Báo cáo khoa học: Escherichia coli cyclophilin B binds a highly distorted form of trans-prolyl peptide isomer doc

Escherichia coli
cyclophilin B binds a highly distorted form
of
trans
-prolyl peptide isomer
Michiko Konno
1
, Yumi Sano
1
, Kayoko Okudaira
1
, Yoko Kawaguchi
1
, Yoko Yamagishi-Ohmori
1
,
Shinya Fushinobu
2
and Hiroshi Matsuzawa
2,
*
1
Department of Chemistry, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan;
2
Department of Biotechnology,
The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan
Cyclophilins facilitate the peptidyl-prolyl isomerization of
a trans-isomer to a cis-isomer in the refolding process of
unfolded p roteins to recover the natural folding state with
cis-proline conformation. To date, only short peptides with a
cis-form proline have been observed in complexes of human

and Escherichia coli proteins of cyclophilin A, which is
present in cytoplasm. The crystal structures analyzed in this
study show two complexes in which peptides having a trans-
form proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide
and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin,
are bound on a K163T mutant of Escherichia coli cyclo-
philin B, the preprotein of which has a signal sequence.
Comparison with cis-form peptides bound to cyclophilin A
reveals that in any case the proline ring i s inserted into the
hydrophobic pocket and a hydrogen bond between CO of
Pro and N
g2
of Arg is formed to fix the peptide. On the other
hand, in the cis-isomer, the formation of two hydrogen
bonds of NH and CO of Ala preceding Pro with the protein
fixes the peptide, whereas in the trans-isomer formation of a
hydrogen bond between CO preceding Ala-Pro and His47
N
e2
via a mediating water molecule allows the large distor-
tion in the orientation of Ala of Ala-Pro. A lthough loss of
double bond character of the amide bond of Ala-Pro is
essential to the isomerization pathway occurring by rotating
around its bond, these peptides have forms impossible to
undergo proton transfer from the guanidyl group o f Arg to
the prolyl N atom, which induces loss of double bond
character.
Keywords: cyclophilin; isozyme; peptidyl-prolyl cis-trans
isomerase; peptide binding; refolding.
Cyclophilins (CyPs) exist abundantly an d ubiquitously in a

broad range of organisms from Escherichia coli to humans
[1–3]. Two cyclophilins, E. coli CyPA and E. coli CyPB,
have been identified [4,5], and at least five cyclophilins in
mammals (human CyPA–D, CyP40) [3] have been identi-
fied. CyPB homologs have a membrane-binding signal
sequence in the amino-terminal, w hereas CyPA homologs
are present in the cytoplasm [1–5]. It has been reported that
E. coli CyPB exhibits almost equal activity of peptidyl-
prolyl isomerization from cis- to trans-form of short
peptides to E. coli CyPA [5]. Because cis-proline conforma-
tion in the polypeptide spontaneously converts to trans-
conformation, acceleration of the isomerization of the
cis-isomer to the trans-isomer is not thought to be the
function of the CyPA and CyPB proteins in these cells. On
the other hand, CyPs facilitate the step of the isomerization
in which a trans-isomerisconvertedtoacis-isomer in the
process of the refolding of unfolded proteins. Although the
natural roles of CyPs are still poorly understood, they may
be related to the fixing of distorted trans-isomers at the
intermediate step of converting the trans-tothecis-isomer
of proteins. It has been reported that in the N-terminal
domain of the HIV-1 capsid protein, a loop containing Gly-
trans-Pro binds to the human CyPA; this occurs at a
position where the Gly residue assumes //w angles in the
regions disallowed for residues with side chains [6]. Because
the distortion of the loop of the c apsid protein is concen-
trated in the torsional angles of the Gly residue, but as most
proteins with a cis-proline, the refolding process of which is
accelerated by CyPs, have no flexible Gly residue at the
position immediately p receding Pro [7–11], this binding

conformation of the capsid p rotein is not sufficient to serve
as a model of t he intermediate formed during isomerization
of the trans-isomer to t he cis -isomer. To date, the b inding
structures of peptides in th e cis-proline form have been
reported for human CyPA [12–15] and E. coli CyPA [16],
but the peptides f orming the disto rted trans-conformation
have not yet been identified.
Based on NMR measurements, Eisenmesser et al.[17]
proposed a possible mechanism by which the C-terminal
peptide segment containing a Pro residue of a tetrapeptide
rotates around the Phe-Pro peptide bond. Their results
indicated that the cis-form in the initial state is inserted
into a hydrophobic pocket of human CyPA, whereas the
trans-form in the final state is released from the pocket. The
Correspondence to M. Konno, Department of Chemistry, Ochanom-
izu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112–8610, Japan.
Fax: +81 359785717, Tel.: +81 359785718,
E-mail:
Abbreviations: CyP, cyclophilin; Suc, succinyl; pNA, p-nitroanilide;
Ac, N-acetyl; AMC, amidomethylcoumarin; PPIase, peptidyl-prolyl
cis-trans isomerase.
*Present address: Department of Bioscience and Biotechnology,
Aomori University, 2-3-1 Kohbata, Aomori, 030-0943, Japan.
(Received 23 March 2004, revised 1 August 2004,
accepted 4 August 2004)
Eur. J. Biochem. 271, 3794–3803 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04321.x
trans-form, the proline of which is bound to the pocket, is
only the loop of the capsid protein. It has been reported that
E. coli CyPA accelerates the conversion of trans- to cis-form
on peptide bonds of not only S er54-Pro55 but also Tyr38-

