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Journal of Bacteriology, May 2000, p. 2453-2460, Vol. 182, No. 9
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Probing the CD Lumenal Loop Region of the D2
Protein of Photosystem II in Synechocystis sp. Strain PCC
6803 by Combinatorial Mutagenesis
Anna T.
Keilty,*
Svetlana Y.
Ermakova-Gerdes, and
Wim F. J.
Vermaas
Department of Plant Biology and Center for
the Study of Early Events in Photosynthesis, Arizona State
University, Tempe, Arizona 85287-1601
Received 14 December 1999/Accepted 13 February 2000
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ABSTRACT |
The CD lumenal loop region of the photosystem II reaction center
protein D2 contains residues involved in oxygen evolution. Since
detailed structural information about this region is unavailable, an
M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 in Synechocystis sp. strain PCC 6803. The CD loop coding
region contains close to 100 nucleotides, and for effective
mutagenesis, it was subdivided into four regions of seven to eight
codons. A gain-of-function selection protocol was employed such that
all mutants that were selected contained a functional D2 protein. In
this way, conservation patterns of residues along with numbers and
types of amino acid substitutions accommodated at each position for
each set of mutants would indicate which residues in the CD loop may
play important structural and functional roles. Results of this study
have substantiated the importance of residues previously studied by
site-directed mutagenesis such as Arg180 and His189 and have identified
other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In
addition, the pliability of the CD loop was further tested using
deletion and D1-D2 substitution constructs in M13. This showed that the
length of the loop was important to its function, and in two cases, D2
could accommodate homologous sequences from D1, which forms a
heterodimer with D2 in photosystem II, but not the other way around.
This study of the CD loop in D2 provides valuable clues regarding the
structural and functional requirements of the region.
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INTRODUCTION |
Photosystem II (PS II), located in
the thylakoid membrane of plants and cyanobacteria, converts light
energy to chemical energy in a unique photochemical process that
results in the splitting of two molecules of water to produce four
reducing equivalents and protons and to release one molecule of oxygen.
The oxygen-evolving complex (OEC), which is located on the donor side
of PS II, oxidizes water via a complex containing manganese. Each
photon of light that oxidizes the primary donor of PS II, P680,
extracts one electron from the OEC, and after four positive charges
have been accumulated, two water molecules are oxidized and oxygen is
released. Cofactors involved with electron transfer in PS II are
associated with the reaction center proteins D1 and D2. The electrons
extracted from the manganese cluster are transferred to
P680+ via the intermediate electron carrier, YZ
(tyrosine 161 of D1). Site-directed mutagenesis studies confirmed that
this tyrosyl residue is in redox contact with the primary donor, P680,
and the OEC (18). Symmetrically arranged in relation to
YZ is YD, tyrosine 160 of D2. This redox-active
Tyr residue forms a dark-stable radical that is not directly involved
in the reduction of P680+ by the water-splitting complex
(5, 32). These functional differences between YZ
and YD may be related to regional structural differences in
the protein environment surrounding the radicals and the OEC.
One of the regions of PS II that possibly interacts with the OEC and
that contains residues close to YZ and YD as
well as to P680 is the CD lumenal loop region of the D1 and D2
proteins. The CD lumenal loop that is present in both of these proteins connects the transmembrane helix C, containing YZ or
YD, with helix D, which contains His residues that are
likely to be ligands to the primary donor P680 and the electron
acceptors QA and QB (reviewed in reference
34). Based on comparison with the reaction center
from purple bacteria, the CD loop in the two proteins is expected to
contain a helical region that interacts with the lumenal edge of the
thylakoid membrane and that may contain a central ligand to accessory
chlorophyll in the reaction center.
The CD loop of D1 and D2 has been studied using site-directed
mutagenesis, and residues Asp170, Glu189, and His190 in D1 along with
Gln164, Arg180, and His189 in D2 have been identified to interact with
the OEC, the redox-active tyrosines, and/or P680. Asp170 is thought to
be a Mn ligand or to be involved with assembly of the cluster (4,
21). Another D1 residue, Glu189, also seems to play a structural
role in stabilizing and assembling the Mn cluster (4),
possibly through its involvement in a network of hydrogen bonds that
influence both YZ and the Mn cluster (7). Alterations in His190 of D1 and His189 of D2 indicate that these residues may serve as proton acceptors upon Tyr oxidation (4, 11,
26, 28). Mutational studies indicate that Arg180 of D2 affects
YD and P680 function and, based on the purple bacterial structure, may be involved in accessory chlorophyll binding
(16). Mutations in Gln164 have been shown to alter the
electron paramagnetic resonance (EPR) signal of YD
(28).
