAM
domain in order to understand the molecular conse-
quences of AEC mutations observed in human p63a.
We have characterized the stability of several addi-
tional mutant p63a SAM domains using chemical
denaturation to investigate the contribution of each
mutation to domain stability and function. The major-
ity of the mutations so far observed in human p63a
SAM domains that lead to AEC syndrome appear to
cause destabilization of the domain.

Results and Discussion
Domain boundary selection
Attempts to crystallize the p63a SAM domain as a
construct comprising residues 543–622 were unsuccess-
ful. The precise domain boundaries of the p63a SAM
domain were subsequently determined by NMR relax-
ation experiments (Fig. S1) and a solution structure
of the domain was obtained. The structure revealed
that only residues 545–611 are involved in secondary
structure elements. A truncated construct comprising
these residues readily crystallized under a number of
conditions.
NMR sample preparation and structure
determination
The initial p63a SAM domain construct (residues 543–
622) was used to determine the NMR solution struc-
ture. Complete
1
H ⁄
13
C ⁄
15
N assignments and structure
determination were carried out using standard methods
as described in the Materials and methods. A summary
of all conformational constraints and statistics is pre-
sented in Table 1. Comparison of our solution struc-
ture with a previously published solution structure of
the p63a SAM domain [21] (PDB
1RG6) revealed that

the global fold and arrangement of helices is essentially
the same (Fig. 1) with an rmsd of 1.36 A
˚
. Of interest
is a short b-sheet that is occasionally absent in the
1RG6 structure, which brings together the N-terminal
region and the third 3
10
-helix, and is defined by the
Table 1. Summary of conformational constraints and statistics for
the 20 accepted NMR structures of p63a SAM domain.
Structural constraints
Intra-residue 609
Sequential 303
Medium range (2 £ |i ) j | £ 4) 195
Long range (|i – j | > 4) 219
Dihedral angle constraints 15
TALOS constraints 108
Distance constraints for 37
hydrogen bonds
74
Total 1523
Statistics for accepted structures
Statistics parameter (± SD)
RMS deviation for distance
constraints (A
˚
)
0.0068 ± 0.0003
RMS deviation for

dihedral constraints (°)
0.232 ± 0.020
Mean CNS energy term (kcalÆmol
)1
± SD)
E (overall) 66.84 ± 2.56
E (van der Waals) 20.44 ± 1.48
E (distance constraints) 4.92 ± 0.42
E (dihedral and
TALOS constraints) 0.81 ± 0.14
RMS deviations from the ideal geometry
(± SD)
Bond lengths (A
˚
) 0.0012 ± 0.00004
Bond angles (°) 0.310 ± 0.0041
Improper angles (°) 0.197 ± 0.0068
Average atomic rmsd from the mean structure
(± SD)
Residues 546–607 (N, C
a
, C atoms) (A
˚
) 0.259 ± 0.068
Residues 546–607 (all heavy atoms) (A
˚
) 0.706 ± 0.067
A. Sathyamurthy et al. Mutants involved in AEC syndrome
FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS 2681
presence of an HA-HA NOE (548–572) and two

HA-HN NOEs (548–573, 572–549) between the two
regions. A corresponding b-sheet region is absent in
the p73a SAM domain [22] (PDB
1DXS).
Crystal structure
The crystal structure of the truncated construct
(545–611) of p63a SAM was solved by molecular
replacement using the NMR structure as a search
model. The crystal structure was refined to 1.6 A
˚
and
is consistent with the NMR structure (rmsd of 0.97 A
˚
).
The crystallographic data are summarized in Table 2.
The crystal structure enabled analysis of the side-chain
atoms of surface residues implicated in AEC muta-
tions, and in particular confirmed the presence of salt
bridges and hydrogen bonds which could only have
been suggested using the NMR structures.
Comparing p63a with p73a and other SAM
domains
The main feature of the p63a SAM domain is a five
helical bundle comprising four a-helices and a short
3
10
-helix. Unlike the other structurally homologous
SAM domains, there is a short and distinct b-sheet,
which brings together the N-terminus and the third
3