Pro39 in the final refolding process of the unfolded RNase
T1, w hereas h uman CyPA accelerates only the conversion
on the Ser54-Pro55 peptide bond [8]. The fact that E. coli
CyP is bound to structurally diverse substrates, and the
notion that CyPA and CyPB should affect d ifferent target
proteins, led us to consider whether E. coli CyPB might
bind to peptides of the disto rted trans-isomer. E. coli CyPB
[4,5] consists of 190 amino acids and has a signal sequence of
24 residues at the N-terminal end. CyPB molecules are
processed and forms of CyPB without the signal peptide are
present in the periplasm of E. coli cells (here, residues
number 25–190 are defined as 1–166).
Analysis of a complex in which a protein is bound to a
peptide containing a Pro residue of trans-form in the initial
state of the isomerization reaction from a trans-tocis-
isomer will be needed. As the isomerization of t he trans-to
cis-isomer of the short peptides proceeds very slowly or does
not occur at all in CyPB molecules, we used short peptides
in order to obtain the complexes with the distorted trans-
isomer. We could obtain no crystals of E. coli CyPB having
suitable size for X-ray analysis. Thus, we designed a K163T
mutant protein of E. coli CyPB that can crystallize under
conditions similar to those for the crystallization of E. coli
CyPA. Finally, we succeeded in crystallizing K163T mutant
proteins complexed with a tripeptide, succinyl-Ala-Pro-
Ala-p-nitroanilide (Suc-Ala-Pro-Ala-pNA), and w ith a
tetrapeptide, acetyl-Ala-Ala-Pro-Ala-amidomethylcouma-
rin (Ac-Ala-Ala-Pro-Ala-AMC), and w e found highly
distorted trans-peptides. We carried out comparisons of
these complexes with a complex of E. coli CyPA and the

tripeptide containing cis-proline , and with a complex of
human CyPA bound to the loop containing the proline in
the trans-form of t he capsid protein. In addition, we
discussed the importance of the conversion from the sp
2
-to
sp
3
-hybrid configuration on the prolyl N atom in the
isomerization of the proline from t he trans-tocis-isomer of
unfolded proteins in the refoldin g process, as well as in the
isomerization of the short peptides containing the proline
from the cis-totrans-isomer.
Materials and methods
Preparation of mutant
E. coli
CyPB
The plasmid pATtrpEPPIa, which has a ClaIandaBamHI
site added at the 5¢ and 3¢ ends, respectively, of the gene
encoding amino acid residues 1–190 of E. coli CyPB, was
kindly supplied by N. Takahashi, Tokyo University of
Agriculture and Technology, Tokyo, Japan [5]. PCR was
carried out using a 32-mer primer of 5¢-AAAAAGAAT
TCATCGATATGTTCAAATCGACC-3¢ with EcoRI
added at the 5¢ end of ClaI, a 23-mer primer of 3¢-CGAGA
CGGCATTCCTAGGTTTTT-5¢, and pATtrpEPPIa as a
template. The P CR fragment was cleaved with Eco RI and
BamHI, and was subcloned into pUC118, producing an
expression vector, pUCPPIb. Mutation was introduced into
pUCPPIb u sing a QuickChange

TM
Site-Directed M utagen-
esis Kit (Strategene, La Jolla, CA). The codon for Lys187
(AAA) was replaced with ACA (as the purified proteins lose
signal sequences of 24 amino acids at the N-terminal, the
mutant protein is designated K163T in this paper). The
sequences of the mutated DNA were checked using an ABI
373 DNA sequencer (Applied Biosystems). The ClaI-
BamHI fragment of each mutant plasmid was replaced
with the ClaI-BamHI fragment of pATtrpEPPIa. This
expression plasmid p ATtrpEPPIa (KT) was transformed in
E. coli HB101 cells. The cells were cultured at 37 °Cin
M9CA medium [0.05% (w/v) NaCl, 0.1% (w/v) NH
4
Cl,
0.2% (w/v) casamino acid, 0.2% (w/v) glucose, 2.0 m
M
MgSO
4
,0.1m
M
CaCl
2
,0.6%(w/v)Na
2
HPO
4
,0.3%(w/v)
KH
2

PO
4
, pH 7.4] and the proteins w ere purified as
described previously [5].
The PPIase activity was measured using the synthetic
peptide Suc-Ala-Ala-Pro-Phe-AMC (Peptide Institute, Inc.,
Osaka, Japan). The synthetic peptide [a 40 lL solution
containing 1.67 m
M
of peptide, 17% (v/v) dimethylsulfoxide
and 35 m
M
HEPES buffer, pH 7.8] was preincubated with
proteins (1.1–4.8 n
M
)in2mLof35m
M
HEPES buffer
containing 5 m
M
2-mercaptoethanol (pH 7.8), and the
assay was started by mechanical mixing with 40 lLof
0.58 m
M
chymotrypsin in a spectrophotometer cell. The
fluorescence of AMC from the cleaved trans-peptide [18]
was measured at 460 nm using a FP-770 fluorescence
spectrophotometer (JASCO Corp., Tokyo, Japan) at 10 °C.
Crystallization, data collection, structure determination
and refinement

Whole crystals of K163T mutant proteins were grown by
vapor diffusion in hanging drops at 20 °C. A 9 lLdrop
containing 0.57 m
M
(12 mgÆmL
)1
)proteinand6m
M
trip-
eptide (Suc-Ala-Pro-Ala-pNA (Peptide Institute, Inc.), or
tetrapeptide (Ac-Ala-Al a-Pro-Ala-AMC (Bachem, Switzer-
land), 38–40% (w/v) saturated ammonium, sulfate, 9%
(v/v) methanol, 0.04% (w/v) NaN
3
,and50m
M
Tris/HCl
buffer (pH 8.0) was equilibrated against a reservoir solution
containing 46% saturated ammonium sulfate, 0.04% (w/v)
NaN
3
, and 100 m
M
Tris/HCl buffer (pH 8.0). When the
peptide dissolved in methanol was added to the drop
containing the protein under the above co nditions, the
solution became turbid, but it became transparent again
when left overnight. The crystals of the free K163T
mutant protein were grown from a drop c ontaining
9.5 mgÆmL

