Structural investigations of redox regulation in ATFKBP13 4

Chapter 4 Results and Discussion

72

CHAPTER 4. RESULTS AND DISCUSSION

4.1 STRUCTURE OF MATURE ATFKBP13 REVEALS UNIQUE DISULFIDE
BRIDGES
4.1.1 Overall Structure of AtFKBP13-S2

The AtFKBP13
-
S2 molecule reveals predominantly β-structures consisting of six
β-strands and two α-helices (Fig. 4-1). The β-strands form an integral antiparallel β-sheet
than constitutes the core of the protein.












Figure 4-1.
Structure of AtFKBP13-S2. The redox active disulfides are shown in
ball-and-stick representation and the sulphur atoms are shown as yellow balls.

Chapter 4 Results and Discussion

73
The secondary structures are arranged in the order: β
1
β
4
β
5a
α
2
β
5b
α
1
β
2
β
6a
β
6b
β
3

(Fig. 4-2). The β
5
strand of AtFKBP13 is split into two fragments, β
5a
and β

5b
, as in
MtFKB17 [Suzuki et al., 2003] and HsFKB12 [Meadows et al., 1993]. However, the β
5a

strand of hFKB12 is formed only when a ligand (ascomycin) is bound [Meadows et al.,
1993]. Otherwise it is disordered [Michnick et al., 1991; Moore et al., 1991]. Between β
5a

and β
5b
is the inserted α
2
helix, similar to that of MtFKB17. The β
6
strand pair, which is
unique to AtFKBP13, is formed by strands β
6a
and

β
6b
connected by a short loop.

5 15 25 35 45
CEFSVSPSGL AFCDKVVGYG PEAVKGQLIK AHYVGKLENG KVFDSSYNR



55 65 75 85 95

GKPLTFRIGVG EVIKGWDQGI LGSDGIPPML TGGKRTLRIP PELAYGDRG



105 115 125
AGCKGGSCLIP PASVLLFDIE YIGKA



Figure 4-2.
Secondary structure elements of AtFKBP-13. The
α
-helix is
represented by a cylinder and the
β
-strand is represented by an arrow.



β1




6a β
β1

β4

β3

β2
β5a

β5b

β6a
α2
β6b

α1

Chapter 4 Results and Discussion

74
4.1.2 Comparison with related structures

A notable feature in the tertiary structure of AtFKBP13-S2 is the presence of two
intra-chain disulfide bonds. These disulfide bonds, Cys5–Cys17 and Cys106–Cys111,
form the two redox active motifs. These disulfides have no counterparts in other animal
or yeast FKBPs. Eukaryotic FKB protein sequences indicate that this region is rather a
unique feature of
Arabidopsis
FKBP13. Also, structural comparisons with other FKBPs
indicate that AtFKBP13 has a conserved PPIase domain with additional strands (β
6a
and

β
6b
) inserted at the C-terminus where Cys106 and Cys111 are located.

Figure 4-3.
Superimposition of the C
α
backbones of AtFKBP13 (residues
5–129, blue), hFKBP12 (residues 1-107, magneta) and
L. pneumophila

FKBP25 (residues 496–612, green). The redox active-site disulfides of
AtFKBP13 are shown in ball-and-stick representation.
N
C

Chapter 4 Results and Discussion

75
Alignment between AtFKBP13 and representatives of other FKBPs, namely
hFKB12 [Wilson et al.,

1995] and the Macrophage Infectivity Potentiator protein from
Legionella pneumophila

, LpMIP [Riboldi-Tunnicliffe et al., 2001] through the Dali
server [Holm, and Sander, 1993] gives the r.m.s deviation values of 1.3 Å (with a Z-value
of 17.5) and 1.4 Å (with a Z-value of 17.2), respectively. The core regions for these
structures are similar (Fig. 4-3). The best matches are found in the regions that are
directly involved in the prolyl isomerase activity. A few residues are conserved among
these structures. These are the residues that form the substrate-binding pocket for the
pipecolinyl ring of FK506 [Van Duyne et al., 1993], namely Tyr37, Phe47, Asp48,
Phe59, Val66, Ile67, Trp72, Tyr99, Ile113, Leu119 and Phe121. These residues are
essential for binding and maintaining the hydrophobic core of FK506 [Radzicka et al.,
1992].
4.1.3 Catalytic domain