10
-helix. Helices 1 and 5 are antiparallel and form a
compact hydrophobic core with the other three helices.
A sequence alignment of p63- and p73-like SAM
domains in different organisms (Fig. 2) highlights the
importance of several residues. The aliphatic isoleucine
and leucine residues (I549, L553, L556, I573, I576,
L584, L587, I589, I597 and I601) that are part of a
compact hydrophobic core are highly conserved in all
SAM domains. G557 is conserved throughout suggest-
ing an important role in forming a turn before the
C-X-X-C motif. Interestingly, this sequence is not
always present and can be replaced by an L-Q ⁄ G-A-Y
motif. Several surface-exposed aspartates, lysines, argi-
nines and serines are also highly conserved. The two
highly conserved residues F552 and F565 participate in
the formation of the hydrophobic core. F593 is par-
tially solvent exposed and can be substituted for either
histidine or tyrosine in other SAM domains. The fully
conserved tryptophan at position 598 in both p63a
and p73a SAM is solvent exposed, whereas in homolo-
gous SAM domains it participates in hydrophobic core
formation. Most of the conserved hydrophobic resi-
dues appear to be involved in stabilizing the fold,
although some of the solvent-exposed residues may
have a functional role.
The structural alignments of the p63a (crystal) and
p73a [22] SAM domains are in good agreement with
an overall rmsd for the backbone atoms of 0.88 A
˚

.
The p63a SAM domain differs from p73a by contain-
A
C
B
D
Fig. 1. (A) Superimposition of the 20
energy-minimized conformers and (B) a
MOLSCRIPT [39] ribbon representation of the
p63a SAM domain solution structure. (C)
The crystal structure of p63a SAM domain,
and (D) the crystal structure of p73a SAM
domain.
Mutants involved in AEC syndrome A. Sathyamurthy et al.
2682 FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS
ing a free cysteine (C547) instead of a proline, possibly
helping in the formation of a small b-sheet region, and
the C-terminus of p63a SAM is significantly longer.
The two conserved cysteines (C558 and C561) in p63a
SAM domain are also reduced as in the p73a SAM
domain. The inclusion of a reducing agent in the crys-
tallization of p63a SAM domain would potentially
have contributed to the reduced state of the cysteine
residues, although the functional significance of this
conserved motif is still unclear. The C-S-S-C region in
p63a forms the beginning of the second a helix and is
consistent in both solution and crystal structure. In the
case of p73a, the C-P-N-C motif forms a loop rather
than a helix, possibly reflecting the effect of a proline
on helix termination.

The p63a and p73a SAM domains form a distinct
subset of the SAM domain family. SAM domains, in
general, were initially thought to be involved in pro-
tein–protein interactions by forming homo- or hetero-
oligomers with other SAM domains or by interacting
with other proteins [23]. The SAM domain of the pro-
tein Smaug, however, has been shown to participate
in RNA binding [24,25]. The exact role of p63a and
p73a SAM domains is still unclear, but it has been
speculated that the SAM domain of both p63a and
p73a have lipid binding properties [26]. The p63a and
p73a SAM domains do not form homo-oligomers or
associate with each other.
Analysis of mutations involved in the AEC
syndrome
In the AEC syndrome, the SAM domain of p63a is
found to undergo a number of mutations. A number of
point mutations have been identified in exons 13 and 14
of the p63 gene. These mutations can be classified into
groups according to their sequence position. I549T,
F552S, L553F, L553V, C561G, C561W, F565L, I576T,
L584P and I597T are present in the hydrophobic core
whereas G557V, G569V, T572P, Q575L, S580F, S580P,
S580Y, D583C, D583Y, P590L, F593S, R594P, G600V
and G600D all occur on the solvent-exposed surface.
The positions of these mutations can be seen in Fig.3.
To analyse the effect of these mutations, the p63a
SAM domain containing some of the point mutations
above were cloned and expressed. The expression of
point mutations found in the AEC syndrome varied

significantly. The wild-type protein and the mutants
L553V and C561W were found to be over-expressed in
the soluble fraction. In contrast, mutants L553F,
C561G, G569V, Q575L and I576T were present only
as inclusion bodies, but could be refolded easily during
purification. The mutants G569V, Q575L and I576T
partially aggregated at the gel filtration stage but suffi-
cient material was purified to allow analysis. The sta-
bilities of the expressed mutants were determined by
analysing their respective denaturation curves (Fig. S2)
using the intrinsic fluorescence of W598 as the probe
of measurement. The measure of stability of these pro-
teins was determined by the amount of the denaturant
required to get 50% of the protein in a denatured state
([D]
50%
). The m-value, a constant related to the change
in the surface-exposed surface area upon denaturation,
gives a very good idea of the degree of unfolding and
was calculated for all mutants. The energetics of some
of the mutations in the p63a SAM domain are shown
in Table 3. The denaturation curves obtained for
mutants were substantially different from that of wild-
type with respect to both the pre-denaturation slope
and the m-value of denaturation. A lower value of
m
D–N
may indicate denaturation from an already par-
tially unfolded structure, as a result of a mutation.
However, it could also reflect unfolding to an incom-