)1
protein, 38% (w/v) saturated ammonium
sulfate, 0.04% (w/v) NaN
3
,and80m
M
Tris/HCl buffer
(pH 8 .0) equilibrated against a reservoir solution of 46%
(w/v) saturated ammonium sulfate, 0.04% (w/v) NaN
3
,and
100 m
M
Tris/HCl buffer (pH 8.0).
Intensity data for crystals were collected using a Weis-
senberg camera for macromolecules [19] installed on the
beam line BL6A of the Photon Factory (PF) at Tsukuba,
Japan. Data sets were reduced using
DENZO
and
SCALEPACK
[20]. The structure for the complex of E. coli CyPB K163T
mutant bound to tripeptide was solved b y m olecular
replacement with
XPLOR
[21] using E. coli CyPA [16] as a
search model. The model was built using
O
software [22],
and refinement was assisted by molecular dynamics using

XPLOR
. The structure of the complex with t he tetrapeptide
was started from the final structure of the complex with the
tripeptide. The crystal structure of the free E. coli CyPB
Ó FEBS 2004 E. coli cyclophilin B/distorted trans-isomer (Eur. J. Biochem. 271) 3795
K163T was solved by m olecular replacement using a model
of the CyPB K163T protein of the complexes, and the final
refinements w ere made using
CNS
[23]. The model buildings
on the basis of electron density maps revealed that in all
three kinds of crystals CyPB proteins do not have 24
residues of s ignal sequence at the N -terminal and were cut
off in the stage of their cultivation or purification. After
refinements of the model containing two proteins and water
molecules for two complexes, model building of the peptide
was tried. Both of these complexes have an asymmetric unit
containing two CyPB molecules and one peptide. The
model of distorted trans-form gave good fitting into the
electron densities in two complexes. The final results are
summarized in Table 1.
Results
Structure of the K163T mutant of
E. coli
CyPB
We constructed a mutant that satisfies the following two
conditions. Firstly, the mutant residue must not disturb the
folding of the overall skeleton and secondly it must not
affect the binding of peptides containing a proline.
A mutant, K163T, in w hich lysine was r eplaced by

threonine at residue 163, was selected under these criteria, as
Lys163 is located in b-strand and has no intramolecular
interaction with any residue s. The k
cat
/K
m
value of the
K163T mutant (1.9 · 10
7
M
)1
Æs
)1
)inthecis-totrans-
isomerization reaction for Suc-Ala-Ala-Pro-Phe-AMC was
almost the same as that of the wild-type protein
(1.8 · 10
7
M
)1
Æs
)1
). Crystals of complexes of mutant
K163T CyPB molecules, and tripeptide (Suc-Ala-trans-
Pro-Ala-pNA) or tetrapeptide (Ac-Ala-Ala-trans-Pro-Ala-
AMC), g rew f rom a solution containing ammonium
sulphate under conditions almost identical to those under
which crystals of a complex of E. coli CyPA and tripeptide
(Suc-Ala-cis-Pro-Ala-pNA) grew. The c rystals of free
K163T CyPB also grew from a protein drop containing

no methanol, which was used for dissolving peptides.
The structural alignments of E. coli CyPB, E. col i CyPA
[16] and human CyPA [6,14] are shown in Fig. 1. The CyPB
molecules, as well as the E. coli and human CyPA molecules,
have a b-barrel structure, consisting of upper and lower
b-sheets of four antiparallel b-strands enclosed by two
a-h elices at the top and bottom (Fig. 2). In both complexes,
CyPB molecule A, i.e. the molecule without a peptide, is
packed along one 3
2
axis, whereas CyPB molecule B, which
is bound to a peptide, is packed along the other 3
2
axis.
CyPB molecules A and B exist in a ratio of 1 : 1 in the
crystal. Molecules A and B, related by a local quasi-twofold
rotation axis, make contacts through the side chains of
Ile159, Ser161, Thr163 (mutant residue) and Leu165 on the
b8 strand, and through Leu8 and Thr10 side chains on the
b1 strand. In the crystal of free CyPB, a crystallographic
twofold rotation axis is observed, and the symmetry of the
space group increases from P3
2
, which is found in the
Table 1. Data collection and refinement statistics f or E. coli CyPB mutant K163T. Values in parentheses are for the highest resolution shell.
R
merge
¼ S
h
S

i
|I
h,i
) <I>
h
|/S
h
S
i
I
h,i
,where<I>
h
is the mea n in tensity. R
factor
¼ S||Fo|-|Fc||/S|Fo|. A subset of the data ( 10%) was excluded from
therefinementandusedtocalculateR
free
.
Peptide Suc-Ala-Pro-Ala-pNA Ac-Ala-Ala-Pro-Ala-AMC none
Space group P3
2
P3
2
P3
2
21
a ¼ ,b¼ (A
˚
) 79.28 78.73 82.55

c ¼ (A
˚
) 56.63 56.16 52.35
Z66 6
Data collection
Number of used crystals 2 2 2
Maximum resolution (A
˚
) 1.7 (1.76–1.70) 1.8 (1.86–1.80) 1.8 (1.86–1.80)
Measured reflections 187361 263488 133992
Unique reflections 40355 35961 19219
Completeness (%) 92.0 (73.3) 99.4 (97.5) 99.0 (96.5)
I/rI 16.7 9.3 14.8
R
merge
(%) 9.0 (23.8) 9.0 (37.1) 6.9 (40.2)
Refinement
Resolution (A
˚
) 6–1.7 (1.78–1.70) 6–1.8 (1.88–1.80) 8–1.8 (1.82–1.80)
Used reflections 39354 34891 18959
Protein atoms 2497 2481 1273
Peptide atoms 35 39 none
Solvent atoms 185 204 81
R
factor
(%) 18.4 (27.0) 18.8 (26.2) 20.1 (28.7)
R
free
(%) 22.1 (29.0) 22.9 (28.4) 22.5 (31.1)

