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.. coli CyPA an
Trang 1Escherichia coli cyclophilin B binds a highly distorted form
Michiko Konno1, Yumi Sano1, Kayoko Okudaira1, Yoko Kawaguchi1, Yoko Yamagishi-Ohmori1,
Shinya Fushinobu2and Hiroshi Matsuzawa2,*
1
Department of Chemistry, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo, Japan;2Department 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 proteins 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 is inserted into the
hydrophobic pocket and a hydrogen bond between CO of
Pro and Ng2of 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
Ne2via a mediating water molecule allows the large distor-tion in the orientadistor-tion of Ala of Ala-Pro Although 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 of 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 and 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, whereas 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-isomer is converted to a cis-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- to the cis-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 capsid 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 preceding Pro [7–11], this binding conformation of the capsid protein is not sufficient to serve
as a model of the intermediate formed during isomerization
of the trans-isomer to the cis-isomer To date, the binding structures of peptides in the cis-proline form have been reported for human CyPA [12–15] and E coli CyPA [16], but the peptides forming the distorted 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: konno@cc.ocha.ac.jp
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)
Trang 2trans-form, the proline of which is bound to the pocket, is
only the loop of the capsid protein It has been reported that
E coliCyPA accelerates the conversion of trans- to cis-form
on peptide bonds of not only Ser54-Pro55 but also
Tyr38-Pro39 in the final refolding process of the unfolded RNase
T1, whereas human 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 different target
proteins, led us to consider whether E coli CyPB might
bind to peptides of the distorted 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- to
cis-isomer will be needed As the cis-isomerization of the 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 with a
tetrapeptide,
acetyl-Ala-Ala-Pro-Ala-amidomethylcouma-rin (Ac-Ala-Ala-Pro-Ala-AMC), and we 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 the capsid protein In addition, we
discussed the importance of the conversion from the sp2- to
sp3-hybrid configuration on the prolyl N atom in the
isomerization of the proline from the trans- to cis-isomer of
unfolded proteins in the refolding process, as well as in the
isomerization of the short peptides containing the proline
from the cis- to trans-isomer
Materials and methods
Preparation of mutantE coli CyPB
The plasmid pATtrpEPPIa, which has a ClaI and a BamHI
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 PCR fragment was cleaved with EcoRI and
BamHI, and was subcloned into pUC118, producing an
expression vector, pUCPPIb Mutation was introduced into
pUCPPIb using a QuickChangeTMSite-Directed
Mutagen-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 pATtrpEPPIa (KT) was transformed in
E coli HB101 cells The cells were cultured at 37C in M9CA medium [0.05% (w/v) NaCl, 0.1% (w/v) NH4Cl, 0.2% (w/v) casamino acid, 0.2% (w/v) glucose, 2.0 mM
MgSO4, 0.1 mMCaCl2, 0.6% (w/v) Na2HPO4, 0.3% (w/v)
KH2PO4, pH 7.4] and the proteins were 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 mMof peptide, 17% (v/v) dimethylsulfoxide and 35 mMHEPES buffer, pH 7.8] was preincubated with proteins (1.1–4.8 nM) in 2 mL of 35 mM HEPES buffer containing 5 mM 2-mercaptoethanol (pH 7.8), and the assay was started by mechanical mixing with 40 lL of 0.58 mM 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 10C
Crystallization, data collection, structure determination and refinement
Whole crystals of K163T mutant proteins were grown by vapor diffusion in hanging drops at 20C A 9 lL drop containing 0.57 mM(12 mgÆmL)1) protein and 6 mM trip-eptide (Suc-Ala-Pro-Ala-pNA (Ptrip-eptide Institute, Inc.), or tetrapeptide (Ac-Ala-Ala-Pro-Ala-AMC (Bachem, Switzer-land), 38–40% (w/v) saturated ammonium, sulfate, 9% (v/v) methanol, 0.04% (w/v) NaN3, and 50 mM Tris/HCl buffer (pH 8.0) was equilibrated against a reservoir solution containing 46% saturated ammonium sulfate, 0.04% (w/v) NaN3, and 100 mM Tris/HCl buffer (pH 8.0) When the peptide dissolved in methanol was added to the drop containing the protein under the above conditions, 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 containing 9.5 mgÆmL)1 protein, 38% (w/v) saturated ammonium sulfate, 0.04% (w/v) NaN3, and 80 mM Tris/HCl buffer (pH 8.0) equilibrated against a reservoir solution of 46% (w/v) saturated ammonium sulfate, 0.04% (w/v) NaN3, and
100 mMTris/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 usingDENZOandSCALEPACK
[20] The structure for the complex of E coli CyPB K163T mutant bound to tripeptide was solved by molecular replacement withXPLOR [21] using E coli CyPA [16] as a search model The model was built usingOsoftware [22], and refinement was assisted by molecular dynamics using
XPLOR The structure of the complex with the tetrapeptide was started from the final structure of the complex with the tripeptide The crystal structure of the free E coli CyPB
Trang 3K163T was solved by molecular replacement using a model
of the CyPB K163T protein of the complexes, and the final
refinements were made usingCNS[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 signal 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 ofE 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 which lysine was replaced by
threonine at residue 163, was selected under these criteria, as
Lys163 is located in b-strand and has no intramolecular
interaction with any residues The kcat/Km value of the
K163T mutant (1.9· 107M )1Æs)1) in the cis- to
trans-isomerization reaction for Suc-Ala-Ala-Pro-Phe-AMC was almost the same as that of the wild-type protein (1.8· 107
