Structural stability and reversible unfolding of recombinant porcine S100A12
A.F. Garcia
a
, W. Garcia
a
, M.C. Nonato
b
, A.P.U. Araújo
a,
⁎
a
Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil
b
Laboratório de Cristalografia de Proteínas, Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto—USP, Brazil
article info abstract
Article history:
Received 21 January 2008
Received in revised form 20 February 2008
Accepted 20 February 2008
Available online 29 February 2008
Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins
characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such
as the regulation of protein phosphorylation, enzymatic activity, protein–protein interaction, Ca
2+
homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of
this family bind Zn
2+
or Ca
2+
with cooperative effect on binding. In this study, the gene sequence encoding
porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report
a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal
titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated
a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the
protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the
physiological responses.
© 2008 Elsevier B.V. All rights reserved.
Keywords
:
S100A12
Calcium-binding protein
S100 family, Circular dichroism (CD)
Fluorescence spectroscopy
Protein unfolding
1. Introduction
Calcium-binding proteins (CaBPs) are involved in the regulation of
several biological processes. The basic structural and functional unit of
CaBPs family is the EF-hand motif which is the target of intracellular
calcium [1,2]. S100 proteins constitute the largest subfamily of EF-
hand proteins. There are at least 25 members of low-molecular-
weight (9–14 kDa) acidic calcium-binding proteins [3,4]. They interact
in a Ca
2+
-dependent or independent way with proteins involved in cell
proliferation and differentiation, cellular architecture, signal trans-
duction, and intracellular metabolism [4,5].
Most S100 proteins occur as non-covalent homodimers [6] and are
characterized by the presence of two EF-hand motifs per monomer,
which are ordered in N-terminal EF-hand (helix I–loop I–helix II) with
a flexible linker region that connects helix II to helix III of the C-
terminal EF-hand (helix III–loop II–helix IV). The linker region and C-
terminal extension show the least amount of sequence conservation
among S100 proteins [7,8]. Different conformational changes are
triggered by calcium binding to S100 proteins in the two EF-hands,
and they exhibit distinctive affinities for calcium [9]. The C-terminal
EF-hand contains the canonical Ca
2+
-binding loop common to all
CaBPs. The N-terminal EF-hand of S100 proteins is distinguished from
all other CaBPs [3,9]. Also, it undergoes a relatively small change in
conformation, whereas the change in the C-terminal EF-hand is much
larger. These large differences in the Ca
2+
affinities indicate that S100
proteins are hetero-bi-functional [10,11]. Furthermore, another char-
acteristic of S100 proteins is their binding to additional divalent metal
ions such as Zn
2+
and Cu
2+
. Binding of these metals can affect the
affinity to Ca
2+
, further contributing to their variable cation binding
properties and their diversified functions [9,12,13].
S100 proteins could act as a calcium mediated intracellular signal
transducers through significant structural changes upon calcium
binding, exposing two hydrophobic target-binding surfaces per
dimer [14,15]. This behavior suggests that S100 proteins might have
a role in the fine regulation of effectors' proteins, specific steps of
signaling pathways, or cellular functions ranging from calcium buff-
ering, cell growth and differentiation, modulation of enzyme ac-
tivities, energy metabolism, motility, secretion, transcription,
apoptosis, neurite extension and chemotaxis [10,16,17].Lackof
regulation of specific S100 proteins is correlated with several human
diseases, including cancer, neurodegenerative and cardiovascular
disorders [16]. The mechanism by which human S100A12 modulates
the course of the inflammatory processes was linked to its interaction
with the receptor for advanced glycated products (RAGE) [18]. Also, in
a recent study, Xie et al. showed that the binding to this receptor
occurs through a hexameric Ca
2
-S100A12 C [19].
Porcine S100A12 (also known as calgranulin C) is a member of
S100 proteins subfamily. This protein consists of 91 amino acids
(approximately 10.7 kDa and an acid pI of 5.8) and is characterized by
the presence of two EF-hands motifs and an additional binding site
(His-X-X-X-His), with high affinity for Zn
2+
on the C-terminal region
[20]. The porcine S100A12 has a lower affinity to bind Ca
2+
than Zn
2+
and the ability for binding calcium is remarkably increased by the zinc
Biophysical Chemistry 134 (2008) 246–253
⁎ Corresponding author. Grupo de Biofísica Molecular “Sérgio Mascarenhas”, Centro
de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, CP 369, 13560-
970 Universidade de São Paulo, São Carlos, SP, Brazil.
E-mail address: anapaula@ifsc.usp.br (A.P.U. Araújo).
0301-4622/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bpc.2008.02.013
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