Exploring the Folding Pathways of Annexin I, a
Multidomain Protein. II. Hierarchy in Domain Folding
Propensities may Govern the Folding Process
FrancËoiseCordier-Ochsenbein
1
,RaphaeÈlGuerois
1
FrancËoiseRusso-Marie
2
,Jean-MichelNeumann
1
andAlainSanson
1
*{
1
De
Â
partement de Biologie
Cellulaire et Mole
Â
culaire
Section de Biophysique des
Prote
Â
ines et des Membranes
and URA CNRS 2096, CEA
Saclay, 91191 Gif sur Yvette
Cedex, France
2
Institut Cochin de Ge
Â
ne
Â
tique
Mole
Â
culaire, Unite
Â
INSERM
U332, 22 rue Me
Â
chain
75014 Paris, France
In the context of exploring the relationship between sequence and folding
pathways, the multi-domain proteins of the annexin family constitute
very attractive models. They are constituted of four $70-residue domains,
named D1 to D4, with identical topologies but only limited sequence
homology of approximately 30%. The domains are organized in a pseu-
dochiral circular arrangement. Here, we report on the folding propensity
of the D1 domain of annexin I obtained from overexpression in Escheri-
chia coli. Unlike the D2 domain, which is only partially folded, the iso-
lated D1 domain exhibits autonomous refolding in pure aqueous
solution. Similarly, the D3 domain and D2-D3 module were obtained
from expression in E. coli but were found to be largely unfolded. No con-
clusion could be drawn for the D4 domain because it was not possible to
extract it from the bacterial inclusion bodies. The data allow us to pro-
pose a plausible scenario for the annexin I folding. This working model
states that ®rstly the D1 domain folds, and the D2 and D3 domains
remain partly unfolded, facilitating the docking of the D4 domain to the
D1 domain. In a second step, the D1 and D4 domains dock, and D4 may
fold if already not folded. The ®nal step starts with the stabilization of
the D1-D4 module. This stabilization is crucial for allowing the non-
native local interactions inside the still partially unfolded D2 domain to
switch to the native long-range interactions involving D4. This switch
allows the complete folding of D2 and D3. The model proposes a sequen-
tial and hierarchical process for the folding of annexin I and emphasizes
the role of both native framework and non-native structures in the
process.
# 1998 Academic Press
Keywords: protein folding; NMR; annexin; stability; non-native structures
*Corresponding author
Introduction
Understanding the process by which proteins
reach their native structure is still a matter of deep
and exciting research. While it is well known and
clearly established that the three-dimensional
structure of proteins is encoded in their sequence,
the way these sequences also control the folding
process is far from understood. The main reason
for this is that there are very few proteins for
which the folding process has been thoroughly
analyzed as compared to the number of proteins
for which the three-dimensional structure has
been solved. A second important reason is prob-
ably that proteins for which the folding process
has been thoroughly studied and better under-
{Also from Universite
Â
P. et M. Curie, 9 Quai Saint-
Bernard, Ba
Ã
t. C, 75005 Paris, France.
F.C.-O. and R.G. contributed equally to this work and
should be considered as joint ®rst authors.
Abbreviations used: COSY, correlated spectroscopy;
DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; FPLC,
fast performance liquid chromatography; GdnHCl,
guanidinium choride; GST, glutathione-S-transferase;
HMQC, heteronuclear multiple-quantum correlation;
HSQC, heteronuclear single-quantum correlation; NOE,
nuclear Overhauser effect; NOESY, nuclear Overhauser
enhancement spectroscopy; PCR, polymerase chain
reaction; TFA, tri¯uoroacetic acid; TOCSY, total
correlated spectroscopy.
Article No. mb981828 J. Mol. Biol. (1998) 279, 1177±1185
0022±2836/98/251177±09 $25.00/0 # 1998 Academic Press