Root-mean-square deviations from ideal values
Bond lengths (A
˚
) 0.006 0.008 0.006
Bond angles (°) 0.874 1.38 0.842
Average B factors (A
˚
2
)
Main chain 17.35 13.46 18.60
Side chain 20.24 15.53 21.11
Solvent 37.24 34.95 30.38
Peptide 39.82 34.26
3796 M. Konno et al.(Eur. J. Biochem. 271) Ó FEBS 2004
complexes, to P3
2
21. In the region from 144 to 151 residues
of the molecule in the crystal of free CyPB (Fig. 3; shown in
green), the turn structure is broken by the strong interaction
with the adjacent molecule. On the other h and, the CyPB
molecules A and B (Fig. 3; red and blue, respectively) of the
complexes possess the T5 turn of Type II¢ and E. coli CyPA
molecule has also the similar Type II¢ turn [16].
Short peptides containing a
trans
-form proline bound
to
E. coli
CyPB
A left-to-right-running cleft is shown in the upper b-sheet of

the b3, b4, b6andb5 strands of the CyPB proteins (Fig. 2).
In the center of the cleft (Fig. 4), there is a hydrophobic
pocket formed by the side chains of Phe53, Met54, Phe112,
Leu113 and Tyr122, and by the side chain of Phe104 at the
bottom. Mainly hydrophilic residue s occupy the l eft half of
the cleft, whereas mainly hydrophobic residues occupy the
right half. The tripeptide Suc-Ala1-Pro2-Ala3-pNA and
the tetrapeptide Ac-Ala1-Ala2-Pro3-Ala4-AMC have the
distorted tra ns-form, allowing them to bury deeply into the
cleft of CyPB m olecules B. In t he tripeptide, the //w
torsional angles of the main chains are )170/100°, )56/118°
and )86/138° for Ala1, Pro2 and Ala3, respectively. In the
tetrapeptide the se angles are )177/91°, )52/ 122° and )79/
132° for Ala2, Pro3 and Ala4, respectively. The Ala-Pro-Ala
segments of the tripeptide and tetrapeptide occupy the
exactly same positions (Fig. 4), when 166 Ca atoms of
CyPB molecules B in the two complexes were superimposed.
The / and w angles of the Ala residue of Ala-Pro are rarely
observed for residues of linear short peptides containing no
glycine.
The Pro2 and Pro3 residues of the tripeptide and
tetrapeptide have an envelope form in which their Cc
Fig. 1. Structural alignments of E. coli CyPB,
E. coli CyPA [16], and human CyPA [6,14].
Pale blue boxes: b-st rands; pink: a-helices;
orange: 3
10
helices; and blue: turns. Amino
acid residues are shown usin g a one-lette r code
and dots indicate deletions. The 14 conserved

residues of the region, in which the peptides
areplaced,arewritteninboldtype.Asterisks
are shown in 10-residue intervals of E. coli
CyPB. In E. coli CyPB K163T mutant, a Lys
at residue 163 is replaced by a Thr.
Fig. 2. The ribbon model of the b-barrel structure of E. coli CyPB
consisting of the upper and the lower b-sheets enclosed by two helices.
The colors of ribbon are shown corresponding to those of Fig. 1. The
loop colored in green is the region expected to affect the selection of the
substrate. T hr163 is located outside of b8 strand. The Suc-Ala-trans-
Pro-Ala-pNA is also shown by ball-and-sticks model. Figures 2,3,4,5,6
and 7 were prepared using the programs
MOLSCRIPT
[35] and
RASTER
3
D
[36].
Fig. 3. Superimposed traces. The superim-
posed traces of Ca of CyPB molecule A (red)
without a peptide and CyPB molecule B (blue)
bound by a tripeptide in the complexes, and
CyPB molecule (green) in the crystal of free
CyPB. The molecules shown in Figs 2 and 3
are in the same orientation.
Ó FEBS 2004 E. coli cyclophilin B/distorted trans-isomer (Eur. J. Biochem. 271) 3797
atoms lie both above the plane of the coplanar Cb-Ca-N-Cd
as shown in Fig. 5 so that their tra ns-proline rings do not
collide with p electron over the ring of Phe104 at the bottom
of the pocket. This envelope form is also observed in

cis-Pro2 of the tripeptide bound to E. coli CyPA and in
trans-Pro90 of the loop of the capsid protein bou nd to
human CyPA but does not belong to any of t hree
conformations shown in a side-chain rotamer library [24].
The Cc atom of most Pro residues lies down the plane of the
coplanar Cb-Ca-N-Cd as observed in Pro51 (w ¼ 141 °),
Pro71 (w ¼ 149°), Pro153 (w ¼ 133°) and Pro156 (w ¼
137°) in the CyPB molecule. There is often another envelope
form observed, in which the Cb atom lies down the plane of
the coplanar Ca-N-Cd-Cc as observed in Pro69 (w ¼ 176°),
Pro72 (w ¼ 161°) and Pro148 (w ¼ )6°).
In the tripeptide (tetrapeptide) (Fig. 4), CO and NH of
the amide bond of Ala-Pro form no hydrogen bond with the
CyPB molecule, and CO of the amide bond of suc-Ala1
(Ala1-Ala2) forms a hydrogen bond with a distance of
2.77 A
˚
(2.59 A
˚
) t o a water molecule, which forms a
hydrogen bond with His47 N
e2
with a distance of 2.87 A
˚
(2.78 A
˚
). The carbonyl o xygen atom of Pro2 of the
tripeptide forms a hydrogen bond to the N
g2
atom o f the

