From the Institut für Kristallographie, Freie
Universität Berlin, Takustraßett 6, D-14195 Berlin, Germany and
Max-Volmer-Institut für Biophysikalische Chemie und
Biochemie, Technische Universität, Straße-des-17, Juni 135, D-10623 Berlin, Germany
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ABSTRACT |
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An improved electron density map of photosystem I
(PSI) calculated at 4-Å resolution yields a more detailed structural
model of the stromal subunits PsaC, PsaD, and PsaE than previously
reported. The NMR structure of the subunit PsaE of PSI from
Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H.,
Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994)
Biochemistry 33, 6052-6062) has been used as a model to
interpret the region of the electron density map corresponding to this
subunit. The spatial orientation with respect to other subunits is
described as well as the possible interactions between the stromal
subunits. A first model of PsaD consisting of a four-stranded In cyanobacteria, green algae, and higher plants, photosystem I
(PSI)1 forms part of the
photosynthetic apparatus of oxygenic photosynthesis. It is a
multisubunit protein complex located in the thylakoid membrane. In
cyanobacteria, PSI consists of 11 subunits, whose nomenclature (PsaA to
PsaF and PsaI to PsaM) has been derived from the encoding genes
psaA to psaF and psaI to
psaM. Most of the subunits are membrane-integral. The
large subunits PsaA and PsaB coordinate the majority of cofactors both
of the electron transfer system and of the antenna system. The
remaining antenna chlorophyll a are bound by the smaller
membrane-integral subunits. Some of these subunits have additional
specialized functions. Thus, for example, PsaL and PsaI are responsible
for the formation of PSI-trimers, while PsaF and PsaJ stabilize PsaE in
the PSI complex (1, 2).
PsaC, PsaD, and PsaE are non-membrane-integral, extrinsic subunits that
may be removed from the membrane-integral core complex by chaotropic
reagents (1). Combinations of electron microscopy studies (3, 4),
cross-linking experiments (5, 6), and x-ray crystallographic
investigations (7-9) have led to progressively more detailed models of
the stromal ridge, showing these subunits to be in close neighborhood
to each other.
Only in the case of PsaC could an x-ray structural model be suggested
based on a previous electron density map (8-10). The model was derived
from the x-ray structure of a 2Fe4S4-ferredoxin from Peptostreptococcus asaccharolyticus (11). Due to the
high degree of 2-fold rotational symmetry inherent to the core of PsaC, a 2-fold ambiguity regarding its orientation in the PSI complex remained (8-10). In contrast to PsaC, the characteristic structural features of PsaE could unexpectedly not be located in the previous electron density map. The NMR structure of PsaE had revealed a five-stranded No structural model has previously been proposed for PsaD. CD- and
NMR-spectroscopical investigations indicate that a central portion of
the PsaD sequence is folded into a An assignment of the transmembrane Here we confirm the NMR structure of PsaE and describe its position and
orientation with respect to the PSI complex. A first structural model
of PsaD is presented. It indicates that PsaD partly covers PsaC
stromally in a clasplike manner. PsaD thereby approaches and interacts
with PsaE. The orientation of the subunit PsaC is discussed in relation
to recent biochemical and spectroscopic experiments. A unique
assignment of the Protein Purification and Crystallization--
Photosystem I was
isolated and purified from the thermophilic cyanobacterium
Synechococcus elongatus; crystals were grown by
microdialysis by lowering the salt concentration in the surrounding buffer under normal gravity and microgravity (14, 15). Details of the
crystallization experiments under microgravity will be published
elsewhere. PSI crystals are highly sensitive to any increase in the
salt concentration of their surrounding medium. Crystals were
consequently soaked with low heavy atom compound concentrations in the
range of 0.5-1 mM. To reduce the loss of crystal quality,
brought about by incubation with heavy atom compounds, PSI protein was
crystallized in the presence of the heavy atom compounds. Only in the
case of sodium-2-ethylmercurithiosalicylate, however, did this result
in crystals suitable for x-ray analysis.
Data Collection and Reduction--
A new native data set with a
resolution of 3.5 Å was obtained by merging several partial data sets.