While these residues appear to be functionally important, little is
known about the structural requirements in the CD loop and about which
other residues in the region are critical for the stable functioning of
the donor side of PS II. One approach that has been used to determine
functionally and structurally important residues in essential regions
of proteins with unknown structure is combinatorial mutagenesis
(3, 22).
Combinatorial mutagenesis is a technique that allows for simultaneous
introduction of many random, degenerate mutations in a large protein
domain. By coupling the mutagenesis with a selection protocol that
screens only for functional proteins, the data obtained can be used to
gain information on the structurally and functionally important
residues in a selected region (8, 20, 36). Various combinatorial techniques have been successfully used to study structure
and function in photosynthetic reaction centers. The residues required
for the QB binding niche and the region surrounding YZ in the D1 protein of Synechocystis sp. strain
PCC 6803 have been studied using a PCR-based combinatorial method
(12, 13). To generate a set of functional mutants for
electron transfer studies, combinatorial cassette mutagenesis was used
to randomly mutagenize regions of the L and M subunits near the
active-branch monomeric bacteriochlorophyll in Rhodobacter
capsulatus (23). Moreover, an M13-based combinatorial
method was recently used to study a conserved region in the E loop of
the CP47 chlorophyll binding protein in Synechocystis sp.
strain PCC 6803 (27).
In this study, we applied combinatorial mutagenesis, also using an
M13-based technique, to develop a set of mutants in the CD loop of the
D2 protein in Synechocystis sp. strain PCC 6803 to gain
insight into structure and function relationships within the CD loop
region of this protein. As a transformable, oxygenic prokaryote,
Synechocystis sp. strain PCC 6803 is a simple model system
for studying PS II structure and function by genetic manipulation (35). Moreover, knowledge gained from this system is
directly applicable to eukaryotic plant systems.
For effective mutagenesis, the CD loop was divided into four regions of
seven to eight amino acids (see Fig. 1). Within each region, the codons
were replaced with random sequences in psbDI (the only D2
gene in the system that was studied) and photoautotrophic mutants were
selected. In addition, seven- to eight-residue regions of the CD loop
of D1 and D2 were replaced by the homologous regions of D2 and D1,
respectively. Since the two proteins have only nine residues in common
in the CD loop and yet appear to have structural symmetry, substituting
homologous D1-D2 sequences provides insight into the residues that
contribute to functional differences between D1 and D2. In addition to
development of combinatorial and substitution mutants in each region,
the CD loop was further probed by attempting to isolate
photoautotrophic deletion mutants for each of the four regions. This
comprehensive genetic study of the CD lumenal loop in the D2 protein of
Synechocystis sp. strain PCC 6803 identifies important
residues not readily determined by primary sequence analysis and
provides a unique set of mutants that contribute valuable information
on regional primary structures that can support oxygen evolution in the
PS II complex.
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MATERIALS AND METHODS |
Acceptor strains.
The genome of Synechocystis sp.
strain PCC 6803 carries two genes that code for the D2 protein,
psbDI and psbDII, and three genes that code for
the D1 protein, psbAI, psbAII, and
psbAIII. To achieve successful mutagenesis and to be able to
utilize gain-of-function selection protocols, it was necessary to
develop acceptor strains for transformation that lacked all but one
psbA and/or psbD gene, and where the remaining
copy carried a CD loop deletion. In the D2
CD strain, the
psbDII gene was replaced by a spectinomycin resistance
cassette (33), while the psbDI gene in this
strain carries a CD loop deletion resulting in the loss of residues
G163 to P195 of D2 and has a kanamycin resistance cassette downstream (33; I. Shalak and W. Vermaas, unpublished data).
Therefore, this strain is unable to make functional D2 protein.
The Synechocystis sp. strain PCC 6803 D1CD strain, which
is unable to make a functional D1 protein, lacks psbAI and
psbAIII and carries a deletion of the region coding for the
CD loop in psbAII. The psbAI gene in the D1CD
strain was replaced by a spectinomycin resistance cassette, and the
psbAIII gene was replaced by a chloramphenicol resistance
cassette (6). The CD loop deletion in psbAII was introduced into this psbAI-psbAIII double-deletion strain in
the following way. First, the plasmid pAIIEr was constructed by cloning a 2.0-kbp genome fragment carrying most of psbAII (starting
at an Eco47III restriction site located 27 bp downstream of
the start codon) and ~900 bp of the downstream region into pUC19.