guanidyl group of Arg48 wit h t he d istance o f 2 .62 A
˚
,and
2.72 A
˚
for t hat o f Pro3 of the tetrapeptide. B ecause the w
angles of Pro2 (w ¼ 118 °)ofthetripeptideandPro3(w ¼
122°) of the tetrapeptide deviate from w angles of 130–150°
observed for trans-Pro residues, it has became possible to
form a hydrogen bond to the side chain of Arg48. The
aromatic ring of p-nitroanilide in the C-terminal end of the
tripeptide overlaps parallel to the ring of Phe112 and the
same is observed for that of coumarin of the tetrapeptide.
Thus the binding of peptides to the cleft of CyPB is mainly
due to the hydrophobic interaction.
In CyPB molecules B bound to the tripeptide and
tetrapeptide (Fig. 4) , t he Arg48 N
e
atom forms a hydrogen
bond to the Gln56 O
e1
atom with the distance of 2.85 A
˚
for
the tripep tide, and 2.94 A
˚
for t he tetrapeptide. The Gln56
N
e2
atom forms a hydrogen bond to the Gln102 O

e1
atom
with the distance of 2.88 A
˚
for the tripeptide, and 2.74 A
˚
for the tetrapeptide. On the other hand, in the CyPB
molecules A without peptide and the CyPB molecule in the
crystal of free CyPB, the conformations of the side chains of
Arg48 differ from those of t he CyPB molecules B bound to
a peptide, and no hydrogen bond between Arg48 N
e
and
Gln56 O
e1
is formed, whereas these Gln56 N
e2
atoms form a
hydrogen bond to Gln102 O
e1
.
Comparison between peptides containing
trans
-Pro
bound to CyPB and the loop containing Gly-
trans
-Pro
of the capsid protein bound to human CyPA
The c rystal structure of the human CyPA complex [6]
revealed that the Ala88-Gly89-trans-Pro90-Ile91-Ala92-

Pro93-Gly94 region of t he most mobile loop in the
N-terminal domain of the HIV-1 capsid protein is bound
to the cleft on the u pper b-sheet. When the cores of E. coli
CyPB and human CyPA were superimposed, the position of
trans-Pro90 of the capsid protein displaces by half of a ring
from that of Pro3 of the tetrapeptide as shown in Fig. 6 . The
main chain of the glycine residue immediately preceding
Pro90 makes the torsional angles of / ¼ 149° and w ¼
158°, which are in t he regions in which o ther amino acids
with side chains are disallowed due to steric hindrance. The
plane of Ca, C and O of Gly89 has the rotation angle x of
 20° from the plane of N, Ca and Cd of Pro90 around the
Gly-Pro amide bond. The //w torsional angles for Pro90
are )78/141° and the ring of Pro90 is inserted into the
hydrophobic pocket. Pro90 C O forms hydrogen bonds to
Arg55 N
g1
and N
g2
atoms with distances of 2.67 A
˚
and
2.91 A
˚
,andtheArg55N
e
atom does not form a hydrogen
Fig. 4. A stereo view of Suc-Ala-trans-Pro-Ala-pNA (green) and Ac-Ala-Ala- trans-Pro-Ala-AMC (y ellow) b ound to superimposed E. coli CyPB
molecules. The hydrogen bonds are shown in broken lines. The CyPB molecules shown in Figs 4, 6 and 7 were rotated by 45° around the horizontal
axis from those shown in Figs 2 and 3.

Fig. 5. The model of the e nvelope form of the proline ring. CO-Ala2-
Pro3-Ala4 portion of the tetrapeptide is shown by ball-and-sticks
model.
3798 M. Konno et al.(Eur. J. Biochem. 271) Ó FEBS 2004
bond. On the other hand, in E. coli CyPB molecules B the
side chain of Arg48 is fixed by formation of a hydrogen
bond between the Arg48 N
e
atom and the Gln56 O
e1
atom
and Pro CO of the tripeptide and tetrapeptide forms a
hydrogen bond only to Arg48 N
g2
. The difference of the
hydrogen bond formation to the side chain of the Arg
residue of E. coli CyPB and human CyPA indicates that
these hydrogen bonds are f ormed after the Pro r esidue of
peptides is fixed in the hydrophobic pocket. A hydrogen
bond is formed between NH of Ala88 and CO of Gly72 of
human CyPA with a distance of 2.84 A
˚
. T he fixing by
hydrogen bonds of Ala88 and Pro90 to CyPA generates the
deviation of x angle of  20° from the plane around the
Gly-Pro amide bond and the distortion of the torsional
angle of Gly89. This fixing and hydrophobic interaction
agree with the finding that the capsid protein binds rigidly to
human CyPA; even i n the presence of a high concentration
of salt or detergent, the binding between them was detected

