Crystal structure of arabidopsis thaliana cyclophilin 38 (atcyp38 2

CHAPTER 4
RESULTS AND DISCUSSION

4.1 EXPRESSION AND PURIFICATION OF NATIVE WILD TYPE
ATCYP38
4.1.1 Expression
Sufficient quantity of soluble AtCyP38 (83-437) was produced as a
Glutathione-S-Transferase (GST) fusion protein at 25
o
C, 4 hrs (Fig. 19).


M123
3
0kDa
45 kDa
66 kDa
97 kDa
AtCyP38-GST
20.1 kDa
14.4 kDa

Figure 19. SDS-PAGE showing the expression of soluble wt
AtCyP38. M – Low Molecular Weight Marker, Lane 1 – Cell
lysate before induction, Lane 2 – Whole cell lysate 4hrs after
induction, Lane 3 – Soluble protein 4 hrs after induction

4.1.2 Affinity chromatographic purification
The produced AtCyP38-GST fusion protein was purified using glutathione-
sepharose beads (Amersham Biosciences), Fig. 20. Due to the slow kinetics of GST
binding, considerable amount of the fusion protein came out along with the flow

79
through. Hence the flow through from the first step was subjected to a second round
of purification process to retrieve the unbound fusion protein.


Figure 20. SDS-PAGE showing the affinity chromatographic
purification of the wt AtCyP38-GST fusion protein. M – Low
Molecular Weight Marker, Lane 1 – Soluble protein before
affinity chromatography, Lane 2 – Flow through from affinity
chromatography column, Lane 3 – Wash 1, Lane 4 – Wash 2,
Lane 5 – Protein bound to glutathione sepharose beads.

M1234 5
97 kDa
AtCyP38-GST
66 kDa
45 kDa
30 kDa
20.1 kDa
14.4 kDa


4.1.3 Thrombin cleavage
Removal of the GST tag was more efficient when cleaved on-column using
the thrombin protease. The cleavage required over-night incubation at 4
o
C for the
reaction to be complete. The cleaved protein had additional 14 residues, which belong
to the linker that connects the tag and the protein, at its N-terminus. The presence of

this linker with the sequence ‘GSPGISGGGGGILL’ is a peculiar feature of the
pGEX-KG vector, which was used for protein expression. This additional glycine-rich
region is expected to improve the thrombin cleavage and probably favored the
complete cleavage. However, probably due to these extra residues from the linker, the

80
band of the tag-removed protein appears at a slightly higher molecular weight than its
original 39 kDa weight in SDS-PAGE, Fig. 21.


M12345
97 kDa
66 kDa
45 kDa
AtCyP38
30 kDa
GST
20.1
kDa
14.4 kDa

Figure 21. SDS-PAGE showing the thrombin cleavage of the wt
AtCyP38-GST fusion protein. M – Low Molecular Weight Marker,
Lane 1 – Protein on resin before cleavage, Lane 2 – Flow through from
the column, after cleavage, Lane 3 – Wash 1, Lane 4 – Wash 2, Lane 5
– GST, bound to the glutathione sepharose beads after cleavage.


The cleaved AtCyP38 protein came out along with the flow through and the
first wash. But it still had some amount of contaminant proteins. The two fractions

were pooled together and subjected to another round of purification by size exclusion
chromatography.

4.1.4 Size exclusion chromatography
HiLoad 16/60 Superdex-75 column (Amersham Biosciences) was used for the
size exclusion chromatographic experiment. Pure AtCyp38 protein was eluted out as a
single peak, Fig. 22, and the corresponding fractions were pooled together. SDS-
PAGE of the pooled fractions, Fig. 23, had no other contaminant proteins.

81

Figure 22. Profile of size-exclusion chromatographic purification of
wt AtCyP38 (83-437). The higher peak at around 60 ml corresponds to
the protein.



M12
97 kDa
66 kDa
45 kDa
Pure AtCyP38
30 kDa
20.1 kDa
14.4 kDa

Figure 23. SDS-PAGE of size-exclusion chromatography fraction of
purified AtCyP38 (83-437). M – Low Molecular Weight Marker,
Lane 1 – Protein before size-exclusion chromatography, Lane 2 –
Pooled fractions of the protein after size-exclusion chromatography.

4.1.5 Analyses for purity and homogeneity
The position of the peak from size-exclusion chromatography indicated the
protein to be a monomer of about 40 kDa size, Fig. 18. This was confirmed on

82
Native-PAGE, Fig. 24, and the protein appeared as a single band that corresponded to
a monomer.


M 1
66 kDa
140 kDa
232 kDa
AtCyP38
Figure 24. Native-PAGE of purified wt AtCyP38 (83-437). M –
High Molecular Weight Native Marker, Lane 1 – Purified wt
AtCyP38.