The catalytic domain of AtFKBP13 is composed of two sub-regions, the PPIase
(residues 26-125) and Cys-X
n
-Cys redox active motifs (residues 5-17 and 106-111). The
PPIase active region follows the general extended PPIase fold, which consists of a four-
stranded β-sheet and an α-helix inserted between them. In AtFKBP13, the corresponding
β-sheet comprises strands β
1
-β
5,
helix α
1
and a short 3
10
helix, α
2 .
The Cys-X
n

-Cys motif
regions are composed of two disulfides, found at the N and C-termini, respectively. The
active site disulfide bond between Cys5 and Cys17 at the N-terminus is located at the β
1
strand. Both Cys5 and Cys17 are partially solvent exposed. The second disulfide bond
between Cys106 and Cys111 (between β
6a
and β
6b
) is fully solvent exposed.

Chapter 4 Results and Discussion

76
These disulfide bonds form additional secondary structures that are located on
either side of the central β-sheet and reflect the intrinsic versatility and flexibility of this
region. The two active sites that are involved in redox regulation exist in their oxidized
state in the crystal structure as shown by their clearly defined electron density (Fig. 4-4).
The theoretical dihedral energy for the N-terminal disulfide is 3.60 kcal mol
-1
and
for the C-terminal disulfide is 4.00 kcal mol
-1
, calculated with the program AMBER
[Weiner et al., 1984], indicate the disulfide bonds are stable with less conformational
strain. The corresponding value varies from 0.5-4.7 for most protein disulfide bonds
[Darby and Creighton, 1995] and rarely reaches values over 5.0. Because the active site
disulfide bonds are extremely stable, they may also act as strong reductants.

Figure 4-4.

Stereoview of the 2
F
o
-
F
c
maps contoured at the 1.2
σ
level at
the C-terminal disulfide region. The amino acid residues in the region are
numbered.


Chapter 4 Results and Discussion

77
Despite this similarity, however, the two redox active disulfides show remarkable
difference in their B-factors. The average B-factor for the side chain atoms (C
β
and S
γ
) of
the two cysteines is 39.32 for the N-terminal disulfide versus 26.22 for the C-terminal
disulfide. Even though, all the residues in both redox active sites have very well defined
electron density in the final 2F
o
-F
c
map, the side chain atoms of the two cysteines in the
C- terminal disulfide have been better defined with more spherically shaped electron

densities than those in the N-terminal disulfide.
4.1.4 Surface of AtFKBP13

In AtFKBP13, the residues around the two redox active sites form two grooves on
the protein surface, the active site Grooves N and C (Fig. 4-5a). Groove C is built
exclusively by residues from the C-terminus and is essentially hydrophobic.

A B C

Figure 4-5.
(
A
) Surface charge distributions (blue for positive and red for negative
charge) for the AtFKBP13 monomer. The dark red region indicates

Chapter 4 Results and Discussion

78
a potential of less than -12 kT/e, while dark blue indicates greater than 12
kT/e. The electrostatic potentials were calculated by GRASP. (
B
) Space-
filling representation in which the sulfur atoms of Cys-106 and Cys-111 are
exposed on the surface of the molecule and (
C
) the S atom of Cys5 is fully
exposed on the surface, and the sulfur atom of Cys17 is buried.

The residues in the vicinity of Cys106 include Gly105, Pro114, Ser110, Leu112,
and Ile113. The S

γ
atoms of Cys106 and Cys111 are exposed on the protein surface (Fig.
4-5b). Unlike Groove C, the formation of Groove N involves residues both from the N
and C-termini. While atom S
γ
of Cys5 is fully exposed on the surface, the sulphur atom
of Cys17 is buried (Fig. 4-5c). The redox active site loops C and N adopt an open
confirmation in the crystal structure so that the S
γ
atom and the substrate binding site are
exposed to solvent. The two redox active grooves are adjacent to each other on the
protein surface. Separately located in the two grooves with different accessibility, the two
redox active sites probably function independently to some extent. The distance between
the two active sites, measured between the S
γ
atoms of Cys5 and Cys106 is 27.6 Å.