They were collected at the EMBL outstation at DESY (Hamburg, beamline
BW7b) and ESRF (Grenoble, beamline ID2). A new
sodium-2-ethylmercurithiosalicylate derivative data set was collected
at LURE (Orsay, beamline DW32). Data collection was performed at
4 °C using Marresearch imaging plates of 30-cm diameter. Data were
processed and reduced with the Denzo/Scalepack package (16). Subsequent
scaling and data analysis was done with programs from the CCP4 suite
(17).
Phasing, Solvent Flattening, Electron Density Map Calculation,
and Interpretation--
To further improve the heavy atom model used
in the calculation of the first electron density map at 4-Å resolution
(9), all heavy atom derivatives were reevaluated using the program SHARP (18), and a new sodium-2-ethylmercurithiosalicylate derivative was introduced. For technical reasons, the number of derivatives used
was limited to five. Statistics of the native and derivative data sets
selected for the calculation of the current electron density map are
shown in Table I.
The heavy atom sites, determined by difference Patterson analyses and
cross-phased isomorphous difference Fourier syntheses, were refined
using the standard modus suggested for SHARP. The relatively low
resolution of the available data prevents heavy atom temperature
factors being refined. The heavy atom model was improved by inspecting
residual log likelihood gradient electron density maps (18). They
permit correctly and incorrectly identified positions to be
distinguished and indicate possible new sites. This procedure was
repeated cyclically until convergence was achieved.
The resulting phases were further improved by the solvent flattening
procedure as implemented in the program SOLOMON (17, 19). The solvent
mask resulting in the best electron density map was calculated assuming
a solvent content of 75% for the PSI crystals.
Electron density maps calculated at various levels of resolution and
displayed at different contour levels were inspected on an Evans and
Sutherland graphic display using the program O (20). Model building was
also performed in O. No amino acid side chains nor phytyl side chains
of the chlorophyll and phylloquinone cofactors were included in the
model. In the case of the stromal subunits PsaC and PsaE (11, 12),
known structures of homologous proteins were used to aid the electron
density interpretation. The present resolution and the low
data:parameter ratio as well as significant errors in the initial
coordinates do not allow for refinement of the structural model of PSI.
All distances should consequently be understood as estimates between
backbone C The Improved Electron Density Map--
The phase information used
in the calculation of the present electron density map has been
provided by the multiple isomorphous replacement method including
anomalous scattering (MIRAS). The low heavy atom compound
concentrations used for soaking the crystals in combination with the
large surface area of PSI generally lead to the formation of multiple
heavy atom sites, each with a relatively low occupancy. This introduces
difficulties to the identification and refinement of the heavy atom
positions and their occupancies, which is crucial in obtaining optimal
phase angles by the MIRAS method.
Previously, major heavy atom sites were identified from isomorphous
difference Patterson maps. Additional minor sites were detected from
cross-phased isomorphous difference Fourier maps. The occupancies and
coordinates of these sites were optimized using vector space refinement
techniques as implemented in VECREF (17). Due to the low resolution,
temperature factors of the heavy atom sites could not be refined. This
heavy atom model was incorporated into MLPHARE (21) for phase
calculation. Further refinement of the heavy atom parameters could not
be achieved, since occupancy optimization resulted in large parameter
shifts without convergence. The calculated electron density maps were not of optimal quality.
Small improvements were achieved by origin correlation refinement along
the c-axis between heavy atom derivatives using the program
HEAVY (22, 23) and reintroducing the refined parameters into MLPHARE
for phasing. (See Fig. 1 for the mean
figures of merit (FOM) for acentric (black dots)
and centric (white) reflections before
(continuous lines) and after (dashed
lines) refinement.) This procedure is seen to slightly increase
the mean FOM in the case of the acentric reflections in the medium
resolution shells and somewhat more in the low resolution shells. The
latter results in an improved final electron density map after solvent
flattening as low resolution terms strongly influence the correctness
of the mask determined from the initial MIRAS electron density map. The
described procedures, VECREF/MLPHARE-DM and VECREF/HEAVY/MLPHARE-DM, produce electron density maps with strong radial quality variations, the highest quality coinciding with the center of the PSI monomer. This
causes the difficulties encountered in interpreting the stromal and
luminal sections of PSI as well as the peripheral membrane integral
regions (9).