[25]. This is the most mobile loop of the capsid protein, and
it will bind to the human CyPA molecule in conjunction
with a conformational change in the loop. The conforma-
tion of acetyl-Ala1 of the tetrapeptide shows a big deviation
from that of His87-Ala88 of the capsid protein.
Comparison between
E. coli
CyPB with a peptide
of the
trans
-proline form and
E. coli
CyPA with a
peptide of the
cis
-proline form
Crystals were obtained in which Suc-Ala-trans-Pro-Ala-
pNA is bound on E. coli CyPB (Fig. 7 A), whereas c rystals
were reported [16] in which Suc-Ala-cis-Pro-Ala-pNA is
bound on E. coli CyPA (Fig. 7 B). In the cis-form of the
tripeptide binding in the cleft of E. coli CyPA, torsional
angles //w for Ala1, Pro2 and Ala3 are )119/146°, )69/143°
and )67/146°, respectively. The cis-proline ring of Pro2 is
also inserted into the hydrophobic pocket, and CO of Pro2
forms a hydrogen bond with N
g2
of the guanidyl g roup of
Arg43, whereas the succinyl group in the N-terminus
protrudes from the cleft, and NH and CO of Ala1 form
hydrogen bonds with CO and NH of Arg87. The p-nitro-

anilide ring in the C-terminal end lies over the side chain of
the Ile45 residue, parallel with the ring of Phe48.
Because all E. coli CyPA molecules are bound to the
peptide with cis-proline in solution, crystals obtained consist
only of complexes. On the other hand, in crystals of E. coli
CyPB analyzed in this study, the CyPB molecule A without
peptide and the CyPB molecule B with a peptide exist in the
ratio of 1 : 1. The findings that the peptides of the distorted
trans-proline form occupy half of the E. coli CyPB mole-
cules can be attributed to the fact that the binding affinity of
peptides of the distorted trans-proline form for CyPB is
smaller than that of peptides of the cis-proline form for
CyPA. Because the concentration of complexes with trans-
peptide may be smaller than that o f free p roteins, crystals
consisting of only complexes were not obtained but crystals
consisting of the complex and t he free protein, the ratio of
which is 1 : 1 due to crystal contact, were obtained. The fact
that crystals of these complexes of E. coli CyPB and CyPA
grew under the same conditions also indicates that the
binding affinity of the trans-form of the tripeptide for CyPB
molecule is higher than that for CyPA, whereas the binding
affinity of the cis-form i s higher for CyPA to the contrary.
We were unable to identify any difference of conformation
in 13 of the 14 conserved residues (CyPB/CyPA; His47/42,
Arg48/43, Ile50/45, Phe53/48, Met54 /49, Gln56/51, Ala91/
86, Arg92/87, Thr100/95, Gln102/97, Phe104/99, Phe112/
107, Leu113/108 and Tyr122/120) of the region in which the
tripeptide is placed (Fig. 7), whereas the nonsubstantial
difference betwe en the CH
3

-group of Met54 in CyPB and
that of Met49 in CyPA r eflects only the presence of steric
hindrance in the case of the complex o f CyPB and the
distorted trans-form. On the other hand, we found that
the different forms of b inding peptides are generated due to
the difference in areas close t o t he peptide-binding region,
i.e. the loop continuing from b5 and the loop connecting the
b4andb5 strands (Figs 2 and 7). Such regions are expected
to determine the orientation of the substrate at the P2 site,
i.e. the second residue of the N-terminal side from proline in
the isomerization reaction of refolding proteins as they
proceed from the trans-form t o the cis-form. The P2 site is
responsible for the difference in the rate of the isomerization
reaction accelerated by CyPA and CyPB molecules. The T4
turns in t he loop between the b5andb6 strands consist o f
Asp95, Lys96, Asp97 and Ser98 in CyPB and Ala90, Pro91,
Fig. 6. Comparison between a loop (His87-Ala88-Gly89-trans-Pro90-Ile91-Ala92-Pro93-Gly94) of the HIV-1 capsid protein bound to human CyPA
[6] (PDB code 1AK4) (yellow) and a distorted tetrapeptide Ac-Ala-Ala-trans-Pro-Ala-AMC bound to E. co li CyPB (green). The cores of the proteins
were superimposed. Residues of E. coli CyPB and human CyP A are shown in gree n and yellow with residue numbers (E. coli CyPB/human CyPA),
respectively.
Ó FEBS 2004 E. coli cyclophilin B/distorted trans-isomer (Eur. J. Biochem. 271) 3799
His92 and Ser93 in CyPA. The difference in these side
chains will generate the difference in conformations of the
side chains of Thr93 in CyPB and Thr88 in CyPA. The side
chains of Thr93 and Ala94 in CyPB and those of Thr88 and
Gln89 in CyPA will come in contact w ith t he su bstrate at
the P2 s ite. The part o f the loop connecting the b4andb5
strands, i.e. L ys68, P ro69, A sn70 and Pro71 in CyPB and
Ala63, Thr64, Lys65 and Glu66 in CyPA occupies the
bottom left side of the cleft and is close to the peptide-

binding region. The difference of hydrophobicity of these
areas between the CyPB and CyPA molecules is expected to
affect the selection of the substrate.
Discussion
Short peptides with a
trans
-proline are distorted
to come to complex with CyPB
CyP proteins accelerate the isomerization reaction f rom cis-
to trans-form of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa
stands for any amino acid residue) [5,17,18,26–29] and
shorter peptides such as Ala-Pro, Ala-Pro-Phe, Ala-Ala-
Pro-Ala and Ala-Ala-Pro-Ala-Ala i nhibit cis to trans
isomerase activity of calf thymus CyPA [27]. It was
suggested that in the mechanism of the observed isomeri-
zation reaction from cis-totrans-form on CyP proteins, the
N atom of Pro receives a proton from the guanidyl group of
Arg55 on the b3 strand for human CyPA [17]. The proton
transfer to the prolyl N atom results in weakening the
double bond character of the amide bond of Xaa-Pro. The
double bond character of this C-N amide bond is due to
the hyperconjugation between the p orbital on the sp
2
-
hybrid type N atom of a proline and the p orbital system
CO of Xaa preceding Pro. The N ato m of a proline with a
proton transferred thereto has an sp
3
-hybrid rather than
sp