M )1Æs)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), grew from 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 crystals 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 coli 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-helices at the top and bottom (Fig 2) In both complexes, CyPB molecule A, i.e the molecule without a peptide, is packed along one 32axis, whereas CyPB molecule B, which
is bound to a peptide, is packed along the other 32axis 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, which is found in the
Table 1 Data collection and refinement statistics for 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 mean intensity R factor ¼ S||Fo|-|Fc||/S|Fo| A subset of the data (10%) was excluded from the refinement and used to calculate R free
Peptide Suc-Ala-Pro-Ala-pNA Ac-Ala-Ala-Pro-Ala-AMC none
Data collection
Maximum resolution (A˚) 1.7 (1.76–1.70) 1.8 (1.86–1.80) 1.8 (1.86–1.80)
Completeness (%) 92.0 (73.3) 99.4 (97.5) 99.0 (96.5)
Refinement
Resolution (A˚) 6–1.7 (1.78–1.70) 6–1.8 (1.88–1.80) 8–1.8 (1.82–1.80)
Root-mean-square deviations from ideal values
Average B factors (A˚ 2 )
Trang 4complexes, to P3221 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 hand, 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 atrans-form proline bound
toE coli CyPB
A left-to-right-running cleft is shown in the upper b-sheet of the b3, b4, b6 and b5 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 residues occupy the left 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 trans-form, allowing them to bury deeply into the cleft of CyPB molecules B In the 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 these 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-strands; pink: a-helices;
orange: 3 10 helices; and blue: turns Amino
acid residues are shown using a one-letter code
and dots indicate deletions The 14 conserved
residues of the region, in which the peptides
are placed, are written in bold type 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 Thr163 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.
Trang 5atoms lie both above the plane of the coplanar Cb-Ca-N-Cd
as shown in Fig 5 so that their trans-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 bound to
human CyPA but does not belong to any of three
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 (w¼ 141),
Pro71 (w¼ 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 (w¼ 176),
Pro72 (w¼ 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˚) to a water molecule, which forms a
hydrogen bond with His47 Ne2with a distance of 2.87 A˚
(2.78 A˚) The carbonyl oxygen atom of Pro2 of the
tripeptide forms a hydrogen bond to the Ng2atom of the
guanidyl group of Arg48 with the distance of 2.62 A˚, and
2.72 A˚ for that of Pro3 of the tetrapeptide Because the w
angles of Pro2 (w¼ 118) of the tripeptide and Pro3 (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), the Arg48 Neatom forms a hydrogen bond to the Gln56 Oe1atom with the distance of 2.85 A˚ for the tripeptide, and 2.94 A˚ for the tetrapeptide The Gln56
Ne2atom forms a hydrogen bond to the Gln102 Oe1atom 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 the CyPB molecules B bound to
a peptide, and no hydrogen bond between Arg48 Neand Gln56 Oe1is formed, whereas these Gln56 Ne2atoms form a hydrogen bond to Gln102 Oe1
Comparison between peptides containingtrans-Pro bound to CyPB and the loop containing Gly-trans-Pro
of the capsid protein bound to human CyPA The crystal structure of the human CyPA complex [6] revealed that the Ala88-Gly89-trans-Pro90-Ile91-Ala92-Pro93-Gly94 region of the most mobile loop in the N-terminal domain of the HIV-1 capsid protein is bound
to the cleft on the upper 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 the regions in which other 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 CO forms hydrogen bonds to Arg55 Ng1and Ng2 atoms with distances of 2.67 A˚ and 2.91 A˚, and the Arg55 Neatom 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 (yellow) bound 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 envelope form of the proline ring
CO-Ala2-Pro3-Ala4 portion of the tetrapeptide is shown by ball-and-sticks
model.
Trang 6bond 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 Neatom and the Gln56 Oe1atom
and Pro CO of the tripeptide and tetrapeptide forms a
hydrogen bond only to Arg48 Ng2 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 formed after the Pro residue 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˚ The 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 in 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 deviaconforma-tion
from that of His87-Ala88 of the capsid protein
Comparison betweenE coli CyPB with a peptide
of thetrans-proline form and E coli CyPA with a
peptide of thecis-proline form
Crystals were obtained in which
Suc-Ala-trans-Pro-Ala-pNA is bound on E coli CyPB (Fig 7A), whereas crystals
were reported [16] in which Suc-Ala-cis-Pro-Ala-pNA is
bound on E coli CyPA (Fig 7B) 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 Ng2of the guanidyl group 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 of free proteins, crystals consisting of only complexes were not obtained but crystals consisting of the complex and the 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 is 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 between the CH3-group of Met54 in CyPB and that of Met49 in CyPA reflects only the presence of steric hindrance in the case of the complex of CyPB and the distorted trans-form On the other hand, we found that the different forms of binding peptides are generated due to the difference in areas close to the peptide-binding region, i.e the loop continuing from b5 and the loop connecting the b4 and b5 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 to 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 the loop between the b5 and b6 strands consist of 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 coli CyPB (green) The cores of the proteins were superimposed Residues of E coli CyPB and human CyPA are shown in green and yellow with residue numbers (E coli CyPB/human CyPA), respectively.