4.2 PAST WORK ON PRECURSOR ATFKBP13
Gupta et al. (2001) identified the first chloroplast FKBP from
Arabidopsis
and the
Rieske protein, a component

in the photosynthetic electron transport, as its putative
target. The interaction

between AtFKBP13 and the Rieske protein probably occurs before

they are imported into the thylakoid because the mature proteins

do not interact with each

Chapter 4 Results and Discussion

79
other. Both yeast

two-hybrid and
in vitro
protein interaction assays demonstrate

that the
full-length precursor proteins (or the cytoplasmic forms)

interact. In addition, the
intermediate (or stromal) forms

of the two proteins also interact well. These results

suggest that AtFKBP13 associates with the Rieske protein both before

and after the
import of the proteins into the chloroplast stroma.

In addition, AtFKBP13 and Rieske
intermediate forms also can

interact after they are imported into the thylakoid lumen but

before the thylakoid signal peptides are cleaved. After the

cleavage of the peptide, the
two matured proteins probably dissociate.
4.2.1 AtFKBP13 is targeted to the thylakoid lumen of chloroplasts by the
∆
pH-
dependent pathway
To determine whether AtFKBP13 is targeted to the chloroplast,

and if so, what its
suborganellar location is, protein import

assays have been performed with isolated
chloroplasts and the AtFKBP13

precursor as a substrate. The translated AtFKBP13
precursor

is

about 27 kDa (estimated by mobility in SDS/PAGE). When isolated

intact,
pea chloroplasts were incubated with the precursor protein

in the presence of ATP, a 13
kDa protein was generated (Fig. 4-6A, lane 2). After incubation with the protease
thermolysin

(which under the used conditions does not penetrate the chloroplast

envelope), intact chloroplasts were reisolated and fractionated.

The resistance of the 13
kDa polypeptide to degradation by

exogenously added thermolysin (Fig. 4-6A, lane 3)
indicates that

it is located within the chloroplast and is a product of the precursor

protein
import. Further analysis revealed that the AtFKBP13 protein

was associated with the
thylakoids (Fig. 4-6A, lane 5) and not

in the stroma (Fig. 4-6A, lane 4).

Chapter 4 Results and Discussion

80





Figure 4-6.
AtFKBP13 is targeted to the chloroplast thylakoid lumen by
the ∆pH-dependent pathway. (
A

) Chloroplast import assay of AtFKBP13:
translation products (lane 1), chloroplasts (lane 2), thermolysin-treated
chloroplasts (lane 3), stromal fraction (lane 4), thylakoid fraction (lane 5),
thermolysin-treated thylakoid fraction (lane 6), sonicated thylakoid
membrane fraction (lane 7), soluble contents of thylakoid lumen (lane 8).
(
B
) Western blot analysis of stromal (lane S) and thylakoid lumen (lane L)
fractions of
Arabidopsis
chloroplasts by Coomassie staining (left), anti-
AtFKBP13 (middle), and anti-Plastocyanin (PC) antibodies (right).
Approximate molecular masses (kDa) are shown on the side.


Sonication of the thylakoid

fraction liberated the 13 kDa polypeptide in a soluble
form

(Fig. 4-6A, lane 8), indicating that the protein is located in

the thylakoid lumen.
From this experiment, it was concluded that

AtFKBP13 is a previously uncharacterized
thylakoid lumen protein.

Chapter 4 Results and Discussion

81
The N-terminal extension of AtFKBP13 has the characteristic

features of a
thylakoid lumen protein presequence (Fig.4-7).

It is bipartite, with the first region being
hydrophilic in

nature and enriched in basic and hydroxylated residues, which

are features
of a chloroplast envelope-transfer signal [Keegstra and Cline, 1999].

The twin arginine
motif is present in

the presequence of AtFKBP13 and is followed by a highly
hydrophobic

domain ending with AXA. These features suggest that AtFKBP13

may be
translocated into the thylakoid lumen by a pH-dependent

pathway.