This was followed by insertion of an erythromycin resistance cassette
into the StuI site ~300 bp downstream of psbAII
(Fig. 2). The plasmid was then linearized by KpnI, which
cuts in psbAII at a site coding for residues in the CD loop,
and digested with exonuclease Bal 31. After ligation, the
plasmid pAIIErDEL, containing a CD loop deletion in psbAII
leading to the loss of residues V157 to G201 of D1, was obtained. This
deletion plasmid was then used to transform the
psbAI-psbAIII deletion strain; after segregation, the
D1CD strain of Synechocystis sp. strain PCC 6803 was
obtained. Since both D1CD and D2CD lack functional PS II, they
are obligate photoheterotrophs.
Primer design.
Since the CD loop region is too large to be
effectively mutagenized as a whole, the region was divided into four
domains of seven or eight amino acid residues each (Fig.
1). Three sets of primers (deletion,
D1-D2 substitution, and combinatorial [degenerate] primers) were
synthesized for each of the eight regions (Fig. 1). Deletion primers of
40 to 45 nucleotides were designed such that approximately half of the
sequence corresponded to the wild-type sequence immediately upstream of
the deletion region and the remaining half corresponded to the
wild-type sequence immediately downstream of the deletion region.
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FIG. 1.
(A) Protein sequence of the CD lumenal loop region of
the D1 and D2 proteins in Synechocystis sp. strain PCC 6803 showing the subdivision of each loop into four regions of seven or
eight codons (regions numbered 1 to 4 for D1 and 5 to 8 for D2). (B)
DNA sequence of psbDI along with an illustration showing
three primers that were designed to introduce changes into region 7 in
psbDI. Similar sets of deletion, D1-D2 substitution, and
combinatorial primers were designed for each region. Nucleotides in the
substitution primer shown in italics are the substituted bases from the
corresponding region of the psbAII gene. Shown underlined
and in boldface is a silent substitution introduced to enhance
primer-template annealing. Degenerate primers were synthesized with an
equimolar mix of all four nucleotides (shown as N) at the first and
second nucleotide positions of each codon and an equimolar mix of
guanines plus cytosines (shown as S) at the third (wobble) position.
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D1-D2 substitution primers ranged in length from 51 to 59 nucleotides.
The substitution primers for each region contained
the wild-type
sequence for
psbAII or
psbDI flanked by 13 to 19
nucleotides upstream and downstream of the wild-type sequence
of the
opposite gene. The substitution sequence in the middle
of the primer
was modified where possible at the third codon position
to improve
hybridization to the other gene sequence without altering
the amino
acid
sequence.
Combinatorial oligonucleotide primers ranging from 62 to 69 nucleotides
were synthesized with 18 to 24 nucleotides of the
upstream and
downstream wild-type sequence flanking a 21- to 24-nucleotide
random
degenerate sequence for each region of the CD loop. The
degenerate
region of the primers was synthesized with an equimolar
mixture of A,
T, C, and G at the first and second codon positions
and an equimolar
mixture of G and C at the third (wobble) position.
By using this
protocol, two of three stop codons were eliminated.
Moreover, this led
to a more equalized probability for the incorporation
of the various
amino acids (
9,
10).
Mutagenesis procedure.
The procedure used to generate
combinatorial DNA for transformation is based on established protocols
for in vitro mutagenesis of single-stranded bacteriophage templates
(2, 27, 33). Two M13mp19 bacteriophage constructs,
M13mp19/psbAII and M13mp19/psbDI, were used as
templates for mutagenesis. M13mp19/psbDI was generated as
described in reference 33. M13mp19/psbAII
was constructed by cloning a 1.2-kbp EcoRI/XbaI
psbAII fragment cut from pAIIEr (Fig.
2) into the polylinker region of M13mp19.
These bacteriophage constructs were propagated in Escherichia
coli strain CJ236 (dut, ung negative) to
allow for incorporation of uracil into the templates as a method for
preferentially selecting against the template strand after mutagenesis
(14). The procedure used for transformation of the
appropriate Synechocystis sp. strain PCC 6803 acceptor strain (D1CD or D2CD) with deletion, D1-D2 substitution, and degenerate combinatorial M13 phage constructs was based on established protocols (27, 31, 37).
