Crystal Structure of Arabidopsis Cyclophilin38 Reveals
a Previously Uncharacterized Immunophilin Fold and
a Possible Autoinhibitory Mechanism
Dileep Vasudevan,a,1,2Aigen Fu,b,1,3Sheng Luan,b,cand Kunchithapadam Swaminathana,4
aDepartment of Biological Sciences, National University of Singapore, Singapore 117543
bDepartment of Plant and Microbial Biology, University of California, Berkeley, California 94720
cDepartment of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
Cyclophilin38 (CYP38) is one of the highly divergent cyclophilins from Arabidopsis thaliana. Here, we report the crystal
structure of the At-CYP38 protein (residues 83 to 437 of 437 amino acids) at 2.39-Å resolution. The structure reveals two
distinct domains: an N-terminal helical bundle and a C-terminal cyclophilin b-barrel, connected by an acidic loop. Two
N-terminal b-strands become part of the C-terminal cyclophilin b-barrel, thereby making a previously undiscovered domain
organization. This study shows that CYP38 does not possess peptidyl-prolyl cis/trans isomerase activity and identifies
a possible interaction of CYP38 with the E-loop of chlorophyll protein47 (CP47), a component of photosystem II. The
interaction of CYP38 with the E-loop of CP47 is mediated through its cyclophilin domain. The N-terminal helical domain is
closely packed together with the putative C-terminal cyclophilin domain and establishes a strong intramolecular interaction,
thereby preventing the access of the cyclophilin domain to other proteins. This was further verified by protein–protein
interaction assays using the yeast two-hybrid system. Furthermore, the non-Leucine zipper N-terminal helical bundle
contains several new elements for protein–protein interaction that may be of functional significance. Together, this study
provides the structure of a plant cyclophilin and explains a possible mechanism for autoinhibition of its function through an
Immunophilins mediate immune suppression and vary consid-
erably in both form and function. They are classified into two
major families (according to their immunosuppressant ligand
partners): the FK-506 binding proteins (FKBPs) and the cyclo-
sporin A binding proteins (cyclophilins [CYPs]). Despite little
sequence similarity, most immunophilins possess peptidyl-
prolyl cis/trans isomerase (PPIase) enzymatic activity, which is
important for proper protein folding. However, not all im-
munophilin functions are explained by the PPIase activity or
cyclosporin A binding alone.
Over 300 cyclophilins have been identified from a wide variety
of organisms, ranging from archaea to human (Andreeva et al.,
1999; Ivery, 2000; Galat, 2003). The abundance and diversity of
single and multidomain immunophilins identified to date un-
derline the functional versatility of this family and are further
exemplified by the presence of multiple immunophilins within
an organism. Compared with other organisms, plants are known
to possess a much larger number of immunophilin isoforms
(Vallon, 2005; Ahn et al., 2010). The Arabidopsis thaliana ge-
nome alone consists of 29 CYP isoforms and 23 FKBP isoforms
(He et al., 2004; Romano et al., 2004). The discovery of plant
cyclophilins has not only demonstrated conservation of these
proteins in a full spectrum of biological systems, but has also
provided clues to their potential functions in plants. The early
works that proposed the distribution of cyclophilins throughout
the plant cell (Breiman et al., 1992; Luan et al., 1994) have been
confirmed and expanded by genomic and proteomic ap-
proaches, which have provided detailed subcellular localization
data for these large gene families (Peltier et al., 2002; Kleffmann
et al., 2004).
In Arabidopsis, up to five cyclophilins and 11 FKBPs are predicted
to reside in the thylakoid lumen (Edvardsson et al., 2007). The im-
munophilin with the highest PPIase activity in the thylakoid lumen
is FKBP13, whose crystal structure has already been reported
(Gopalan et al., 2004, 2006), followed by CYP20-2. The spinach
(Spinacia oleracea) homolog thylakoid lumen protein40 (TLP40)
(82% sequence identity to At-CYP38) is known to be an active
thylakoid lumenal PPIase with enzymatic activity observed in vitro
(Fulgosi et al., 1998). The spinach thylakoid lumen has TLP20 (a
homolog of At-CYP20-2) and TLP40 as the active PPIases. How-
ever, PPIase activity of the Arabidopsis thylakoid lumen has been
shown to be restricted to FKBP13 and CYP20-2 (Shapiguzov et al.,
2006; Edvardsson et al., 2007; Ingelsson et al., 2009). Hence, it
is very likely that the remaining 14 immunophilins in the thylakoid
1These authors contributed equally to this work.
2Current address: Disease Biology Unit, Novartis Institute for Tropical
Diseases, 10 Biopolis Road, #05-01, Chromos, Singapore 138670.
3Current address: College of Life Sciences, Northwest University, Xi’an,
Shaanxi 710069, People’s Republic of China.
4Address correspondence to firstname.lastname@example.org.
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) are: Sheng Luan
(email@example.com) and Kunchithapadam Swaminathan (dbsks@nus.
WOnline version contains Web-only data.