MSSLGFSVGT CSPPSEKRKC RFLVNNSLNK AEAINLRNKQ KVSSDPELSF 50
▼
AQLSSCGRRE AIIGFGFSIG LLDNVSALAE TTSCEFSVSP SGLAFCDKVV 100

GYGPEAVKGQ LIKAHYVGKL ENGKVFDSSY NRGKPLTFRI GVGEVIKGWD 150

QGILGSDGIP PMLTGGKRTL RIPPELAYGD RGAGCKGGSC LIPPASVLLF 200

DIEYIGKA

Figure. 4-7
Sequence of precursor
AtFKBP13
protein. The amino acid
sequence of the AtFKBP13 protein deduced from the cDNA. The double
arginine motif is shown as bold and highlighted. The thylakoidal
processing peptidase site (AxA) is shown in italics and the arrow indicates
the putative cleavage site. The mature protein sequence is shown in black.





Chapter 4 Results and Discussion

82
4.2.2 AtFKBP13 precursor interacts with the Rieske protein

To function as chaperone or other regulatory entities, immunophilins

often
interact with their target proteins [Pratt et al., 2001; Harrar et al., 2001].

To identify a
putative target for AtFKBP13, a yeast

two-hybrid screening procedure was used. The
precursor of AtFKBP13 was

used as bait in fusion with the Gal4 DNA-binding domain to
screen

an
Arabidopsis
cDNA library in a Gal4 activation-domain vector.

Among the
AtFKBP13 interacting clones that were sequenced,

a number of them encoded the Rieske
protein of various lengths

fused in frame with the activation domain. The longest one

had
four amino acid truncations in the N terminus. The Rieske protein

is an essential subunit
of the cytochrome
bf

complex in the photosynthetic

electron transport chain [Hope,
1993]. It is located in the thylakoid

with a transmembrane domain and a soluble region in
the lumen

[Hope, 1993]. As AtFKBP13 is located in the lumen, the Rieske protein may

serve as a physiological target for AtFKBP13.


A search of the
Arabidopsis
Expressed Sequence Tag (EST) database identified
an EST encoding

the full-length precursor of the Rieske protein. This EST39G11T7
(GenBank

accession no. AJ243702) was sequenced on both strands to confirm

its identity
and was used in subsequent experiments to determine

the interacting domains of the
AtFKBP13 and the Rieske proteins. A

number of cDNA fragments encoding various

domains of the AtFKBP13

or Rieske protein were fused in frame with either the DNA-
binding

or activation domain of the Gal4 protein in the vectors. Different

combinations of
these constructs were co transformed into yeast

strain Y190. The interactions were
studied

Chapter 4 Results and Discussion

83
by the growth on selection

medium and filter lift assay and quantified by the β-
galactosidase

activity. The precursor of AtFKBP13 interacts with the precursor,

mature,
and lumen domain but not with the transit peptide of the

Rieske protein. When the
chloroplast envelope signal peptide

was deleted from AtFKBP13, the truncated protein

retained interaction

with the Rieske protein. However, the mature form of AtFKBP13
lacking

the entire transit peptide did not interact with the full-length

or any domain of the
Rieske protein. The transit peptide

of AtFKBP13 interacted with the precursor and mature
form of Rieske,

although the interactions were significantly weaker compared

with the
interaction between the AtFKBP13 precursor and the Rieske

protein. The chloroplast
envelope signal or thylakoid-targeting

signal alone did not interact with the full-length or
any domain

of the Rieske protein. The transit peptide

of AtFKBP13 is required and, to a
certain degree, sufficient

for interaction with the Rieske protein. The mature region,

more
specifically, the lumen domain, of the Rieske protein is sufficient

for interaction.








Figure 4-8.
Interaction between AtFKBP13 and the Rieske protein
determined by
in vitro
protein interaction assays. GST (lanes 1 and 5),
GST-Rieske precursor (lanes 2 and 6), GST-Rieske mature form (lanes 3

Chapter 4 Results and Discussion

84
and 7), and GST-Rieske lumen domain (lanes 4 and 8) were immobilized
on glutathione beads and used to "pull-down" the purified precursor (lanes
1–4) or mature (lanes 5–8) form of AtFKBP13. The approximate
molecular masses (kDa) are shown on the right side. (
A
) An immunoblot
of co purified products probed with anti-AtFKBP13. The arrows on the

left and right sides indicate the position of the precursor and mature form
of AtFKBP13, respectively. (
B
) A Coomassie blue stained gel shows the
amount of each bait protein used in the pull-down experiment.