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FIG. 2.
Map of the pAIIEr plasmid used in developing the D1CD
strain. The plasmid carries a Synechocystis sp. strain PCC
6803 genomic fragment (shown in black) of ~2.0 kbp extending from the
Eco47III site located 27 bp downstream of the
psbAII start codon to the HindIII site ~900
bp downstream of the psbAII stop codon. The genome fragment
is interrupted downstream of psbAII by an erythromycin
resistance cassette which was introduced into the StuI site
~300 bp downstream of the coding sequence. The CD loop deletion
introduced into this plasmid to make pAIIErDEL, which was used to
transform Synechocystis sp. strain PCC 6803 to develop the
D1CD strain, is indicated.
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To introduce seven- to eight-codon deletions or D1-D2 substitutions,
deletion and D1-D2 substitution primers were hybridized
to the
appropriate uracil-containing M13mp19 template containing
the wild-type
psbAII or
psbDI gene. The second strand was
synthesized
in vitro using methylated dCTP, which results in a
double-stranded,
hemimethylated M13 recombinant phage. The recombinant
M13 is subsequently
nicked with restriction enzyme
MspI or
Sau3AI, which recognizes
hemimethylated DNA and cuts only
the nonmethylated wild-type strand
(
29). Upon introduction
of the heteroduplex M13 into
E. coli strain DH10B for
amplification, the nicked template strand is
preferentially destroyed,
providing a second selection against
the template strand, thus
increasing the yield of M13 mutants
among the plaques. Single-stranded
M13 was then isolated from
individual plaques and prepared for
sequencing to verify the presence
of the deletion or substitution.
Subsequently, double-stranded
M13 DNA carrying the desired deletions
and substitutions was isolated
for transformation into the appropriate
Synechocystis sp. strain
PCC 6803 acceptor
strain.
Degenerate combinatorial primers were hybridized to single-stranded
uracil-labeled M13mp19/
psbDI deletion templates. Following
in vitro synthesis with unmethylated dCTP and phage amplification
in
E. coli, the isolated pools of double-stranded phages were
directly transformed into the
Synechocystis sp. strain PCC
6803
D1
CD acceptor strain. The deletion template strands for each
region were unable to sustain photoautotrophic growth, thus obviating
the need for further selection at the level of M13 and allowing
for
direct selection of photoautotrophically competent transformants
in the
Synechocystis sp. strain PCC 6803 acceptor strain. Colonies
of photoautotrophic transformants were visible within 1 to 2 weeks
after
transformation.
DNA sequencing.
The sequences of the
Synechocystis sp. strain PCC 6803 photoautotrophic
combinatorial mutants were determined by PCR amplification of the CD
loop region of psbDI from genomic DNA preparations of the
mutants. Single-stranded M13 deletion and substitution constructs were
prepared for sequencing using established protocols (14, 17). The region coding for the CD loop and its flanking regions (both as PCR products and as single-stranded M13 deletion and substitution constructs) were sequenced using an ABI 377 DNA sequencer.
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RESULTS |
Deletions and D1-D2 substitutions in the CD loop.
Eight
deletion and eight substitution constructs were introduced into the
appropriate acceptor strains of Synechocystis sp. strain PCC
6803 (Fig. 1). The aim of this part of the project was to answer two
basic questions: (i) is the length of the loop region important to its
function, and (ii) can a homologous region from D1 function in D2 and
vice versa? Photoautotrophic growth could not be restored by
transformation with any of the eight deletion constructs, indicating
that removal of seven to eight amino acids in the CD loop of either D1
or D2 does not allow for formation of a functional PS II complex. In
addition, replacement of any of the four D1 regions with corresponding
domains of D2 did not lead to photoautotrophic mutants. This indicates
that the primary sequence of the CD loop of D1 cannot accommodate major changes without a loss of PS II function. However, D2 was found to
functionally accommodate substitution (Fig. 1) by the homologous region
from D1 in regions 6 (Pro171 to Ile178) and 8 (Gly187 to Asn194),
whereas regions 5 (Gln164 to Ala170) and 7 (Phe179 to Gln186) could not
be replaced by the D1 analogs without a loss of photoautotrophic capacity.
The substitution mutant S6, which carried a homologous D1 sequence in
region 6 of the D2 protein, displayed a photoautotrophic
growth rate
similar to that of wild type (Table
1)
over a range
of light intensities. These data suggest that region 6 may
be
a linker region in D2 that has no functional role in redox activity
and oxygen evolution.