The theoretical molecular weight of wt AtCyP38 (83-437) protein is 39.25
kDa. MALDI-TOF mass spectrometry of the recombinant wt AtCyP38, carried out
using an Applied Biosystems 4700 Proteomics Analyzer 86, showed a mass of about
40389.69 + 1.217, Fig. 25. The additional stretch of 14 residues at the N-terminus of
the protein makes its theoretical molecular weight 40.38 kDa. The molecular weight
determined by mass spectrometry is quite close to this value.
A Dynamic Light Scattering (DLS) experiment showed a dispersity index
of 0.16 and this assured that the protein was monodispersed. The molecular weight
predicted by DLS was 39.6 kDa, which again is quite close to the actual molecular
weight and hence the protein was confirmed to be a monomer.

83

40389.69
Figure 25. Mass spectrometry for wt AtCyP38 (83-437).

4.2 EXPRESSION AND PURIFICATION OF SELENOMETHIONYLATED
WILD TYPE ATCYP38
4.2.1 Expression
Feedback inhibition of methionine biosynthesis (Doublie, 1997) at the protein
expression stage was used for the incorporation of selenomethionine into the
AtCyP38 protein. The expression of selenomethionylated protein in the minimal
medium M9 resulted in slightly lower yield compared to the native protein which was
expressed in nutrient-rich LB medium.

4.2.2 Purification
2 mM DTT was maintained throughout in all the buffers to avoid the oxidation
of selenomethionine during the purification process. The protocol that was used for
native AtCyP38 purification was used for the purification of selenomethionylated

84
AtCyP38. MALDI-TOF analysis on the derivatized protein gave a mass of 40839.95
+ 1.205. The mass difference between the native and selenomethionylated proteins
indicates that 9 out of the 10 methionine residues of AtCyp38 have been replaced by
selenomethionine.

4.3 CRYSTALLIZATION AND DATA COLLECTION FOR WILD TYPE
ATCYP38

4.3.1 Crystallization
Initial crystallization screening of the native AtCyP38 protein by the hanging
drop vapor diffusion method yielded crystals in two different conditions within 2 days.
Poly ethylene glycol (PEG) was the common precipitant in both the conditions and in
addition to PEG, the conditions also had a volatile precipitant solution. One condition
had 10% isopropanol and the other condition had 2.5% t-butanol. The presence of
these volatile precipitants was indispensable for crystal formation. However, their
volatile nature caused disintegration of the crystals as soon as the cover-slip was
opened. Optimization of these hanging drop conditions did not show any
improvement.
The vapor batch method of crystallization was attempted in order to avoid the
problem of excessive evaporation of volatile precipitants, as suggested by Mortuza
and co-workers (2004). The volatile precipitant was provided in the reservoir and was
allowed to diffuse slowly into the drops that were overlaid with paraffin oil. Crystal
formation took slightly longer time than in the hanging drop method, probably due to
the slow diffusion rate. Crystals were larger in size, Fig. 26, and fewer in number. The
presence of oil prevented the fast evaporation rate of volatile precipitants out of the

85
drop when the cover was opened. The paraffin oil on the top of the drop also served as
an additional cryo-protectant during the cryo-cooling of crystals for data collection.


Figure 26. Crystal of AtCyP38 obtained by vapor batch method in a
condition having PEG6000 and t-butanol as precipitants.




Figure 27. Crystal of selenomethionylated AtCyP38 obtained by vapor

batch method in a condition having PEG6000 and t-butanol as
precipitants.


Crystals of selenomethionylated AtCyP38 protein was obtained in the same
condition as that of native AtCyP38 by the vapor batch method, Fig. 27. The native
protein was required at an initial concentration of 5 mg ml
-1
for crystallization,
whereas, the selenomethionylated protein was required only at 3 mg ml
-1
, probably
due to its lower solubility.