The interaction between AtFKBP13 and the Rieske protein
in vitro
was also
tested by protein–protein interaction assay. As shown

in Fig. 4-8, the precursor, mature,
or lumen domain of the Rieske protein

co purified with the precursor but not with the
mature form

of AtFKBP13 (Fig. 4-8A). When GST was used as an affinity agent,

neither
the precursor nor the mature form of AtFKBP13 was co purified

(Fig. 4-8A, lanes 1 and
5). The AtFKBP13 antibodies also reacted

with an unknown 33 kDa protein, associated
only with the preparation

of the GST-Rieske mature protein (Fig. 4-8A, lanes 3 and lane

7). The amount of GST and other bait proteins used in the experiment

are shown in Fig.
4-8B. Some GST was detected in the preparations

of Rieske precursor and mature protein
fusions, suggesting

that a small portion of GST fusion proteins were degraded (Fig. 4-8B,
lanes 2, 3, 6, and 7).


Because the precursor, but not the mature form, of AtFKBP13

interacted with the
Rieske protein, and the precursor of AtFKBP13 was

not detectable by the western blot in

Chapter 4 Results and Discussion

85
Arabidopsis
plants, it was

difficult to study the
in vivo
interaction of the two partner
proteins

by using immunoprecipitation. Indeed, the AtFKBP13 mature protein was
precipitated by using an AtFKBP13 antibody but did

not detect any Rieske protein that
was co purified. In a similar

manner, a Rieske antibody was used to precipitate the Rieske
protein

but AtFKBP13 was not co purified. This further

supports the idea that the two
mature proteins most probably

do not interact with each other.
4.2.3 AtFKBP13 down-regulates accumulation of Rieske protein

AtFKBP13 is localized in the thylakoid lumen, and the Rieske protein

is localized
in the thylakoid membrane with a soluble domain

in the lumen, making it physically
possible for the two mature

proteins to interact. However, our results suggest that these

proteins probably interact before they are targeted to their

final destination.

Figure 4-9.
Rieske protein accumulation in AtFKBP13 silenced plants.
Western blot analysis of AtFKBP1
3
RNAi (lanes 1 and 3) and control
plants (lanes 2 and 4) with anti-Rieske (lanes 1 and 2) or anti-PC (lanes 3
and 4) antibody.

Chapter 4 Results and Discussion

86
What is the functional relevance of interaction

between the two protein partners?
To address this question, a "reverse genetics" approach was undertaken to suppress the
expression

of AtFKBP13 in transgenic plants and the consequence

of this manipulation to
the Rieske protein was examined.
If the AtFKBP13 interaction with the Rieske protein is functionally

relevant, it is
expected that AtFKBP13 silencing would alter

the level or function of the Rieske protein.
Because the interaction

may occur before the proteins are imported to the final
destination,

we suspected that AtFKBP13 might regulate the accumulation

of the Rieske
protein. As the Rieske protein is an essential subunit

of the cytochrome
bf
complex in the
photosynthetic electron-transport

chain, changes in the Rieske protein may affect the
photosynthetic

electron transport. It is possible that the Rieske

protein is modified in a
more subtle manner.
The

level of Rieske protein was examined in the control and RNAi plants by

western

blot analysis and found a significant difference in the accumulation

of the Rieske
protein. As shown in Fig. 4-9, the RNAi plants produced

higher levels of the Rieske
protein (Fig. 4-9, lane 1) as compared

with the control plants (Fig. 4-9, lane 2). Statistical
analysis

on eight RNAi lines, eight empty vector control lines, and wild-type

plants
indicated that the Rieske protein in the AtFKBP13-silenced

plants produced about 70%
more Rieske protein (Fig. 4-9, lanes 1 and 2). As a control western