2
-hybrid orbital; this change in electronic configuratio n is
essential to the transient state of cis to trans isomerization
pathway of short peptides catalyzed by CyP proteins. As the
mutation of Arg t o Ala in human CyPA loses the above-
mentioned pathway via the proton transfer from the
guanidyl group of Arg to the prolyl N atom, the mutant
wasreportedtoretainlessthan1%ofwildtypecatalytic
activity [30]. The local energy d iagram of the rotating C-N
bond of Xaa-Pro for two distinct pathways contributing to
cis to tra ns isomerization o f short peptides, which explains
the enzymatic activity difference between the wild-type and
the mutant, is illustrated in Fig. 8A. If the isomerization
from a cis-totrans-isomer occurs by rotating around the
Xaa-Pro amide bond while preserving the planarity of three
bonds around the N atom of the Pro residue, the energy
barrier DE
1
for the rotation corresponds to loss of stability
of energy due to the hyperconjugation of the Xaa-Pro amide
bond having sp
2
-hybrid configuration on the N a tom. On
the other hand, if the peptide rotates after the N atom of Pro
in the C-N amide bond of Xaa-Pro accepts a proton to be
convertedintoansp
3
-hybrid c onfiguration, the barrier for
Fig. 7. The stereo views of clefts of (A) E. coli CyPB complexed w ith Suc-Ala-trans-Pro-Ala-pNA and ( B) E. coli CyPAcomplexedwithaSuc-Ala-cis-
Pro-Ala-pNA [ 16] (PDB code 1LOP). These were viewed from the same direc tion using coordination of CyPA superimposed on CyPB. The

hydrogen bonds are shown in broken lines. The conserved residues of the region, in which the tripeptide are placed, are shown in light gray, and the
residues, which is expected to affect the selection of th e substrate , are shown in green.
3800 M. Konno et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the rotation due to only steric hindrance is low (DE
2
), as sp
3
-
hybrid configuration on the N atom has no interaction with
CO of Xaa. For the reverse conversion from a trans-isomer
having the sp
2
-hybrid configuration on the N atom of Pro to
a cis-isomer having also the sp
2
-hybrid configuration, in
addition to DE
1
, a difference in the energy between these
states, DE
3
, is also required, and the activation e nergy for
short peptides is too high for the reverse conversion to
occur.
However, in the observed complex of E. coli CyPA
with Suc-Ala-cis-Pro-Ala-pNA (Fig. 7B), CO of cis-Pro2
forms a hydrogen bond with the distance of 2.75 A
˚
to the
N

g2
atom of the guanidyl group of the Arg43 on the b3
strand, but the N atom of Pro2 is 4.02 A
˚
away from the
N
g2
atom of Arg43. T he N
g1
atom of Arg43 i s not within
5.5 A
˚
from the N atom of Pro2. In addition, NH and CO
of Ala1 of the cis-form have hydrogen bonds with the CO
and NH of Arg87, respectively. The amide bond of
Ala-cis-Pro is planar and t he N atom of Pro has the
sp
2
-hybrid configuration.
In E. coli CyPB molecules B bound to Suc-Ala-trans-Pro-
Ala-pNA and Ac-Ala-Ala-trans-Pro-Ala-AMC (Figs 4 and
7A), the N
g2
atom of Arg48 forms a hydrogen bond with
the distance of 2.62 A
˚
and 2.72 A
˚
to CO of trans-Pro of the
tripeptide and tetrapeptide, respectively. In these complexes,

the distance of the N atom of Pro and the N
g2
atom of
Arg48 is 3.08 A
˚
and 3.02 A
˚
, and the angle between the N
g2
-
N vector and normal vector to the amide bond plane of Ala-
Pro is 16° and 17°, respectively. The N
g1
atom of Arg48 i s
not within 5.0 A
˚
from the N atom of Pro. For both
peptides, the amide bond of Ala-trans-Pro is planar and the
NatomofProhasansp
2
-hybrid configuration.
In conclusion, the proton transfer from the guanidyl
group of Arg to the prolyl N atom no longer occurs in
the complex of E. coli CyPA, as hydrogen bonding to
CO of Ala1 gives rise to the increased double bond
character of the Ala1-Pro2 amide bond and the guanidyl
group of Arg43 is far from the N atom of Pro2. In the
case of the observed complex of CyPB, because the
hydrogen bond between the guanidyl group of Arg48
and CO of Pro plays an essential role in the binding of

the peptide, it is impossible for the guanidyl group of
Arg48 to be involved in proton transfer to the prolyl N
atom. These facts show that in both cases the enzyme
cannot carry o ut the isomerization of these peptides
observed in crystals of complexes. Similarly, a crystal
structure [15] of the tetrapeptide Ac-Ala-Ala-cis-Pro-Phe-
pNA staying in unreacted form on human CyPA also
demonstrated that Arg55 forms a hydrogen bond with
the C O of cis-Pro of the tetrapeptide, and that this
peptide retains still planarity of the Ala-cis-Pro amide
bond.
In the case of CyPB in complexes with Suc-Ala-trans-Pro-
Ala-pNA and Ac-Ala-Ala-trans-Pro-Ala-AMC, the large
distortion in the orientation of Ala1 and Ala2 of Ala-Pro
with //w torsional angles of )170°/100° and )177°/91°
allows the fixing of CO of Suc and Ala1 to His47 N
e2
via a
mediating water molecule. T he fixing t o Arg48 and His47
makes a major contribution to binding of peptides in the
trans-form. In contrast, as for CyPA in complex w ith Suc-
Ala-cis-Pro-Ala-pNA, two hydrogen bonds of NH and CO
of Ala1 are formed, and the orientation of Ala1 has a little
distortion.
When CO of Xaa preceding Pro has no formation of a
hydrogen bond, the double bond character of this C-N
amide bond is due only to the hyperconjugation. Proton
transfer to the prolyl N atom results in weakening the
double bo nd c haracter o f the amide bond of Xaa-Pro. As
result, the N atom of a proline with a proton transferred