86
A few large crystals were picked-up, washed thoroughly in the crystallization
buffer, dissolved in water and subjected to MALDI-TOF mass spectrometric analysis.
The determined mass for the native and selenomethionylated crystals were the same
as that of the corresponding purified protein, indicating that the proteins are intact in
the crystal and no part of them has been cleaved off during the crystallization process.
Crystals were tested at an in-house X-ray imageplate detector facility for
diffraction quality and optimization of cryo-condition. Cryo-conditions with glycerol
concentration less than 20% always resulted in the formation of ice-rings in
diffraction images. Suitable cryo-protecting conditions were identified for crystals
obtained from both the conditions. Essentially, the chosen cryo-conditions had 5%
additional PEG in addition to 20-25% glycerol.
Crystals from the vapor batch droplets were carefully picked-up with cryo-
loops and immediately transferred to the respective cryo-protectant. Crystals were
either directly tested on the in-house system or flash-cooled in liquid nitrogen for later
experiments. The native crystals from the condition containing PEG 6000 and t-

butanol as precipitants diffracted up to 3 Å whereas those from the condition
containing PEG 4000 and isopropanol diffracted only up to 4 Å. The diffraction
quality of all selenomethionylated crystals was poor. But a few of these crystals from
the condition containing PEG 6000 and t-butanol diffracted up to about 4 Å.

4.3.2 Data collection and analysis
Data collection was done at the X25 beamline, National Synchrotron Light
Source, Brookhaven National Laboratory (Upton, NY, USA) with a Q315 charge-
coupled device detector (Area Detector Systems Corporation). Prior to data collection,
a fluorescence scan was carried out to identify the peak, remote and inflection

87
wavelengths for MAD dataset based on selenium absorption spectrum. 360 frames of
the native and 360 frames for each of the 3 MAD wavelengths were collected with an
oscillation one degree for the respective crystals. The crystal parameters and data
collection statistics are given in Table 1 below.

Table 1. Crystal parameters and data-collection statistics for wt
AtCyP38. Values in parentheses are for the highest resolution shell
(Native: 2.59-2.50 Å and Se-Met: 3.63-3.50 Å)

Unit-cell parameters
Space group C222
1

Native Se-Met
a (Å) 58.2 58.1
b (Å) 95.9 96.0
c (Å) 167.5 167.2
No. of molecules in ASU 1 1

Data collection
X-ray source and detector BNL (X-25) / ADSCQ-315 CCD
Resolution (Å) 2.5 3.5

Inflection Peak Remote

Wavelength (Å) 0.9500 0.9799 0.9798 0.9640
Total observations 109,448 38,377 38,181 37,311
Unique reflections 16,710 6,213 6,259 6,181
Completeness (%) 97.8 (99.8) 98.4 (93.3) 98.2 (91.9) 98.3 (92.3)
Redundancy 6.7 (6.7) 6.3 (6.0) 6.2 (5.7) 6.1 (5.6)
1
R
sym
0.071 (0.29) 0.059 (0.09) 0.065 (0.10) 0.050 (0.07)
〈I/σ(I)〉 21.3 (5.4) 30.2 (18.3) 30.7 (17.5) 20.5 (12.1)

1
R
sym
= ∑
hkl
∑
i
[|I
i
(hkl) - <I(hkl)>| / ∑
hkl
∑

i
I
i
(hkl)]



88
All data sets were processed and scaled using the HKL2000 (Otwinowski and
Minor, 1997) program package. The Matthew’s coefficient (Matthews, 1968) of the
crystal indicated a solvent content of 57% and the asymmetric unit contained one
molecule. The structure was determined by the three-wavelength (Multi-wavelength)
Anomalous Dispersion (MAD) method using the selenomethionine data. Even though
mass-spectrometric analysis indicated that 9 out of the 10 methionine residues of the
protein had been replaced by selenomethionine, the BnP program (Weeks et al., 2002),
which was used to solve the selenium

substructure, found only seven selenium sites.
The sites were confirmed

by comparison with the anomalous difference Patterson map.
Automated model building with the use of the ARP/wARP program (Perrakis et al.,
1999) was not very successful, except for tracing a few short stretches in the C-
terminal region.
The first electron density map that was calculated from the MAD

phases was
clean and well interpretable. The overall structure could very well be seen, wherein
the N-terminus had a bundle of 5 helices and the C-terminus had the typical β-barrel
cyclophilin domain, as expected from bioinformatics predictions. However, the early

maps did not permit rapid model building. Certain loop regions connecting the five
helices of the N-terminus were missing. The first three helices of the N-terminus had
no long or bulky aromatic side-chain residues that could help in identifying the region
and the missing density in the connecting loop regions made it all the more difficult.
Also, the extreme N-terminus was not quite clear in the density which was later found
to form part of the C-terminal cyclophilin β-barrel domain. In spite of these
difficulties, an initial model was built with all available maps in the graphics suite O
(Jones et al., 1991). This partial model was refined using the CNS program (Brunger
et al., 1998). But the R-factor did not change from the high 40’s, even after repeated

89
attempts of refinement. This clearly indicated some serious error in the model which
could not be easily identified and rectified due to the low resolution of the maps.