Trang 7His92 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 with the substrate at
the P2 site The part of the loop connecting the b4 and b5
strands, i.e Lys68, Pro69, Asn70 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 atrans-proline are distorted
to come to complex with CyPB
CyP proteins accelerate the isomerization reaction from
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-Ala-Ala-Pro-Ala-Ala inhibit 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- to trans-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 sp2 -hybrid type N atom of a proline and the p orbital system
CO of Xaa preceding Pro The N atom of a proline with a proton transferred thereto has an sp3-hybrid rather than
sp2-hybrid orbital; this change in electronic configuration is essential to the transient state of cis to trans isomerization pathway of short peptides catalyzed by CyP proteins As the mutation of Arg to 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 was reported to retain less than 1% of wild type catalytic activity [30] The local energy diagram of the rotating C-N bond of Xaa-Pro for two distinct pathways contributing to cisto trans isomerization of 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- to trans-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 DE1for the rotation corresponds to loss of stability
of energy due to the hyperconjugation of the Xaa-Pro amide bond having sp2-hybrid configuration on the N atom 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 converted into an sp3-hybrid configuration, the barrier for
Fig 7 The stereo views of clefts of (A) E coli CyPB complexed with Suc-Ala-trans-Pro-Ala-pNA and (B) E coli CyPA complexed with a Suc-Ala-cis-Pro-Ala-pNA [16] (PDB code 1LOP) These were viewed from the same direction 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 the substrate, are shown in green.
Trang 8the rotation due to only steric hindrance is low (DE2), as sp3
-hybrid configuration on the N atom has no interaction with
CO of Xaa For the reverse conversion from a trans-isomer
having the sp2-hybrid configuration on the N atom of Pro to
a cis-isomer having also the sp2-hybrid configuration, in
addition to DE1, a difference in the energy between these
states, DE3, is also required, and the activation energy 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
Ng2atom of the guanidyl group of the Arg43 on the b3
strand, but the N atom of Pro2 is 4.02 A˚ away from the
Ng2atom of Arg43 The Ng1atom of Arg43 is 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 the N atom of Pro has the
sp2-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 Ng2atom 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 Ng2 atom of Arg48 is 3.08 A˚ and 3.02 A˚, and the angle between the Ng2
-N vector and normal vector to the amide bond plane of Ala-Pro is 16 and 17, respectively The Ng1atom of Arg48 is 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
N atom of Pro has an sp2-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 out 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 CO 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 Ne2via a mediating water molecule The fixing to Arg48 and His47 makes a major contribution to binding of peptides in the trans-form In contrast, as for CyPA in complex with 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 bond character of the amide bond of Xaa-Pro As result, the N atom of a proline with a proton transferred thereto has an sp3-hybrid rather than an sp2-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
sp2-hybrid orbital on the N atom of Pro is lost owing to the small rotation around the amide bond of Xaa-Pro, the process for the conversion from sp2- to sp3-hybrid confi-guration is enhanced
The above mentioned mechanism for short peptides, where the conversion from sp2- to sp3-hybrid configuration has a critical contribution to in vitro cis 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.
Trang 9prolyl isomerization involved in the reported enhancement
by CyP proteins in the refolding process of unfolded
proteins (e.g RNase A, carbonic anhydrase II, and
RNase T1 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 direct path
requiring larger activation energy DE5 where the
trans-isomer with the sp2-hybrid configuration on the N atom of
Pro is directly isomerized to the cis-isomer with the sp2
-hybrid configuration (Fig 8B) On the other hand, in the
in vitrosystem 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 DE4 The
conversion from the sp2- to the sp3-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 sp3
-hybrid configuration to the cis-isomer with the sp3-hybrid
configuration At the stage of the enzymatic conversion
from the sp2- to the sp3-hybrid configuration on the N atom
of Pro of the trans-isomer, the guanidyl group of Arg should
be used only for the proton transfer to the N atom of Pro
rather than the hydrogen-bonding to the CO of 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
sp3-hybrid configuration to the cis-isomer with the sp3
-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 were disrupted survived [31–33] Therefore, CyPs
appear to be irrelevant to the in vivo folding process for
native proteins that possess a cis-proline conformation 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 causes (for example, 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 extrinsic
cause is removed 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 function of CyPs to block
the extensive denaturing course of proteins promoted by
extrinsic causes may 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, and accession code 1J2A for the
E coliCyPB 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 supported in part by a grant from the National Project on Protein Structural and Functional Analyses
to M K.
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