Ca2+ transporting fast twitch 1 (ATP2A or SERCA). As a consequence, the efficiency of the transfer of the calcium ions between
ER and mitochondria increases, as well as the propensity to
apoptosis.
It becomes now essential to link all these emerging mechanisms. Indeed, for example, anti-apoptotic members of the Bcl-2
protein family can also localize at the ER and modulate Ca2+
homeostasis, but their role in the p53-SERCA-mediated process
is unknown, along with the structural domain of p53 for
p53-SERCA interaction [25].
1.2 p53 Protein
Architecture
and DNA-Binding
Domain (DBD)
P53 forms a homotetramer with a dimer-of-dimers topology,
where each monomer accounts for 393 amino acids and include
multiple domains. In particular, a p53 monomer is composed of: an
intrinsically disordered N-terminal transactivation domain (TAD,
residues 1-42), a proline (Pro)-rich region with multiple copies of
the PXXP sequence (residues 61-94), a core DNA-binding domain
(DBD, residues 101-292), a tetramerization domain (324-355)
connected via a flexible linker, and an intrinsically disordered
C-terminal regulatory domain (356-393) [12, 26]. P53 modular
structure is typical of signaling proteins, and it provides conformational plasticity to adapt to the interaction with a myriad of different
partners and be modulated by a diverse range of PTMs [27].
A recent review article focused on the property and function of
the tetramer [12], whereas this protocol focuses on the
DNA-binding domain in the context of conformational ensemble
and long-range communication, as well as transcription-dependent
and -independent functions.
The p53 DBD folds into an immunoglobulin-like β-sandwich
architecture with an extended DNA-binding surface (Fig. 1), which
is formed by a loop-sheet-helix motif (including loop L1, F113 to
T123) and two large loops (L2, i.e., K164-C176 and L3, i.e.,
M237-P250) that are held together by zinc coordination
[29]. The L1 loop can adopt an extended conformation and interacts directly with DNA via lysine (Lys) 120 [28]. The L1 loop
conformations can also explore recessed conformations without
direct DNA contacts [28] (Fig. 1).
Compelling evidence suggests that p53 DBDs evolved to be
only marginally stable, and there is a clear correlation between their
thermodynamic stability and the corresponding optimum temperature of the organism of origin [30].
Most cancer-associated mutations are located in the DBD
[10]. All but seven residues of p53 have been the target of at least
one mutation in human cancer [10, 31, 32].
The effects at the structural and functional levels of p53 cancer
mutations, as stated above, can be very different. Some of them are
likely to remove important DNA interaction sites, and other can
Dynamics of p53
225
ER and mitochondria increases, as well as the propensity to
apoptosis.
It becomes now essential to link all these emerging mechanisms. Indeed, for example, anti-apoptotic members of the Bcl-2
protein family can also localize at the ER and modulate Ca2+
homeostasis, but their role in the p53-SERCA-mediated process
is unknown, along with the structural domain of p53 for
p53-SERCA interaction [25].
1.2 p53 Protein
Architecture
and DNA-Binding
Domain (DBD)
P53 forms a homotetramer with a dimer-of-dimers topology,
where each monomer accounts for 393 amino acids and include
multiple domains. In particular, a p53 monomer is composed of: an
intrinsically disordered N-terminal transactivation domain (TAD,
residues 1-42), a proline (Pro)-rich region with multiple copies of
the PXXP sequence (residues 61-94), a core DNA-binding domain
(DBD, residues 101-292), a tetramerization domain (324-355)
connected via a flexible linker, and an intrinsically disordered
C-terminal regulatory domain (356-393) [12, 26]. P53 modular
structure is typical of signaling proteins, and it provides conformational plasticity to adapt to the interaction with a myriad of different
partners and be modulated by a diverse range of PTMs [27].
A recent review article focused on the property and function of
the tetramer [12], whereas this protocol focuses on the
DNA-binding domain in the context of conformational ensemble
and long-range communication, as well as transcription-dependent
and -independent functions.
The p53 DBD folds into an immunoglobulin-like β-sandwich
architecture with an extended DNA-binding surface (Fig. 1), which
is formed by a loop-sheet-helix motif (including loop L1, F113 to
T123) and two large loops (L2, i.e., K164-C176 and L3, i.e.,
M237-P250) that are held together by zinc coordination
[29]. The L1 loop can adopt an extended conformation and interacts directly with DNA via lysine (Lys) 120 [28]. The L1 loop
conformations can also explore recessed conformations without
direct DNA contacts [28] (Fig. 1).
Compelling evidence suggests that p53 DBDs evolved to be
only marginally stable, and there is a clear correlation between their
thermodynamic stability and the corresponding optimum temperature of the organism of origin [30].
Most cancer-associated mutations are located in the DBD
[10]. All but seven residues of p53 have been the target of at least
one mutation in human cancer [10, 31, 32].
The effects at the structural and functional levels of p53 cancer
mutations, as stated above, can be very different. Some of them are
likely to remove important DNA interaction sites, and other can
Dynamics of p53
225