4.4 SELECTIVE MUTATION OF RESIDUES TO AID PHASING
The first three helices of the N-terminus did not have any methionine residues,
which, if substituted by selenomethionine, can help in correct model building. A
strategy of selective mutation of a few residues to methionine by site-directed
mutagenesis and subsequent selenomethionylation was planned for. The choice of
residues to be mutated to methionine was mainly based on the studies by Gassner and
Matthews (1999). In their studies, leucine was found to be the optimal amino acid to
be substituted by methionine. Furthermore, this is in complete agreement with the
ranking suggested by the Dayhoff mutation probability (Dayhoff et al., 1978), i.e. by
the frequency of amino-acid substitutions in the sequences of related proteins.
Amongst closely related proteins, leucine is the most frequent amino acid that is
replaced by methionine, without any disruption of local structure and change in the
overall fold (Jones et al., 1992; Leahy et al., 1994). The methionine side chain can, to
a great extent, adopt a conformation so as to occupy the space vacated by leucine.
Leucine has a side chain volume of 76 Å
3

, which is the same for methionine. It has
been shown that up to 10 such mutations to methionine are tolerated without any
change in overall structure or fold for T4 lysozyme (Gassner et al., 2003).
As mentioned earlier, while refining the AtCyP38 structure, assignment of
correct sequence was difficult for the first three helices of the N-terminus. AtCyP38
has several leucines in this region. We decided to mutate five selected leucines in this
region to methionine. Using the currently available model and secondary structure
predictions for the N-terminal helical region, adequate care was taken in the design

90
that the replacement would not lead to any structural disturbance and hence would not
interfere with the growth or quality of crystals. The selected leucines were likely not
to occupy internal sites within the protein; thereby disrupting a zipper formation (if
any). Leucines 107, 111, 125, 140 and 154 of AtCyP38 (83-437) were chosen to be
mutated to methionine. Individual replacement mutant genes as well as a mutant with
all the 5 leucines mutated to methionine were produced.
The mutant genes were inserted between the XbaI/XhoI sites of the pGEX-KG
vector. The sequence of AtCyP38 (83-437) showing the 10 inherent methionine
residues as well as the five mutated residues is given in Fig. 28.

083 VANPVIPDVS VLISGPPIKD PEALMRYAMP 112
113 IDNKAIREVQ KPMEDITDSL KIAGVKAMDS 142
143 VERNVRQASR TMQQGKSIIV AGFAESKKDH 172
173 GNEMIEKLEA GMQDMLKIVE DRKRDAVAPK 202
203 QKEILKYVGG IEEDMVDGFP YEVPEEYRNM 232
233 PLLKGRASVD MKVKIKDNPN IEDCVFRIVL 262
263 DGYNAPVTAG NFVDLVERHF YDGMEIQRSD 292
293 GFVVQTGDPE GPAEGFIDPS TEKTRTVPLE 322
323 IMVTGEKTPF YGSTLEELGL YKAQVVIPFN 352
353 AFGTMAMARE EFENDSGSSQ VFWLLKESEL 382

383 TPSNSNILDG RYAVFGYVTD NEDFLADLKV 412
413 GDVIESIQVV SGLENLANPS YKIAG 437

Figure 28. The sequence of AtCyP38 (83-437). The native methionine
residues are highlighted in grey and the leucine residues that were
mutated to methionine are highlighted in yellow.


4.5 CRYSTALLOGRAPHY OF ATCYP38 MUTANTS
4.5.1 Expression, purification and crystallization
The mutants and selenomethionine derivatives were expressed and purified
exactly as wild type AtCyP38 and its selenomethionine derivative, respectively.
Optimal crystallization conditions varied slightly for the mutant proteins. Suitable

91
cryo-conditions were used and the crystals were tested at the in-house facility for
diffraction quality. The mutant crystals were found to be of better diffraction quality
compared to the wild type. On an average, these crystals diffracted to about 2.8 Å at
the in-house facility and MAD data collection at the NSLS synchrotron was planned
for.
MALDI-TOF mass spectrometric analysis of the crystals gave a mass of
40470.24 +
1.06 for the native multiple mutant and 41102.15 + 1.16 for its
selenomethionine derivative. The mass difference indicated the incorporation of at
least 13 selenium atoms. The same mass was obtained for the respective proteins
before crystallization as well. This indicated that the whole protein is present in the
crystal and no part of it has got cleaved off during the crystallization process.