pletely denatured state which would also reduce the
change in surface-exposed surface area upon denatur-
Table 2. Crystallographic summary of p63a SAM domain.
Data set Native
Symmetry P2
1
2
1
2
1
Wavelength (A
˚
) 0.9795
Resolution range (A
˚
) 27.4–1.6
Unique reflections 8205
Completeness (%)
a
98.9 (96.0)
R
merge
b
0.108 (0.302)
Multiplicity
c
5.3 (5.5)
I ⁄ rI
a
14.0 (4.7)

a = 34.681 A
˚
, b = 38.336 A
˚
,
c = 44.471 A
˚
Model refinement
Resolution range (A
˚
) 22.0–1.6
No. of residues A: 545–611
No. of water, ligand molecules 81, 1 sulphate
R
work
⁄ R
free
(%)
d
0.185, 0.206
B average
e
20.29 A
˚
2
Geometry bonds ⁄ angles
f
0.009 A
˚
, 1.192°

Ramachandran
g
95.1%, 0.0%
PDB ID
h
2Y9U
a
Signal to noise ratio of intensities, highest resolution bin in brack-
ets.
b
R
m
: RhRi |I(h, i ) ) I(h)| ⁄ RhRiI(h, i ) where I(h, i ) are symmetry-
related intensities and I(h) is the mean intensity of the reflection
with unique index h.
c
Multiplicity for unique reflections.
d
5% of
reflections were randomly selected for determination of the free R
factor, prior to any refinement.
e
Temperature factors averaged for
all atoms.
f
RMS deviations from ideal geometry for bond lengths
and restraint angles (Engh and Huber).
g
Percentage of residues
in the ‘most favoured region’ of the Ramachandran plot and per-

centage of outliers (
PROCHECK).
h
Protein Data Bank identifiers for
coordinates.
A. Sathyamurthy et al. Mutants involved in AEC syndrome
FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS 2683
ation. The pronounced slopes of the pre-transition
phase could be consistent with the former explanation,
indicating non-cooperative partial unfolding over this
concentration of guanidinium hydrochloride or reflect-
ing a more open and dynamic structure as a starting
point.
Compared with the wild-type, mutations L553F,
C561G, C561W, G569V, Q575 and I576T are all signifi-
cantly destabilized, as judged by the reduction in [D]
50%
and DG
H
2
O
DÀN
. A possible explanation for the instability of
each of these point mutations was obtained by looking
at the structural features of the wild-type protein. Muta-
tion L553F, which is close to F552, would probably
cause a severe steric clash between the two phenylala-
nine rings and result in overcrowding in the hydropho-
bic core. The tryptophan ring in C561W would possible
result in a steric clash with the aromatic ring of F593.

** **
T
Y
PT
** * * **
TPWVFVL
*
α4
α5









α1
α2 α3
β1
β2
NN
P PYP T D CSI V S FLA R L GCSS C LDYF T TQGL T TIYQ
P PYP T D CSI V S FLA R L GCSS C LDYF T TQGL T TIYQ
P PYP T D CSI V S FLA R L GCSS C VDYF T TQGL T TIYH
P PYP M D NSI S S FLL R L GCSA C LDYF T AQGL T NIYQ
P PYH A D PSL V S FLT G L GCPN C IEYF T SQGL Q SIYH
P PYH A D PSL V S FLT G L GCPN C IECF T SQGL Q SIYH
K CEP T E NTI A Q WLT K L GLQA Y IDNF Q QKGL H NMFQ