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TABLE 1.
Photoautotrophic doubling times at various light
intensities of substitution mutants where corresponding D1
sequences have been incorporated into region 6 (S6) and region 8 (S8) of D2 (Fig. 1)
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In contrast, the substitution mutant S8, carrying a homologous D1
sequence at residues 187 to 194 of D2, had a significantly
lower growth
rate than that of the control; the photoautotrophic
doubling time of
the S8 strain was ~60 to 80% longer than that
of the control (Table
1). The altered D2 region in the S8 mutant
includes the functionally
important residue His189, which is conserved
in D1 (His190) and was
therefore not changed in S8. However, the
decreased photoautotrophic
growth rate of S8 indicates that the
surrounding environment is
important for PS II function as
well.
Combinatorial mutants.
Photoautotrophic combinatorial mutants
in the CD loop of D2 were generated in regions 5, 7, and 8. Since the
S6 mutant showed a growth rate similar to that of the wild type, we
chose to focus our efforts on the remaining three regions in the CD
loop of D2. The combinatorial mutants isolated for regions 5, 7, and 8 along with their interesting features will be presented and discussed in the paragraphs below.
Region 5 (Gln164 to Ala170).
The primary sequences of the 45 combinatorial mutants that were obtained for region 5 are listed in
Table 2. This region is immediately
adjacent to helix C and, in terms of primary sequence, is closest to
YD. The most highly conserved residues in this set of
combinatorial mutants are Ser166 and Phe169. Both positions are 76%
conserved and have strict replacement requirements accommodating only
six and five different residues, respectively. The 166 position shows a
preference for small uncharged but mostly polar residues (Ser, Thr,
Asn, and Gly) or Ala or His. Position 169 strongly prefers a
hydrophobic residue (Phe, Leu, or Val), with His or Thr being found in
a few cases.
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TABLE 2.
Amino acid sequence of wild-type and combinatorial mutant
strains in region 5 of the CD lumenal loop of the
D2 proteina
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Another interesting residue in this region is Ala170. This residue is
conserved in 53% of the mutants, but at this position,
only small
uncharged but somewhat hydrophilic residues (Cys, Ser,
and Gly) can be
functionally accommodated. While Gln164 has been
shown to alter the EPR
signal of oxidized Y
D (
28), it is conserved
in
only 20% of our mutants, and the position can accommodate 10
different
residues, Gln, His, Met, Ser, Leu, Val, Gly, Ala, Thr,
and Phe.
Positions 165 and 167 are the least conserved in region
5, accommodating 16 and 14 different amino acids, respectively,
from large
to small and from hydrophobic to charged. Interestingly,
position 165 is unique in that it is the only position in the
CD loop in any of the
combinatorial mutants analyzed thus far
that has accommodated a Pro
residue.
Region 7 (Phe179 to Gln186).
Combinatorial mutants obtained in
region 7 have been listed in Table 3.
This region makes up part of the proposed lumenal helix of the CD loop
and contains Arg180, which was identified by site-directed mutagenesis
as interacting with both YD and P680 and which could not be
replaced by other residues without major functional consequences
(16). Surprisingly, this residue is conserved in only 43%
of the mutants shown in Table 3, but if Arg is absent at position 180, an Arg appears in position 184. This position is approximately one full
turn of the proposed 
-helix away from position 180.
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TABLE 3.
Amino acid sequence of wild-type and combinatorial mutant
strains in region 7 of the CD lumenal loop of the
D2 proteina
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The most conserved residue in the region is Phe179, which appears in
64% of the mutants and is replaced only by two residues,
Leu and Tyr.
The least conserved residue in region 7 is Gln186;
in none of the
combinatorial mutants of region 7 was a Gln residue
found at position
186, and a mild preference for hydrophobic residues
was
observed.
Region 8 (Gly187 to Asn194).
The primary sequences of the 34 combinatorial mutants in region 8 of D2 are listed in Table
4 and clearly substantiate the importance
of His189 in this region for sustaining photoautotrophic growth. This
residue is 100% conserved in the 34 mutants in this region. However,
note that the site-directed mutants H189L, H189Y, and H189Q can grow
photoautotrophically (26, 28), suggesting the importance of
neighboring residues in supporting the stable functioning of this
region of the CD loop.
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TABLE 4.