4.5.2 Data collection and analysis
The native and selenomethionylated crystal data sets for all the five single

mutants and the multiple mutant were collected at the X12B beamline, National
Synchrotron Light Source, Brookhaven National Laboratory (Upton, NY, USA) with
a Q210 charge-coupled device detector (Brandies). However, the dataset from the
multiple mutant alone was used to solve the structure. The crystal parameters, data
collection and initial refinement statistics are given in Table 2.
All data sets were processed and scaled using the HKL2000 program package.
The Matthew’s coefficient of the crystal indicated a solvent content of 57% and the
asymmetric unit contained only one molecule. The structure of the five LÆM mutant
protein was attempted to be solved by the three-wavelength method, making use of
the selenomethionine data.


92
Table 2. Crystal parameters and data-collection statistics for the
multiple L→M mutant. Values in parentheses are for the highest
resolution shell (Native: 2.73-2.64 Å and Se-Met: 2.57-2.46 Å)


Unit-cell parameters
Space group C222
1

Native Se-Met
a (Å) 58.0 57.7
b (Å) 96.0 96.7
c (Å) 166.7 166.8
No. of molecules in ASU 1 1

Data collection
X-ray source and detector BNL (X-12B) / ADSC Q210 CCD

Resolution (Å) 2.64 2.46

Inflection Peak Remote

Wavelength (Å) 0.9500 0.9797 0.9794 0.9500
Total observations 230,354 94,780 91,174 96,420
Unique reflections 14,209 17,556 17,609 19,441
Completeness (%) 97.8 (99.8) 98.1(93.3) 98.2 (90.9) 98.6 (91.6)
Redundancy 6.2 (5.7) 6.3 (6.0) 6.2 (5.7) 6.3 (5.9)
1
R
sym
0.069 (0.29) 0.059 (0.30) 0.065 (0.25) 0.067 (0.23)
〈I/σ(I)〉 21.40 (5.4) 20.2 (5.3) 20.7 (6.5) 19.21 (5.1)

1
R
sym
= ∑
hkl
∑
i
[|I
i
(hkl) - <I(hkl)>| / ∑
hkl
∑
i
I
i

(hkl)]

4.6 STRUCTURE DETERMINATION OF MUTANT ATCYP38
Mass-spectrometric analysis had predicted replacement of 13 out of 15
methionine residues of this mutant protein by selenomethionine. However, the BnP
program found only twelve selenium sites. These sites were manually confirmed

by
comparison with the anomalous difference Patterson map. Also, heavy atom site

93
refinement by density modification and solvent flattening was performed by using the
RESOLVE (Terwilliger, 2000) program. The 5 refined selenium sites of the mutations
L107M, L111M, L125M, L140M and L154M were significantly helpful in model
building.
Due to the higher resolution limits, the electron density map calculated from
the combined

phases was much better than that of the wild type protein. The native
data for the mutant was of lower resolution (2.64 Å) compared to that of the
derivative (2.46 Å) and hence was not used at all. The remote data from the derivative
was used for all subsequent map calculations and refinement steps. The maps were
very clear at the loop regions that connect the N-terminal helices. Also, the extreme
N-terminus (residues 83-99) was clearly modeled and found to form part of the C-
terminal cyclophilin β-barrel domain. However, density for two long loop regions
within the C-terminal cyclophilin domain was not clearly observed.
The initial R-factor was 0.43. However, ARP/wARP could build only a few
short stretches and did not dock any sequence. Manual model building was performed
using the program O. Crystallographic refinement was carried out with the program
CNS as well as Refmac5 of the CCP4 suite (Murshudov et al., 1997). Iteratively, 2F

o
-
F
c
and F
o
-F
c
maps were computed to check any misfit in the model. Temperature
factors were refined isotropically in the later stages of refinement and water was
picked up using the Water-Pick program from CNS based on the F
o
-F
c
map at the 3.0
σ level and was checked with the 2F
o
-F
c
map at the 2.0 σ level.
The final R-factor and R
free
(with 10% of reflections that were not included for
refinement) were 0.22 and 0.28, respectively using reflections with |F| > 3.0 σ(|F|).
The geometry of the final model was checked with the program PROCHECK
(Laskowski et al., 1993). All parameters were within acceptable ranges, except for a

94
few residues in the two long loop regions which were found to be in the disallowed
region of Ramachandran plot. These long loops were quite flexible, with high B-

factors. All figures were prepared using the PYMOL (DeLano, 2002) program. A
summary of the refinement statistics is shown in Table 3.

Table 3. Structure refinement statistics for mutant AtCyP38.