Q NDM Q D NSV S T WLN A L GLGA Y IDGF H EQNL Y SLLQ
N GEM T D ISV A A WLN H L GLGA Y IDSF H EHNL Y SVIQ
DGA DL LSIS R WLSN IMEKY TQE F I KHG F K VCGH
AV M
IEH Y S MDD L A SLK I P EQFR H AIWK G ILDH R QL
IEH Y S MDD L A SLK I P EQFR H AIWK G ILDH R QL
IEH Y S MDD L V SLK I P EQFR H AIWK G ILDH R QL
IEN Y N LED L S RLK I P TEFQ H IIWK G IMEY R QT
LQN L T IED L G ALK I P EQYR M TIWR G LQDL K QG
LQN L T IED L G ALK V P DQYR M TIWR G LQDL K QS
LDE F T LED L Q SMR I G TGHR N KIWK S LLDY R RL
LDD F S LDD L A KMK I G NSHR N KIWK S LLEL R NQ
H EF
H DF
H DF
M EF
H DY
H DC
L SS
G FT
LDD F S LDD L A KMK I G NAHR N KIWK S VLEL R NE
G LT
L N S Y SD KKII K NMED C K KIS A Y LLE S N FS
S GN
Q9H3D4
O88898
Q9DEC7
Q8JHZ6
O15350
Q9JJP2

Q27937
Q9NGC7
Q8T7V3
B3RZS6
HUMAN P63 576
MOUSE P63 576
GAL G P63 478
DAN R P63 482
HUMAN P73 520
MOUSE P73 514
LOL F P53 486
MYA A P73 520
SPI S P53 492
TRI A P53 501
Q9H3D4
O88898
Q9DEC7
Q8JHZ6
O15350
Q9JJP2
Q27937
Q9NGC7
Q8T7V3
B3RZS6
HUMAN P63 541
MOUSE P63 541
GAL G P63 443
DAN R P63 447
HUMAN P73 485
MOUSE P73 479

LOL F P53 451
MYA A P73 485
SPI S P53 457
TRI A P53 466
610
610
512
516
554
548
520
554
526
535
575
575
477
481
519
513
485
519
491
500
*
*
*
VP
L
*

*
V
L
*
S
F
P
*
S
V
G
D
Y
Fig. 2. CLUSTALW sequence alignment of
SAM domains. Residues that are absolutely
conserved are shown in red. Conserved and
partially conserved hydrophobic residues are
shown in black and grey, respectively. Con-
served and partially conserved charged resi-
dues are shown in blue and light blue,
respectively. The positions and type of AEC
mutations are shown above the sequence
alignment. A diagrammatic representation of
the domain is shown below the alignment.
I549
L553
G557
C561
G569
T572

Q575
S580
I576
R594
I597
F565
D581
L584
F593
P590
F552
Fig. 3. Ribbon representation of the p63a SAM domain showing
the position of mutations that are associated with AEC syndrome.
Table 3. Thermodynamic values of p63a SAM domain AEC mutants. DG
H
2
O
DÀN
is an estimate of the stability of the proteins in buffer calcu-
lated from DG
H
2
O
DÀN
¼ m½D
50%
, where m
D–N
is the m value for denaturation of the protein. The mutants G534V and I541T were highly destabi-
lized and their denaturation curves could not be fitted reliably. These mutants proved problematic to express and purify (see Results and

Discussion) consistent with this destabilization.
Protein Location of mutation [D]
50%
(M) m
D–N
(kcalÆmol
)1
) DG
H
2
O
DÀN
(kcalÆmol
)1
) DDG
H
2
O
DÀN
(kcalÆmol
)1
)
Wild-type – 3.50 (± 0.02) 2.31 (± 0.1) 8.11 (± 0.35) –
L553V Core 3.38 (± 0.09) 1.31 (± 0.17) 4.43 (± 0.59) 3.68
L553F Core 1.79 (± 0.16) 0.96 (± 0.06) 1.72 (± 0.19) 6.39
C561G Core 1.61 (± 0.17) 1.11 (± 0.08) 1.79 (± 0.23) 6.32
C561W Core 2.59 (± 0.12) 1.12 (± 0.13) 2.90 (± 0.36) 5.21
G569V Surface Highly destabilized Highly destabilized Highly destabilized Highly destabilized
Q575L Surface 2.66 (± 0.04) 1.85 (± 0.17) 4.92 (± 0.46) 3.19
I576T Core Highly destabilized Highly destabilized Highly destabilized Highly destabilized