Amino acid sequence of wild-type and combinatorial mutant
strains in region 8 of the CD lumenal loop of the
D2 proteina
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Gly187 is proposed to be at the end of the CD lumenal helix (
25,
38). In our mutants, it is only 24% conserved, but in
our
collection of mutants, only five residues (Gly, Ala, Val,
Ser, and Thr)
were found to function at position 187. This shows
a distinct
preference for a small, uncharged amino acid at this
position, thus
supporting the idea that the helix may end at position
187.
Two other residues in the region, Trp191 and Thr192, are not highly
conserved in the combinatorial region 8 mutants, but they
can be
replaced by a limited number of amino acids. The least
conserved
position in region 8 is Trp191 (conserved in only one
of the
combinatorial mutants), and this position was found to
accommodate
either another aromatic residue (Phe or Tyr) or a
hydrophobic residue
(Val, Ile, Leu, or Met). Thr192 also shows
low conservation but
stringency in replacement. This position
is 10% conserved, and it
accommodates only seven different residues
being replaced by
hydrophobic amino acids (Leu, Ile, Val, Ala,
and Met) or by
Ser.
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DISCUSSION |
Combinatorial mutagenesis, which typically uses gain-of-function
selection protocols, allows for rapid, efficient identification of
structural or functional amino acid residues in proteins of unknown
structure (8, 22, 27). In this study, combinatorial mutagenesis was used to probe the CD lumenal loop region of the D2
protein in Synechocystis sp. strain PCC 6803. The CD loop is located on the donor side of PS II, and little detailed structural information is available for this region, as the primary structure similarity to the purple bacterial reaction center is particularly poor
in this protein domain. The selection method utilized the concept of
functional complementation whereby obligate photoheterotrophic Synechocystis sp. strain PCC 6803 acceptor strains were
restored to photoautotrophy by transformation with M13 constructs,
resulting in a pool of functional mutants from which structure-function information could be gained.
The CD loop was investigated from three perspectives aimed at
addressing these questions: (i) is the length of the CD loop region
important to its function, (ii) can a homologous region from D1
function in D2 and vice versa, and (iii) which residues in the CD loop
of D2 can be functionally replaced? The first two questions were
answered by experiments in which Synechocystis sp. strain
PCC 6803 acceptor strains were transformed with deletion and
substitution M13 constructs. Since no deletion constructs led to
restoration of photoautotrophy, no major (seven- to eight-residue) deletions can be introduced without a loss of function and/or stable
assembly of PS II. Results of transformation with substitution constructs indicate that the sequence of the CD loop in D1 is subject
to much stricter requirements than that of D2, since no D2 sequence
could be functionally accommodated in D1, whereas introduction of two
of the four D1 regions into D2 led to functional PS II centers.
Frequency of functionally active sequence combinations.
Each
CD loop region studied in this report displayed different degrees of
difficulty in isolation of photoautotrophic combinatorial mutants. In
region 5, a single transformation experiment yielded all 45 mutants,
while the 34 mutants in region 8 were isolated from two transformation
experiments. Region 7 was the most difficult region in which to obtain
transformants, requiring four separate transformation experiments to
yield only 14 photoautotrophic mutants.
Furthermore, the rather strict structural requirements imposed by the
selection for stable PS II function were apparent, since
all
combinatorial mutants in D2 retained at least one, and usually
multiple, wild-type residues in the combinatorial region. However,
it
is clear that we have by no means exhausted the number of possible
sequence combinations that can be functionally accommodated, as
no
sequences that strongly resembled wild type were picked up
in our
analysis. Therefore, the frequency with which functionally
active
sequence combinations occur can only be
approximated.
One method of approximation is to do an estimation of the number of
functionally competent sequences based on the assumption
that most
combinations of those residues that were found at a
particular position
in any of the combinatorial mutants yield
photoautotrophic mutants. A
multiplication of the number of different
residues that can be
accommodated at each position yields values
of 10
6 for
regions 5 and 8. The value for region 7 is an order of magnitude
lower
(10
5); a contributing factor is likely to be the smaller
number of
mutants in this case. Comparing this to the total complexity
expected
for each domain (2.56 × 10
10 different
combinations for the eight-residue domains [regions
7 and 8];
1.28 × 10
9 for region 5, which is a seven-residue
domain), the probability
of a sequence being functionally active is on
the order of 10
3 for region 5, 10
4 for
region 8, and 10
5 for region 7. Of course, these numbers
assume that all combinations
of amino acids that can be accommodated at
a position will lead
to a photoautotrophic phenotype regardless of the
identity of
other allowed residues in the combinatorial region. This
assumption
does not consider the effects of sequence context on the
functionality
of any one position, a factor that may account for the
100% conservation
of His189 in our mutants in light of the existence
of photoautotrophic
site-directed mutants at this position. Thus, while
these probability
numbers are likely to be overestimates, they do
confirm the observation
mentioned previously that region 5 showed the
least difficulty
in obtaining transformants followed by region 8, with
region 7
being the least flexible of the CD loop regions
tested.