Mutant AtCyP38
1
R
cryst
/ R
free
0.22/0.28
Resolution range (Å) 8 - 2.46
Reflections (working/test)

14317/1588
Final model:
Non-hydrogen atoms 2765
Waters 214
Average B-factors (Å
2
):
• Protein (all atoms) 64.36
• Protein (with two loops removed) 54.86 (2186 atoms)
• Waters 49.48
R.M.S.D. in bond lengths (Å) 0.013
R.M.S.D. in bond angles (°) 1.605

1

R-factor = ∑
hkl
||F
o
(hkl)| – |F
c
(hkl)|| / ∑
hkl
|F
o
(hkl)|









95
4.7 THREE-DIMENSIONAL STRUCTURE OF ATCYP38 (83-437)
The secondary structural organization of AtCyP38 is illustrated in Fig. 28.
AtCyP38 (83-437) has two distinct domains as seen from its crystal structure. The N-
terminus is a helical domain made up of 5 helices (α1 – α5) of varying lengths. This
domain is followed by a typical cyclophilin domain, made up of an eight-stranded β-
barrel that is capped by one α-helix at each end. There are differences in the
cyclophilin domain of AtCyP38 and other known cyclophilin structures (section
4.9.2). The helical domain and the cyclophilin domain of the structure are connected

GGILLVANPVIPDVSVLISGPPIKDPEALMRYAMPIDNKAIREVQKPME
α
β2
1
β1
α2


DITDSLKIAGVKAMDSVERNVRQASRTMQQGKSIIVAGFAESKKDHGNE


MIEKLEAGMQDMLKIVEDRKRDAVAPKQKEILKYVGGIEEDMVDGFPYE


VPEEYRNMPLLKGRASVDMKVKIKDNPNIEDCVFRIVLDGYNAPVTAGN


FVDLVERHFYDGMEIQRSDGFVVQTGDPEGPAEGFIDPSTEKTRTVPLE


IMVTGEKTPFYGSTLEELGLYKAQVVIPFNAFGTMAMAREEFENDSGSS


QVFWLLKESELTPSNSNILDGRYAVFGYVTDNEDFLADLKVGDVIESIQ


VVSGLENLANPSYKIAG

Figure 29. Secondary structural organization of AtCyP38. β-strands
are shown as yellow arrows and α-helices are shown as red cylinders.
The two β-strands from the N-terminus which form part of the β-barrel
are shown in blue.
β5
α4
α
5
β3
β
4 α6
β
6
180
280
330
90
100
110
120
130
140
150
160
170
230
240
250
260 70
190

200
210
220
290
300
310
320
340
350
360
370
380
390
0
410
420
430
80
α
3
2
40
α
4
α
3
α2
α6
β
8

β
9
α
7

β7

β9
α8

96
by a loop which has an excess of negatively charged residues. Most interestingly, the
extreme N-terminus of the protein gets into the C-terminal cyclophilin domain and
forms part of the β-barrel, Fig. 30. This feature is quite novel and observed for the
first time. The functional implication of this feature will become very clear when we
solve the structure of mature AtCyP38 (93-437). The loops in the cyclophilin domain
are quite disordered. The first 10 of 14 amino acids of the N-terminal tag-linker and
the last four C-terminal residues were not observed in the electron density map.


CyP domain
Helical bundle
β
1
β
2
α
1
α
2

α2
α3
α3
α4
α5
α6
α
7
α
8
β
3
β
4
β5
β6
β
7
β
8
β9

Figure 30. Overall structure of AtCyP38 (83-437). α-helices are
shown in red, loops in green and β-strands as yellow arrows. The N-
terminus, which forms part of the β-barrel, is shown in cyan (figure
made with PYMOL).


97

Figure 31. Overall structure of AtCyP38 (83-437) in stereo view.

4.7.1 N-terminal domain
The N-terminus consists largely of two β-strands and five α-helices which are
connected by loops. The two β-strands of the N-terminus get into the C-terminus and
form part of the anti-parallel cyclophilin β-barrel. Helix α1 is the smallest of the five
helices in the N-terminus and it lies close to the cyclophilin domain. The remaining
four helices form a bundle. Helices α2 and α3 are split into two fragments each and
the fragments are connected by very short loops. Helices α4 and α5 are long and are
connected to each other by a longer loop. The sequence
102
DPEALLR
108
of the short
helix (α1) is about 57% identical to DPNYLHR and DPVANVR, the regions of the
regulatory subunit A that is responsible for binding the catalytic subunit of protein
phosphatase 2A (PP2A) (Ruediger et al., 1994). Phosphatase binding proteins are
known to have such short stretches which are important for the interaction.
The region between residues 125 and 161 has been predicted to be a leucine-
zipper, based on the fact that leucine or isoleucine residues are present almost at every
7
th
position (He et al., 2004). But the experimental 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

98
characteristic feature of the leucine zipper architecture. Also, some of the leucine
residues fall within the loop region between the helices and that is quite unlikely for a
leucine zipper.