Mutants involved in AEC syndrome A. Sathyamurthy et al.
2684 FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS
The instability of C561G is most probably caused by the
formation of a hydrophobic cavity resulting from the
loss of a bulky thiol group. The cavity created would
potentially cause some rearrangement of the hydropho-
bic core and the associated instability. Introduction of a
polar residue into the hydrophobic core, as happens
with mutation I576T, would potentially lower the stabil-
ity of the domain. G569 adopts a positive angle of phi in
the wild-type domain, something rarely seen for ali-
phatic residues. As such, mutation G569V may cause
instability due to the residues in the loops adopting a
more unfavourable conformation. Mutation Q575L
occurs in a solvent-exposed position and the side-chain
carbonyl of Q575 forms a hydrogen bond with the
main-chain amides of both T571 and T572 (Fig. S4). As
such, any mutation, with the exception of Q575E, would
potentially destabilize the domain. Mutations G569V
and T572P were analysed by another group using molec-
ular simulations due to protein instability and degrada-
tion during purification [21]. The side-chain hydroxyl of
T572 forms an N-cap hydrogen bond with the main-
chain amide of Q575 and thus a mutation to proline
would abolish the potential to form this hydrogen bond.
L553V was the only conservative AEC-inducing muta-
tion analysed but it also produced a significant change
in DG
H
2

O
DÀN
at 25 °C. The location of the L553V mutation
in the core of the domain precludes this being a func-
tional mutation and hence the mutation also appears to
be structural, possibly by creating a small hydrophobic
cavity in the hydrophobic core.
The different pre-denaturation slope and m
D–N
val-
ues for all the mutants may indicate that they were
partially denatured or unfolded even under non-dena-
turing conditions. As such any attempt to crystallize
the domains would be almost impossible, and indeed
we were unable to crystallize any of the mutant
domains. The change in stability at 25 °C may be
enough to result in sufficient instability at 37 °Cto
cause complete unfolding of the domain and hence a
loss of domain function in vivo. Similar effects have
been observed with the human p53 core domain that
has a stability of 7.5 kcalÆmol
)1
at 25 °C and 3.0 kcalÆ-
mol
)1
at 37 °C [27]. Structural stability mutants were
found with p53 core domain with changes in stability
in a similar range to those for p63a SAM domains.
Potential effects of other mutations
Mutations I549T, F552S, G557V, F565L, S580F, S580P,

S580Y D583C, D583Y, L584P, P590L, F593S, R594P,
I597T, G600V and G600D were not tested experimen-
tally, but analysis of the wild-type crystal structure
potentially explains how some of the mutations might
cause significant loss of stability. Mutations I549T and
I597T would also introduce a polar residue into the
hydrophobic core, and presumably these mutants would
be destabilized in a similar manner to mutation I576T.
Mutations F552S and F565L would both result in the
loss of a large aromatic residue from the hydrophobic
core. G557 adopts a positive angle of phi in the wild-type
domain, and hence mutation G557V may cause instabil-
ity due to the residues in the loops adopting a more unfa-
vourable conformation in a similar manner to that
observed for mutation G569V. Mutations S580F, S580P
and S580Y would all result in the loss of the N-cap
hydrogen bond from the main-chain amide of D583 to
the side-chain hydroxyl of S580, whilst mutations
D583C and D583Y would disrupt a hydrogen bond
from the main-chain amide of S580 to the side-chain car-
bonyl oxygen of D583 (see Fig. S3). Mutation R594P
would disrupt a standard i–i+4 hydrogen bond in helix
5. Mutations G600V and G600D would probably result
in a steric clash between a-helix 5 and L556. Finally, the
in-frame 3 bp insert (573–574 inserting TTC) encoding
an additional phenylalanine residue would be expected
to be destabilizing as it probably disrupts the packing of
the 3
10
-helix to the rest of the protein. Of all the muta-

tions only P590L and F593S could not easily be
explained as mutations that would either disrupt the
hydrophobic core or result in a loss of a stabilizing
hydrogen bond or salt bridge. Indeed F593 is a solvent-
exposed aromatic residue and a mutation to a polar resi-
due might even be expected to increase stability.
Several charged and hydrophilic residues are
conserved amongst the p63a and p73a SAM domains
(Fig. 2). Some of these charge residues may contribute
to the stability of the domain by forming hydrogen
bonds or salt bridges (D546, E577, R594, R605,
H608), whilst others (D563, Q568, D582, K588, Q592,
K599 and D603) are solvent exposed and could poten-
tially be involved in domain function. It is interesting
to note that the two mutations that appear not to be
structural mutations, P590L and F593S, are close in
sequence to two of the highly conserved hydrophilic
residues (K588 and Q592) and may indicate that this
interface plays some role in domain function.
It is becoming clear that the SAM domain is a very
versatile protein module and more work is clearly
needed to elucidate its function in p63. The develop-
mental malformations in AEC syndrome are primarily
caused by mutations in the p63a SAM domain that
appear to cause a significant destabilization of the SAM
domain. So far, no equivalent mutations have been
identified in the p73a isoform. The residues involved in
p63 mutations are conserved in p73a SAM domains,
and yet only the p63a gene is found to undergo hetero-
A. Sathyamurthy et al. Mutants involved in AEC syndrome

FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS 2685
zygous in-frame insertion and point mutations in nat-
ure. The results presented here provide an insight into
the role of domain stability in the developmental mal-
formations observed in AEC. Our high resolution crys-
tal structure will contribute to an understanding of the
potential destabilizing or functional effects of AEC
mutations within the p63a SAM domain.
Materials and methods
Cloning, expression and purification of the p63a
SAM domain
All reagents were purchased from Sigma (Sigma-Aldrich
Corp., St Louis, MO, USA) and were Anal-R grade
or higher, with the exception of ultrapure guanidine
hydrochloride which was purchased from ICN Biomedicals
Inc. (Aurora, OH, USA). Human p63a SAM domain (543–
621) gene was codon optimized and synthesized using
overlapping primers and cloned into a modified pRSETa
expression vector [28]. The shortened construct (545–611)
was subcloned from the full-length gene into the same vec-
tor. Seven mutants involved in the AEC syndrome – L553F,
L553V, C561G, C561W, G569V, Q575L and I576T – were
made using the Quickchange kit. The plasmids were trans-
formed into Escherichia coli C-41 host cells and grown in
2YT medium containing 50 lgÆmL
)1
ampicillin. These cul-
tures were induced with 1 m
M isopropyl thio-b-D-galactoside
with A

600
= 0.8 and harvested after 4 h at 37 °C by centrifu-
gation. Isotopically labelled p63 SAM domain was prepared
by growing cells in K-MOPS minimal media [29] containing
15
NH
4
Cl and ⁄ or [
13
C]-glucose. The resulting protein was
purified by Ni
2+
-nitrilotriacetic acid affinity chromatogra-
phy, TEV protease digestion and a second Ni
2+
-nitrilotri-
acetic acid affinity chromatography to remove the lipoyl
domain fusion tag. Final purification was performed with gel
filtration using a HiLoad 26 ⁄ 60 Superdex 75 column.
SDS ⁄ PAGE and MALDI-TOF mass spectrometry were
used to confirm proteins were of the expected mass and to
assess their purity. The mutants L553F, C561G, G569V,
Q575L and I576T were present as inclusion bodies. The pro-
tein was solubilized in 8
M guanidinium hydrochloride and
refolded on the Ni
2+
-nitrilotriacetic acid affinity column.
The homogeneity of the refolded material was confirmed by
gel filtration and fluorescence measurements.

Folding analysis
All folding experiments were performed at 298 K using a
buffer of 50 m
M sodium phosphate pH 7.0 and 10 mM
dithiothreitol. Guanidium hydrochloride was used as
denaturant in the unfolding experiments. Tryptophan fluo-
rescence was used as a monitor for protein denaturation of
p63a SAM domain upon the addition of guanidinium
hydrochloride. The tryptophan residue W598 of p63a SAM
domain (and tryptophan W561 in the case of mutant
C561W) was used as a probe to monitor folding. Excitation
was at 280 nm using a 4 nm bandwidth and emitted light
was collected after passage through a 325 nm bandpass.
Denaturation experiments were performed on a Hitachi
F4500 spectrofluorimeter. Data from denaturation experi-
ments were fitted to equations assuming two-state kinetics.
Fitting of data was performed using the
KALEIDAGRAPH
version 3.6 and PRISM version 4.0a softwares.
NMR spectroscopy and structure determination
Protein samples prepared for NMR spectroscopy experi-
ments were typically 1.5 m
M in 90% H
2
O, 10% D
2
O, con-
taining 50 m
M potassium phosphate, pH 6.5, 200 mM NaCl
and 5 m