The Plant Cell, Vol. 24: 2666–2674, June 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.
lumen have developed functions other than PPIase activity. Se-
quence analysis reveals that some of the lumenal immunophilins are
so divergent that they have lost most of the conserved active site
residues that are essential for PPIase activity (He et al., 2004; Lima
et al., 2006). It is known that CYP38 plays a critical role in the as-
sembly and maintenance of photosystem II (PSII) supercomplexes
in Arabidopsis. Mutant plants lacking CYP38 display stunted growth
and are hypersensitivetolight due to defective PSIIsupercomplexes
(Fu et al., 2007). This protein may be essential for the correct folding
of the D1 protein and CP43 of PSII and successful assembly of the
oxygen evolving complex, whereas the absence of CYP38 renders
the PSII complexes extremely susceptible to photoinhibition (Sirpiö
et al., 2008). Even though it has been predicted that CYP38 might
function as a totally new entity of chaperone or might have de-
veloped some other enzymatic activity, it is not clear why it does not
have PPIase activity in spite of its having a putative CYP domain. To
address this question, we determined the crystal structure of
CYP38. We tested and confirmed that CYP38 does not possess
PPIase activity, and the structure presented in this article clearly
explains why CYP38 is not an active PPIase.
RESULTS AND DISCUSSION
The crystal structure of the Arabidopsis CYP38 protein (residues
83 to 437 of 437 amino acids) is reported here at 2.39-Å resolu-
tion. The structure reveals two distinct domains (Figure 1A). The
N terminus consists of a short helix (a1), followed by a helical
bundle domain (residues 102 to 216), made up of four helices (a2
to a5) of varying lengths. This domain is followed by a typical
cyclophilin domain (residues 238 to 423), made up of a b-barrel
that is capped by an a-helix at each end. The two domains are
connected by a loop, which has an excess of negatively charged
residues. Most interestingly, the extreme N terminus of the protein
(residues 83 to 96) gets into the C-terminal cyclophilin domain
and forms part of the b-barrel. This feature has not been observed
before in a cyclophilin. The loops in the cyclophilin domain are
quite disordered. Also, the first 10 of the 14 amino acids of the
N-terminal tag linker and the last four C-terminal residues are not
observed in the electron density map.
Helix a1 lies close to the cyclophilin domain. Helices a2 and a3
are split into two shorter fragments each, and the fragments are
connected by very short loops. Helices a4 and a5 are long and
are connected to each other by a long loop. The a2 and a3
region (residues 125 and 161) was predicted to be a Leu zipper
(He et al., 2004), based on the fact that Leu or Ile residues are
present almost at every 7th position. However, the present
structure does not support this prediction. The helices do not
intertwine to form a coiled coil and do not show a well-defined
hydrophobic surface with the Leu/Ile residues facing on one
side, which is the characteristic feature of the Leu zipper ar-
chitecture. Also, some of the 7th position Leu residues fall in the
loop region between the helices, which is unlikely for a Leu
zipper. Even the low-resolution (3.5 Å) electron density map
obtained for the wild-type CYP38 clearly indicated that the he-
lical region does not form a zipper domain. This alleviates the
doubt that the mutation of three Leu residues (which was
needed for structure determination) could have disturbed the
Leu zipper arrangement. The four helices of the helical bundle
are packed together mainly through hydrophobic interactions.
The internal surface of the bundle is highly hydrophobic and is
mainly occupied by Leu, Ile, Ala, and Val residues. Furthermore,
the helices are rich in charged residues, and all charged and
hydrophilic residues protrude toward solvent. The four-helix
bundle motif is relatively common in proteins. A DALI search
(Holm and Sander, 1993) with the helical bundle (residues 115 to
217) shows that the closest structural homologs are spinach
photosystem b Q (PsbQ) (PDB code 1NZE; Z score, 10.1) and
Escherichia coli cytochrome b562 (PDB code 256B; Z score,
10.0) (see Supplemental Figure 1 online). PsbQ is a 16-kD ox-
ygen evolving subunit of PSII, and cytochrome b562 is a heme
binding protein involved in electron transport as well as DNA-
dependent transcription regulation. These structures include
a four-helix bundle with up-and-down topology. However, no
region of the above two proteins shows any strong sequence
similarity to CYP38. PsbQ is only ;11% identical and aligns with
the helical bundle of CYP38 with a root mean square deviation
of 1.71 Å over a stretch of 88 residues. Cytochrome b562 is
;10% identical and aligns with a root mean square deviation of
1.73 Å for 83 residues.
A Previously Undiscovered Cyclophilin Domain
The cyclophilin domain of CYP38 has the typical b-barrel
structure, closed at both ends by a-helices. Eight antiparallel
b-strands make the barrel. There are differences between the
cyclophilin domain of CYP38 and other known cyclophilin
structures. Divergent cyclophilins, which form a separate class
of cyclophilins, have an additional loop of five to several amino
acids at about residue 50 (human cyclophilin A [hCYPA] num-
bering). Also, another common feature of this group of proteins
is the presence of two reduced Cys residues, which come to-
gether in close proximity and hence might be involved in a redox
signaling process. Even though the CYP38 cyclophilin domain
has extensive loops, they do not have any conformation similar
to the loops of other known divergent cyclophilins. In fact, this
domain does not even have the conserved Cys residues that are
found in other divergent cyclophilins, making it a new kind of
divergent cyclophilin. CYP38 got its name due to the presence
of a putative CYP domain in the C terminus, and cyclophilins are
known to be active PPIases. However, the current structure
does not quite support CYP38 being a PPIase, even though it
satisfies the requirements of a CYP fold.
CYP38 and PPIase Activity
When the CYP38 cyclophilin sequence (residues 232 to 437) is
aligned with that of the well-studied hCYPA and other known
cyclophilins (see Supplemental Figure 2 online), the residues
corresponding to the b5 and b6 region align well, suggesting that
CYP38 could also be a PPIase. At least three of the critical
Crystal Structure of Cyclophilin382667
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