The four-helix bundle motif is relatively common in proteins. A DALI search
(Holm & Sander, 1993) with the helical bundle of AtCyP38 covering residues 115-
217 showed that the closest structural homologues are spinach PsbQ (1nze; Z score,
10.1) and E. coli cytochrome b562 (256B; Z score, 10.0). PsbQ is a 16 KDa oxygen
evolving subunit of Photosystem II and cytochrome b562 is a heme-binding protein
involved in electron transport as well as DNA-dependant transcription regulation.
These structures include a four-helix bundle with up-and-down topology. However,
no region of the above proteins shows a strong sequence similarity to AtCyP38. PsbQ
is only about 11% identical and aligns with the helical bundle of AtCyP38 with an
R.M.S.D. of 1.71 over a stretch of 88 residues. Cytochrome b562 is about 10%
identical and aligns with an R.M.S.D. of 1.73 for 83 residues, Fig. 32.

A B

Figure 32. Structure overlap for the helical bundle of AtCyP38 with
(A) PsbQ and (B) cytochrome b562. AtCyP38 is drawn in red and
green whereas PsbQ and cytochrome b562 are shown in cyan and pink.
The overlap was generated with the MULTIPROT server and the
figure was prepared using PYMOL.


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The four helices of AtCyP38 helical bundle pack together mainly through
hydrophobic interactions. The internal surface of the bundle is highly hydrophobic,
being occupied mainly by Leu, Ile, Ala and Val residues. The helices are rich in
leucine and isoleucine residues. The functional implication of this property is to be
further verified. Also, the helices are rich in charged residues. All the potentially
charged and hydrophilic residues point towards solvent. The region between Glu167
and Asp255 has clusters of charged residues with a net surplus of acidic residues (21
acidic, 16 basic). In other high molecular weight immunophilins, such as CyP40 and

FKBP52, similar clusters of charged residues are involved in protein-protein
interactions, such as interaction with Hsp90 (Ratajczak and Carrello, 1996).
A short acidic linker region (~15 residues) connects the N-terminal domain
with the C-terminal cyclophilin domain. It consists of 1 aspartate and 3 glutamate
residues. Unlike bovine CyP40, there are no β-turns in this linker region.

4.7.2 Cyclophilin domain
The cyclophilin domain of AtCyP38 (83-437) has the typical β-barrel structure,
closed at both ends by α-helices. Eight anti-parallel β-strands make the barrel. Even
though the AtCyP38 cyclophilin domain has quite extensive loops, they do not have
any conformation similar to the loops of other known divergent cyclophilins. Also,
this domain does not have the conserved cysteine residues of other divergent
cyclophilins. Fig. 33 shows the structural overlap of AtCyP38 CyP domain with
hCyPA. A structure based sequence alignment of AtCyP38 with all these cyclophilins
can be seen in Fig.34.



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Figure 33. Structural overlap of cyclophilin domains of AtCyP38 (red)
and hCyPA (green) in two different views. The N-terminal region of
AtCyP38 that forms part of the CyP domain is shown in orange. The
overlap was generated with the MULTIPROT server and the figure
was prepared using PYMOL.

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Several variations can be seen in the AtCyP38 (83-437) cyclophilin domain,

when compared to other known cyclophilin structures. When the AtCyP38 sequence
is aligned with that of other cyclophilins, it can be seen that the sequence in β5 and β6
region of other cyclophilins aligns well with the respective region of AtCyP38 and
many of the active site residues can be seen in this region. But when structurally
superimposed, it is the β1-β2 region of the current intermediate AtCyP38 that
occupies the position of the β5-β6 region of other cyclophilins. This results in the
displacement of a long stretch with the residues that must make the active site, in the
form of a flexible loop. Strands β3 and β4 of AtCyP38 correspond to β1 and β2 of
other cyclophilins. Strand β5 of AtCyP38 which corresponds to β3 of other
cyclophilins, appears to be much longer. The current AtCyP38 structure lacks the β-
strand, corresponding to the short β4 of other cyclophilins. Instead there is a β-strand
(β6) in AtCyP38 in the later part of the structure. The corresponding region in other
cyclophilins normally forms part of a loop. The β-strand β7 of AtCyP38 sits directly
over the active site and does not form part of the barrel. In other cyclophilins the
corresponding region forms a 3
10
helix, contributing to the active site. This β-strand of
AtCyP38 is so oriented that it makes a cavity perpendicular to the β-barrel in the
active sire region. β8 and β9 of AtCyP38 are slightly longer than the corresponding
β7 and β8 of other cyclophilins. The helix α6 (between β4 and β5) of AtCyP38 caps
one end of the β−barrel and this helix corresponds to α1 in other cyclophilins.
α7 which caps the other end of the barrel is much shorter than the corresponding
α−helix in other cyclophilins. The extreme C-terminus of AtCyP38 has a short 3
10