blot analysis with a
plastocyanin antibody did not reveal any significant

difference in the accumulation of PC
in the RNAi

and control plants (Fig. 4-9, lanes 3 and 4). RNA gel blot analysis

did not

detect any significant difference in the Rieske mRNA

levels among RNAi and control

Chapter 4 Results and Discussion

87
plants, suggesting

that AtFKBP13 affects the level of the Rieske protein by a post-
transcriptional

process.
4.2.4 Mature FKBP13 is a target for reduction by thioredoxin

The possibility of thioredoxin-linked reduction of AtFKBP13 arose from the
finding of two solvent exposed disulfides in its structure and from recent evidence that
AtCYP20-3, a functionally related protein present in the chloroplast stroma, has been
found to be a thioredoxin target [Motohashi et al., 2001, 2003]. We tested this possibility
by applying the NADP/thioredoxin reducing system of
E.coli
combined with
fluorescence gel analysis using mBBr as a thiol-specific probe. In this assay, AtFKBP13
was incubated with thioredoxin and NTR in the presence of NADPH, a source of
reducing equivalents. The newly formed –SH groups were labeled with mBBr, the
proteins separated by SDS-PAGE and the fluorescence was recorded. The reduction of
disulfide(s) by the NADP/thioredoxin system was reflected by an increase in the
fluorescence of the treated protein (Fig. 4-10). Owing to the absence of additional free
cysteines in the sequence of AtFKBP13, the protein alone did not react with mBBr (Fig.
4-10, lane 1, control treatment).

Chapter 4 Results and Discussion

88







Figure 4-10.
Reduction of AtFKBP13 by the NADP_thioredoxin system
from
E. coli
. Ctrl- control (FKBP13 alone); Cpl- complete thioredoxin
system (NADPH+NTR+Trx) plus FKBP13; Trx- thioredoxin; -Trx, -NTR,
and -NADPH lanes indicate the complete thioredoxin system in which
each of these components was individually omitted. Protein refers to the
complete treatment in which the gel was stained with Coomassie blue.

By contrast, incubation of AtFKPB13 with the NADP/thioredoxin system for 20
minutes resulted in marked fluorescent labeling indicating disulfide reduction (Fig. 4-10,
lane 2, complete treatment). Reduction was not observed when any one of the
components of the system was omitted, i.e., thioredoxin, NTR or NADPH (Figure. 4-10,

lanes 3-5).
A similar experiment performed with mutant forms of AtFKBP13 in which either
the N- or C-terminal disulfide (Cys5/17 or Cys106/111) was replaced by serine
established that both S-S bonds are fully reduced by thioredoxin. Additional assays using

Chapter 4 Results and Discussion

89
a low concentration of DTT (0.5 mM) as hydrogen donor and different types of
thioredoxin (
E.coli
, chloroplast
f
and
m
, extraplastidic
h
) indicated the chloroplast
m
-type
to be the most efficient. DTT alone was a poor reducing agent under these conditions,
especially for cleaving the C-terminal disulfide.
4.2.5 Mature AtFKPB13 PPIase activity is regulated by redox status
Having structurally resolved the position of the disulfide bridges and established
their reducibility by thioredoxin, we sought to investigate the effect of redox status on
catalysis of the PPIase reaction by AtFKBP13. In contrast to AtCYP20-3, the isolated
oxidized form of AtFKBP13 efficiently catalyzed the peptidyl-prolyl
cis-trans

isomerization of a chromogenic synthetic pentapeptide in a standard PPIase assay (Table

4-2).

Table 4-1.
Requirement for both disulfide (S-S) groups for the PPIase
activity of FKBP13.
FKBP13 kcat_Km, s_1
_M_1
WT

11.6

No Cys

3.1
+DTT

10.2
+Trx
m

9.2
DTT + Trx
m
5.3


Chapter 4 Results and Discussion

90
To ascertain the importance of each disulfide bridge to total PPIase activity,

mutant forms of AtFKBP13 were constructed which lacked either one or both cysteine
pairs. AtFKBP13 proteins containing mutations in the amino (C5S/C17S) or carboxy
(C106S/C111S) terminal cysteine pairs showed, respectively, about 30 and 55%
reduction in catalytic efficiency (
k
cat
/K
m
). This result suggests that the surface-exposed C-
terminal disulfide bridge may be more important for catalysis and may constitute the
major site of regulation by thioredoxin.
Activity was reduced almost 80% in the quadruple cysteine mutant (C5S/C17S
and C106S/C111S). Following incubation and reduction of AtFKBP13 by
Arabidopsis

thioredoxin
m
and DTT, the PPIase activity was reduced by 55%, whereas DTT alone
had a minimal effect. These results suggest that AtFKBP13 PPIase activity can be
modulated by the redox state
in vitro
.