Another approximation approach is to compare the number of unique
photoautotrophic transformants obtained with the number
of different
M13s with which
Synechocystis sp. strain PCC 6803
was
transformed. The facts that (i) M13 was amplified about
10
5-fold between
E. coli transformation and
phage harvesting-DNA
isolation 4 h later and (ii) the frequency of
Synechocystis sp.
strain PCC 6803 transformation was such
that about 1 out of 10
4 to 10
5 M13 DNA
molecules led to a transformant under control conditions
imply that in
principle all M13 sequences that initially were
present in transformed
E. coli will be represented in the collection
of
Synechocystis sp. strain PCC 6803 transformants. Thus, the
number of unique photoautotrophic transformants per experiment,
divided
by the number of different combinatorial M13 phages used
for the
transformation of the
Synechocystis sp. strain PCC 6803
acceptor strain approximates the probability of an M13 clone leading
to
restoration of photoautotrophic growth. As the number of different
combinatorial phages per experiment was estimated to be 4 × 10
5 (based on 2 × 10
6 transformants per
experiment without M13 amplification and a
20% probability that the
combinatorial strand rather than the
deletion strand is amplified),
this yields a frequency of 10
4 for region 5, 10
5 for region 8, and 10
6 for region 7. These numbers are comparable within an order of
magnitude with the
frequencies calculated using the first approach,
which may have yielded
an overestimate of the
probabilities.
The N-terminal part of the CD loop.
In the D2 protein, the CD
loop region immediately adjacent to helix C has never been assigned a
critical role in PS II function. A surprising observation in this study
was that nonetheless three residues in region 5 (Gln164 to Ala170),
Ser166, Phe169, and Ala170, remained highly conserved in the
combinatorial mutants. This observation may support a previous
suggestion that the backbone of Phe169 may hydrogen bond with
YD (38). The high conservation and the limited
number of residues that can be functionally accommodated at these three
positions imply that this region may interact with other proteins not
considered by the models and/or that PS II and the bacterial reaction
center are quite different in this region. The latter would not be
surprising since there is poor primary sequence conservation between
the two; moreover, the bacterial reaction center lacks an OEC. While
this region of the loop may have fewer structural constraints than the
other regions, as evidenced by the relative ease by which
photoautotrophic mutants were isolated, nonetheless, the strong
preference for specific residues in part of region 5 seems to imply a
stringent structural requirement around these residues. Interestingly,
the apparently important residues in the region at positions 166, 169, and 170 are spaced as if they were in a -helical arrangement (i.e.,
one turn of the helix apart). This spacing may be coincidental or may
indicate that the CD helix in the D2 protein of PS II is much longer
than that in purple bacteria.
The region around residue 180 in D2.
The most noteworthy
feature of region 7 is the conservation pattern of Arg180. In the
mutants that lack Arg at position 180, an Arg residue always appears at
position 184. Since this region of the CD loop has been proposed to be
at the center of the lumenal helix, the shifting of Arg from 180 to 184 corresponds to one turn of the helix downstream. This suggests that the
functional side groups of Arg retain the same relative position in the
protein at either position 180 or 184. If an Arg residue is present at position 184, the residue at position 180 (Gly, Val, or Thr) generally is rather small; on the other hand, if Arg180 is present, at position 184 a large hydrophobic residue (Phe) is strongly preferred. It is
likely that these residues are involved in proper positioning of the
Arg residue.
Arg180 has been studied by site-directed mutagenesis, and of the
mutants that were analyzed, only R180Q can sustain photoautotrophic
growth, albeit at marginal rates (
16). Arg180 mutants show
altered
Y
Dox EPR signals as well as altered
kinetics of charge recombination
between Q
A
and the donor side (
16). The suggestion that Arg180 may
function
as a critical bifunctional residue interacting with
Y
D and providing
a ligand to chlorophyll (
16)
and/or modifying P680 redox characteristics
(
16) is
supported by the pattern observed in our combinatorial
mutants.