M d-dithiothreitol. All spectra were acquired using
either a Bruker DRX800 or DRX600 spectrometer
equipped with pulsed field gradient triple resonance
at 25 °C, and referenced relative to external sodium
2,2-dimethyl-2-silapentane-5-sulfonate for proton and car-
bon signals, or liquid ammonium for that of nitrogen.
Assignments were obtained using standard NMR methods
using
13
C ⁄
15
N-labelled,
15
N-lablled, 10%
13
C-labelled and
unlabelled p63 NMR samples [30,31]. Backbone assign-
ments were obtained using the following standard set of 2D
and 3D heteronuclear spectra: 1H-15N HSQC (Fig. S4),
HNCACB, CBCA(CO)NH, HACACO, HNCO, CCCONH
and 1H-13C HSQC. Additional assignments were made
using 2D TOCSY and DQF-COSY spectra. A set of dis-
tance constraints were derived from 2D NOESY spectra
recorded from a 1.5 m
M p63 domain sample with a mixing
time of 150 ms. Hydrogen bond constraints were included
for a number of backbone amide protons whose signals
were still detected after 10 min in a 2D
1
H-

15
N HSQC spec-
trum recorded in D
2
O at 278 K (pH 5.0). For hydrogen
bond partners, two distance constraints were used where the
distance
(D)
H–O
(A)
corresponded to 1.5–2.5 A
˚
and
(D)
N–
O
(A)
to 2.5–3.5 A
˚
. Torsional angle constraints were
obtained from an analysis of C¢,N,C
a
,H
a
and C
b
chemical
shifts using the program
TALOS [32]. The stereospecific
assignments of H

b
resonances determined from DQF-COSY
and HNHB spectra were confirmed by analysing the initial
ensemble of structures. Stereospecific assignments of H
c
and
H
d
resonances of Val and Leu residues, respectively, were
assigned using a fractionally
13
C-labelled protein sample
[33]. Stereospecific assignments were identified for resolved
resonances when the side-chain atoms were sufficiently well
defined in the ensemble of structures. The 3D structures of
the p63a SAM domain were calculated using the standard
torsion angle dynamics simulated annealing protocol in the
program
CNS 1.2 [34]. Structures were accepted where no
distance violation was greater than 0.25 A
˚
and no dihedral
angle violations were greater than 5°. The final coordinates
have been deposited in the Protein Data Bank (PDB
2Y9T).
Mutants involved in AEC syndrome A. Sathyamurthy et al.
2686 FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS
Crystallization and structural determination
Crystals were obtained using sitting drops containing 2 lLof
14 mgÆmL

)1
wild-type p63a SAM domain (510–576) protein
in 100 m
M sodium citrate, pH 6.4, 500 mM lithium sulphate,
500 m
M ammonium sulphate and 5 mM dithiothreitol. Clus-
tered plate-like crystals were obtained in 2 days at 4 °C. Sin-
gle crystals were obtained by seeding. The crystal contained
one molecule per asymmetric unit and grew in the space
group P2
1
2
1
2
1
(a = 34.64 A
˚
, b = 38.30 A
˚
, c = 44.52 A
˚
,
a = b = c =90°). Crystal was cryo-protected in reservoir
buffer with an additional 20% glycerol prior to stream freez-
ing. The data set was collected at the European Synchrotron
Radiation Facility on beam line ID14-2 to 1.6 A
˚
. Data pro-
cessing and integration was done using
CCP4 [35]. The struc-

ture was solved by molecular replacement using
PHASER [36]
with the NMR solution structure as a starting model. Struc-
ture calculations were done using
PHENIX [37]. The model
refinement was done using
MAIN [38]. The final coordinates
have been deposited in the Protein Data Bank (PDB
2Y9U).
Acknowledgements
We would like to thank the Nehru Trust and the
Cambridge Commonwealth Trust for the scholarship
award and financial support.
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Supporting information
The following supplementary material is available:
Fig. S1. Comparison of the B-factors obtained for
the backbone amide atoms of the p63a crystal struc-
ture with the NMR dynamics of the same domain in
solution.
Fig. S2.
KALEIDOGRAPH plots of the denaturation
curves of wild-type protein and mutants L553V,
L553F, C561G, C561W and Q575L.
Fig. S3. Close up views of several of the residues
involved in side-chain hydrogen bonds and salt
bridges.
Fig. S4.
15
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1
H HSQC spectrum of p63a SAM domain.
This supplementary material can be found in the
online version of this article.
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should be addressed to the authors.
Mutants involved in AEC syndrome A. Sathyamurthy et al.
2688 FEBS Journal 278 (2011) 2680–2688 ª 2011 The Authors Journal compilation ª 2011 FEBS