4.2.6 Effect of pH and Mg
2+
on the Activity
Interestingly, unlike certain enzymes of the Calvin cycle (e.g., fructose
bisphosphatase), neither pH nor Mg
2+
appeared to be a factor in determining the extent of

activation by thioredoxin [Buchanan, 1980]. AtFKBP13 was found to remain
catalytically active down to pH 5, suggesting that this enzyme is functionally adapting to
the acidic conditions of the thylakoid lumen (Table 4-2).




Chapter 4 Results and Discussion

91

Table 4-2.
Effect of Mg
2+
and pH on the AtFKBP13 activity

FKBP13

kcat_Km, s_1
_M_1
WT 11.6

WT+ Mg
2+
11.5
11.4
11.2
10.5

5.6



4.3 NEED FOR THE REDUCED STRUCTURE
In general, redox potential and protein stability are thermodynamically linked for
the thiol-disulfide oxidoreductases [Creighton, 1986]. The effect of stabilization or
destabilization of the reduced and oxidized forms as well as the enzyme-substrate
complexes is to modulate the redox potentials of these proteins. The analysis of the
molecular details of AtFKBP13 demonstrates that the importance of the active site
residues is dependent on the local interactions and small conformational changes in the
active site modulate the redox potential by affecting the stability of each of the oxidized,
reduced and mixed-disulfide forms.
Apart from this, a structural comparison of oxidized and reduced AtFKBP13 will
shed light on the physicochemical properties of the enzyme that will help us understand if
there is any disulfide mediated structural changes. On the other hand, the structural

Chapter 4 Results and Discussion

92
comparison of the oxidized and reduced forms of human and
E. coli
thioredoxin and
E.
coli
glutaredoxin revealed only slight local differences [Katti et al., 1990; Qin et al.,
1994; Dyson et al., 1990; Jeng et al., 1994]. Overall, knowledge of the three-dimensional
structure of reduced AtFKBP13 is required for understanding the extraordinary properties
of this enzyme at a molecular level.

4.3.1 AtFKBP13-(SH)2 Redox active site
DDT (0.5mM) treated form of this enzyme has almost native activity (similar to
oxidized form) but when crystallized at higher concentration of DTT used interestingly,
the redox active cysteines (Cys5, Cys17, Cys106, Cys111)are in the reduced form and do
not make any disulfide bonds between the corresponding S atoms. The conclusions of
this work depend on the detailed structure of the redox active site, especially the distance
between the S atoms of Cys5–Cys17 and Cys106–Cys111.


A B


Figure 4-11.
Comparison of 2
F
o
-
F
c
maps contoured at the 1.2
σ
level at
the N-terminal disulfide region of oxidized (
A
) and reduced (
B
) structures.
The amino acid residues in the region are numbered.

Chapter 4 Results and Discussion

93
The average distance between the sulphur atoms of Cys5 and Cys17 from 5
molecules is 5.18 Å and the same for Cys106 and Cys111 is 2.81 Å. (as opposed to the
ideal disulfide-bond distance of 2.03 Å). They are clearly separated in the electron
density map (Fig. 4-11). The observed average distances were obtained after refinement
was carried out without any bonding restraints between the two S atoms.
The distances indicate that there is no covalent bond and that the cysteine residues
are in the reduced state. The difference Fourier map, calculated with the
F
o
-
F
c

coefficients, show no significant features in the vicinity of the S atoms. In order to
confirm this result, 15 cycles of refinement were carried out under a disulfide-bonding
restraint between the S atoms. The restraint caused the atoms to move within 2.1 Å of
each other, while a negative peak appeared at the 2σ level in the
F
o
-
F
c
map between the
two atoms. Corresponding positive peaks were observed on the opposite sides of the
atomic centers. Upon releasing the bonding restraint and further refinement the S atoms
moved back to their original non-bonded positions and the difference map no longer
showed any significant peaks in the vicinity.
4.3.2 Comparison with the oxidized AtFKBP13 structure

The oxidized form of AtFKBP13 [Gopalan et al., 2004] has been discussed
previously. Comparison of the reduced model and the oxidized model reveals large shifts
in the overall shape of the active site regions. Superimposition of the reduced AtFKBP13
onto the corresponding residues of the oxidized structure gives an r.m.s.d. of 1.22 Å. The
backbone fold is identical in the structures of AtFKBP13-(SH)2 and AtFKBP13-S2, as
shown in Fig. 4-12. We emphasize that these structures have been superimposed on the

Chapter 4 Results and Discussion

94
backbone of the entire molecule (residues 5–129) and thus the excellent correspondence
among the secondary structure elements in the two structures is not the product of local
superposition. The major contribution to this significant difference is coming from the
redox active C-terminal loop. The catalytic domains are significantly shifted and twisted
compared with the oxidized structure and this results in differences of up to 10 Å.