thereto has an sp
3
-hybrid rather than an sp
2
-hybrid orbital,
and thus the planar configuration of the N atom of proline
is converted to an ammonium type configuration. As the
contribution of the hyperconjugation into the stability by
sp
2
-hybrid orbital on the N atom of Pro is lost owing to
the small rotation around the amide bon d of Xaa-Pro, the
process for the conversion from sp
2
-tosp
3
-hybrid confi-
guration is enhanced.
The a bove mentioned mechanism for short peptides,
where t he conversion from sp
2
-tosp
3
-hybrid configuration
has a critical contribution to in vitro c is to trans peptidyl-
prolyl isomerization activity of CyP proteins, may be
extended to understand the in vitro trans to cis peptidyl-
Fig. 8. The local energy diagram of the rotating C-N bond of Xaa-Pro.
(A) Two distinct pathways contributing to cis to trans isomerization of
short peptides. (B) Two distinct pathways contributing to trans to cis

isomerization of unfolded proteins, which possess the cis-proline
conformation in the natural folding state.
Ó FEBS 2004 E. coli cyclophilin B/distorted trans-isomer (Eur. J. Biochem. 271) 3801
prolyl isomerization in volved in t he reported e nhancement
by CyP proteins in the refolding process of unfolded
proteins (e.g. RNase A, carbonic anhydrase I I, and
RNase T
1
proteins [7–11]), which possess a cis-proline
conformation in the natural folding state. In the in vitro
refolding experiment without CyP proteins, the in vitro trans
to cis peptidyl-prolyl isomerization was observed to progress
slowly, which may be attributed to such a d irect path
requiring larger activation energy DE
5
where the trans-
isomer with the sp
2
-hybrid configuration o n the N atom of
Pro is directly isomerized to the cis-isomer with the sp
2
-
hybrid configuration (Fig. 8B). On the other hand, in the
in vitro system with CyP proteins, the in vitro trans to cis
peptidyl-prolyl isomerization was enhanced remarkably,
which may be reasonably understood to be due to such an
indirect path requiring a smaller activation energy DE
4
.The
conversion from the sp

2
-tothesp
3
-hybrid configuration on
the N atom of Pro of the trans-isomer is enzymatically
conducted by means of CyP proteins, being followed by the
quick isomerization from the trans-isomer with the sp
3
-
hybrid configuration to the cis-isomer with the sp
3
-hybrid
configuration. At the stage of the enzymatic conversion
from the sp
2
-tothesp
3
-hybrid configuration on the N atom
of Pro of the trans-isomer, the guanidyl group of Arg should
be used only for the proton t ransfer to t he N atom o f Pro
rather than the hydrogen-bonding to the CO o f Pro so that
the guanidyl group of Arg may be positioned much closer to
the N atom of Pro than that observed in CyPB in complex
with Suc-Ala-trans-Pro-Ala-pNA or Ac-Ala-Ala-trans-Pro-
Ala-AMC analyzed here. Thus, in place of hydrogen
bonding of the guanidyl group of Arg to CO of Pro,
another amino acid residue located somewhat behind the
Pro concerned is predicted to be involved in the binding of
unfolded proteins to CyP proteins. In such a predicted
binding form, the proline containing the peptide portion of

the unfolded proteins embedded in the cleft of CyP proteins
may have not only the distortion of Xaa of Xaa-Pro but also
additional distortion in the C-terminal region next to the
Pro. These distortions may be effectively used as driving
force for quick isomerization from the trans-isomer with the
sp
3
-hybrid configuration to the cis-isomer with the sp
3
-
hybrid configuration in the enhanced refolding process of
unfolded proteins observed in the the in vitro system with
CyP proteins.
It has been reported that strains of the yeast Saccharo-
myces cerevisiae in which all eight identified CyP family
genes w ere d isrupted survived [31–33]. Therefore, CyPs
appear to be irrelevant to the in vivo folding process for
native proteins that possess a cis-proline c onformation. As
explained above, CyPA and CyPB may have an advanta-
geous potential for binding distorted peptide portions of
partially unfolded proteins in its cleft. If such a distorted
peptide portion of a partially unfolded protein resulting
from extrinsic c auses (for e xample, heat shock) is bound in
the cleft of CyPA or CyPB protein, further progress of the
protein denaturation induced by the extrinsic causes would
be successfully blocked. In such a case, when the e xtrinsic
cause is r emoved from the partially unfolded proteins held
in the CyPA or CyPB protein, successful refolding of this is
achieved to make a quick recovery from damage due to the
extrinsic causes. Such a possible f unction of CyPs to block

the extensive denaturing course of proteins promoted by
extrinsic c auses m ay provide a more probable explanation
for previous reports [34] that yeast strains lacking CyPA and
CyPB are sensitive to heat shock, and that both of these
proteins facilitate the survival of cells exposed to high
temperatures.
Protein Data Bank access codes
Coordinates of the structures have been deposited in the
Protein Data Bank (accession codes 1V9T and 1VAI for the
two kinds of complexes of the E. coli CyPB K163T mutant
bound by Suc-Ala-trans-Pro-Ala-pNA and Ac-Ala-Ala-
trans-Pro-Ala-AMC, a nd accession code 1J2A for the
E. coli CyPB K163T mutant).
Acknowledgements
We thank Dr Mamoru Suzuki and Dr Noriyoshi Sakabe at the High
Energy Accelerator Research Organization, KEK, for their help in
the data collection. This work was su pported in part by a grant from
the N ational Project on Pro tein Structural and Func tional Analyses
to M. K.
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