Of the three regions studied here, the smallest number of
photoautotrophic transformants were isolated in region 7, and for
those
isolated, there appeared to be less flexibility in the number
of
residues that could be accommodated at each position. In the
set of
combinatorial mutants encompassing this region, not more
than seven
different amino acid residues were found at any given
position, whereas
regions 5 and 8 contain several positions that
can accommodate 10 or
more different residues. In addition, this
region shows a stronger
global preference for aromatic residues
and large hydrophobic residues,
probably reflecting the strict
requirements imposed by the proposed
-helical structure of the
region.
The
-helical structure of region 7 may also explain why tryptophan
occurs in this set of mutants at a higher frequency than
that in the
other CD loop mutant sets. Trp is incorporated into
region 7 mutants in
four instances even though Trp is absent from
the wild-type sequence of
the region and the frequency of Trp
incorporation is expected to be
lower than most other residues
since it is encoded by only one codon.
While the region 5 set
of mutants also has four tryptophan residues,
three are conserved
at the wild-type position, leaving only one
tryptophan accommodated
outside of this position in 45 mutants. In
region 8, only one
mutant contains a Trp, and it is at the wild-type
position. The
Trp content has been found to be higher on average in
transmembrane
proteins and preferentially located near the ends of
-helices
(reviewed in references
1 and
24). Trp has the capability
of forming hydrogen
bonds, making this residue suited for stabilization
of helices at
boundaries between lipid and polar environments
(reviewed in references
15 and
24). These properties may
account
for the increased presence of Trp in this set of mutants
because
region 7 makes up a large part of the proposed CD loop helix
and
the helix is believed to interact with the membrane
(
25).
The C-terminal domain of the CD loop.
The absolute
conservation of His189 in the region 8 combinatorial mutants was
unexpected, since site-directed mutations at this position can be
accommodated. Many of the combinatorial mutants grow slowly and
appeared on plates weeks after transformation of the D2CD strain
(the wild type comes up within a week), arguing against a bias toward
more photoautotrophically competent strains as an explanation for the
conservation of His189. Instead, the finding implies that the presence
of an altered environment around position 189 (as occurs in the
combinatorial mutations) may decrease the ability of the region as a
whole to accommodate a proton originating from YD, thereby
increasing the stringency of the requirement of His at position 189.
Close examination of the photoautotrophic single-site mutants in
His189, H189L, H189Y, and H189Q hints at the importance of
the
relationship between hydrogen bonding of Y
D and His189 and
efficient operation of the OEC. Of the three single mutants, H189Y
shows the least impairment (
26,
28), which may fit with the
fact that the replacing tyrosine has hydrogen-bonding capabilities
(reviewed in reference
24). If the neighboring
wild-type residues
are able to support limited photoautotrophic growth
in the absence
of His189, then the potential candidates should have the
ability
to partially substitute for His189 in its hydrogen
binding-acceptor
role with Y
D. One potential candidate for
this role from the wild-type
sequence in region 8 is Trp191. Other than
Trp191 serving as a
potential hydrogen bond partner, a recent model of
PS II proposed
that Trp191 might play a structural role in the region
by providing
ring-stacking forces to the chlorophyll special pair P680
as well
(
38).
The results of this study demonstrate that combinatorial mutagenesis is
an efficient method for obtaining a substantial pool
of different
functional mutants with amino acid changes in a large
domain of a
protein with unknown structure. Additionally, sequence
comparisons of
sets of combinatorial mutants can provide insight
into otherwise
unremarkable primary sequence elements that may
have structural and/or
functional roles in the protein, thus opening
new avenues for research.
One example of this is the analysis
of several combinatorial mutants in
region 7, in which introduction
of a Trp residue at position 181 of the
D2 protein led to a large
quenching of variable fluorescence
(
30).
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the National Institutes
of Health to W.F.J.V. (GM 51556). A.T.K. was supported by a Graduate
Research Training (GRT) grant from the National Science Foundation
(DGE-9553456).
We thank Richard Debus for providing us with the
psbAI-psbAIII double-deletion strain of
Synechocystis sp. strain PCC 6803 used in making the D1CD
strain for these experiments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant Biology and Center for the Study of Early Events in
Photosynthesis, Arizona State University, Box 871601, Tempe, AZ
85287-1601. Phone: (480) 965-3698. Fax: (480) 965-6899. E-mail:
a.keilty{at}asu.edu.
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Abstract
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