Figure 4-12.
Superimposition of the backbone C
α
traces of AtFKBP13-
(SH)2 and AtFKBP13-S2, colored blue and magenta, respectively, for
residues 5–129. The redox active site Cys sulfur atom is shown as a
yellow sphere.
C
N

Chapter 4 Results and Discussion

95

There are no major rearrangements within the catalytic domains. Superimposition
of only the catalytic domains of the reduced structure and the corresponding parts of the
oxidized structure gives an r.m.s.d. of 1.478 Å for 16 C
α
atoms (residue numbers 100-
116).
In Fig. 4-12, the largest changes are in the position of the S atoms of the key
cysteine residues: each S atom in the two thiol groups is shifted by about 0.5 Å compared
with the disulfide form. The hydrogen-bond network in the active site is affected by the
oxidation state of the thiol groups. The oxidized Cys5 S atom forms a strong hydrogen
bond with the Cys17 amide, while the thiol S atom in the present reduced structure is
located farther away. Cys17 makes other short contacts: as a proton donor with the
carbonyl O atom of Thr90 (3.0 Å) and Cys17 Sγ with N of Lys19, Arg89 and Glu6. The
reduction of the active site is also accompanied by a shift of the Cys17 S atom, which
forms a short hydrogen bond with the O atom of the Thr90 side chain. The side chain
conformations of the two cysteines are the same in AtFKBP13-(SH)2 and AtFKBP13-S2.
Reduction of the disulfide bond requires an increase in the distance between the sulfur
atoms of the two cysteines: the observed S–S distance in AtFKBP13-S2 Cys5–Cys17 and
Cys106–Cys111 are 2.04±0.01 and 2.01±0.01Å, respectively as expected for a disulfide
bond, whereas the corresponding distance is 5.18 Å and 2.81 Å for AtFKBP13-
(SH)2.The corresponding distance seen in reduced human thioredoxin is, surprisingly, 3.1
Å [Forman-Ka et al., 1991]. The backbone conformational change that occurs to
accommodate the increase in the radius of sulfur upon reduction and the breakage of the
disulfide bond also causes an increase in the C
α
–C
α
distances between the two cysteines,

Chapter 4 Results and Discussion

96
6.208 (Cys5-Cys17) and 3.83 Å (Cys106-Cys111) for AtFKBP13-S2, compared with
6.988 (Cys5-Cys17) and 3.91 Å (Cys106-Cys111) for AtFKBP13-(SH)2, and the thiol of
Cys5 is more solvent exposed in AtFKBP13-(SH)2. All surface features identified in the
oxidized structure are also present in reduced AtFKBP13. Their general properties are
nearly the same within allowable differences.
There are some apparent changes in the local structure in the active site region
upon the change of the reduction state. Small dihedral angle differences are also observed
in the region of residues 107-109, which form a loop structure that closely contacts the
active site.
A significant difference in secondary structures between oxidized AtFKBP13-S2
and AtFKBP13-(SH)2 is found in α2, which in oxidized AtFKBP13 includes residues 50
to 53 and is absent in AtFKBP13-(SH)2. This region is converted to a loop. Another
region where there is a difference in secondary structure is α1, which is one residue
shorter in AtFKBP13-(SH)2. Interestingly, α2 in the oxidized structure contains the
PPIase conserved residues namely Ser50 and Arg53. These shifts in secondary structures
may account for the loss of PPIase activity. However, both variants occur in each of the
conformers although with different proportions, notably, the structure of oxidized
AtFKBP13 is better defined in this region.
In addition to these changes in secondary structures, a few side-chains have large
average displacement, in particular Asp48. In AtFKBP13-S2, Asp48 forms a salt bridge
with Arg53. In reduced AtFKBP13, Asp48 is no longer at a suitable distance for such