helix (α8), which is not seen in other cyclophilins. The current AtCyP38 intermediate
structure has two long loops. The loop that connects β5 and β6 of AtCyP38 is about

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26 residues longer than the corresponding loop in other cyclophilins. The other long

loop in the structure between β6 and β7 is formed by the displacement due to the
incorporation of the β1-β2 region into the β-barrel.


AtCyP38 232 MPLLKGRASVDMKVKIKDNPNIEDCVFRIVLDGYNAPVTAGNFVDLVER
Bov CyP40 011 SNPSNPRVFFDVDIGG ERVGRIVLELFADIVPKTAENFRALCTGEKGIGPTTGKP
hCyPA 001 MVNPTVFFDIAVDG EPLGRVSFELFADKVPKTAENFRALSTGEKGFG
hCyPB 007 GPKVTVKVYFDLRIGD EDVGRVIFGLFGKTVPKTVDNFVALATGEKGFG
Eco CyPB 001 AKGDPHVLLTTSA GNIELELDKQKAPVSVQNFVDYVNSG


AtCyP38 281 HFYDGMEIQRS-DGFVVQTGDPEGPAEGFIDPSTEKTRTVPLEIMVTGEKTPFYGSTLEE
Bov CyP40 066 LHFKGCPFHRIIKKFMIQGGDFSNQNG TGGESIY
hCyPA 048 YKGSCFHRIIPGFMCQGGDFTRHNG TGGKSIY
hCyPB 056 YKNSKFHRVIKDFMIQGGDFTRGDG TGGKSIY
Eco CyPB 040 -FYNNTTFHRVIPGFMIQGGGFTEQMQ QKKPNPP


AtCyP38 078 GG

ILLVA
NPVIPDVSVLISGPP 099
AtCyP38 340 LGLYKAQVVIPFNAF GTMAMAREEFENDSGSSQVFWLL-KESELTPSNSNILDGRYAV
Bov CyP40 100 GEKFEDEN-FHYKHDKEGLLSMANAGSNT NGSQFFITTVPTPHL DGKHVV
hCyPA 080 GEKFEDEN-FILKHTGPGILSMANAGPNT NGSQFFICTAKTEWL DGKHVV
hCyPB 88 GERFPDEN-FKLKHYGPGWVSMANAGKDT NGSQFFITTVKTAWL DGKHVV 0
Eco CyPB 073 IKNEADNG LRNTRGTIAMARTADKD-SATSQFFINVADNAFLDH GQRDFGYAV


AtCyP38 397 FGYVTDN EDFLADLKV GDVIESIQVVSGLENLANPSYKIAG 437

Bov CyP40 149 FGQVIKGMGVAKILEN EVKG EKPAKLV CVIAECGELKEGDDWGIFPK 195
hCyPA 129 FGKVKEGMNIVEAMERFGSRN GKTSKKITIADCGQLE 165
hCyPB 137 FGKVLEGMEVVRKVESTKTDS RDKPLKDVIIADCGKIEVEKPFAIAKE 184
Eco CyPB 125 FGKVVKGMDVADKISQVPTHDVG-PYQNVPSKPVVILSATVLP 166

Figure 34. Structure based sequence alignment of the cyclophilin
domains of AtCyP38, bovine CyP40, human CyPA, human CyPB and
E. coli CyPB. Residues corresponding to β-strands are underlined and
those corresponding to helices are shown in bold. The conserved
residues in all the 5 sequences are highlighted in yellow. The residues
that are known to be important for CsA binding are shown in red. The
N-terminal region of AtCyP38, which enters into the cyclophilin
domain, is shown in blue at the corresponding position.

When compared with human CyPA, the cyclophilin domain of AtCyP38
shows the lowest degree of identity (17 %) to any known cyclophilin. This may be
due to the functional role of this protein in plants. The cyclosporin A (CsA) binding of
hCyPA involves 13 residues. In AtCyP38, only 9 of these residues are conserved in
the active site. This could be the reason for the inability of its spinach homolog

103