The most significant improvement in the quality of the electron density
map was, however, achieved by introducing the programs SHARP (18) for
refinement of heavy atom parameters and phase calculation and SOLOMON
(19) for density modification including solvent flattening. Starting
with the existing heavy atom model determined from isomorphous
difference Patterson maps, the analysis of the residual log likelihood
gradient maps increased the number of minor sites for each derivative
used. The earlier heavy atom sites nevertheless generally remained
consistent with the newly optimized models (see Table
II). Due to the low resolution of the
diffraction data, the isotropic temperature factor of all heavy atom
sites was fixed at B = 30 Å2.
By visual judgment, the electron density map, calculated with phases
obtained from SHARP using five heavy atom derivatives, is generally of
poorer quality than the best map resulting from phasing with MLPHARE
using heavy atom models for 10 derivatives. The difference in the
number of derivatives used is based on program-inherent parameter
limitations in the current version of SHARP. The quantity of
information introduced in MLPHARE produced a better multiple isomorphous replacement electron density map than the more accurately defined heavy atom parameters of the limited selection of derivatives used in the SHARP refinement (see Fig. 1 and Table II). The nominal mean FOM-values for both centric and acentric reflections reported by
SHARP are significantly lower than those from MLPHARE, although this
may be due to small differences in FOM definitions. Furthermore, SHARP determines similar average FOM values for both centric and acentric reflections, whereas centric reflections have significantly better values as reported by MLPHARE. On the other hand, comparing phasing statistics for individual derivatives indicates an overall improvement for each derivative (higher phasing power and
lowerRcullis values) during refinement by SHARP,
except, that is, for the platinum derivative. This confirms the
improvement of the heavy atom models by using SHARP, whereas the lower
quality of the MIRAS phases calculated with SHARP seems simply to be
due to the smaller number of derivatives used.
None of the MIRAS electron density maps allow PsaE or the second
phylloquinone molecule (two previously undetected structural motifs) to
be identified (Fig. 2). Comparing the
SHARP and MLPHARE electron density maps, the
The situation is dramatically reversed following solvent
flattening by SOLOMON (19). The electron density map obtained by combination of MLPHARE and SOLOMON is of higher quality than that obtained with MLPHARE and DM (data not shown). Consequently, SOLOMON was used for all further solvent flattening procedures. Fig. 1, b and c, depicts the FOM and mean phase
differences following solvent flattening for three procedures
combining VECREF/HEAVY/MLPHARE-DM, VECREF/HEAVY/MLPHARE-SOLOMON,
and SHARP-SOLOMON.
Clearly, the combination SHARP-SOLOMON provided the best phases,
followed by VECREF/HEAVY/MLPHARE-SOLOMON. Presumably, the more
elaborately defined phase probability distributions of SHARP allow for
optimal combination of phase information during solvent flattening
procedures, leading finally to more accurately defined phases.
Furthermore, little or no additional information is made available by
phase extension to between 4.0 and 3.5 Å. The limit of resolution was
therefore taken to be 4 Å. To avoid being misled by statistics, the
subjective visual impression (compactness and continuity) of each
electron density map was taken as the final quality criterion. An
example of the variations in the quality of the electron density maps
is given in Fig. 2, which shows identical sections of differently
calculated electron density maps corresponding to the subunit PsaE.
Location of the Three Stromal Subunits of Photosystem I--
PSI
possesses three small stromal subunits, PsaC, PsaD, and PsaE of
molecular masses 8.7, 15.2, and 8.3 kDa in S. elongatus, respectively (24). Together, they form a stromal ridge that extends
~30 Å beyond the membrane-integral regions (Fig.
3a), as first revealed by
electron microscopy studies (3). PsaC is positioned such that the
2-fold pseudorotation axis C2(AB), which relates PsaA and
PsaB, intersects the local 2-fold axis C2(C) of PsaC at an
angle of 62° (cf. Fig. 6). PsaE is located adjacent to
PsaC, facing away from the trimeric axis C3, whereas PsaD
faces toward this axis (9). In projection onto the
a,b-plane, connecting vectors between the
approximate "centers of mass" (COM) of PsaD, PsaC, and PsaE form an
obtuse angle of ~160° (Fig. 3a); the angle between
vectors C3-C2(AB) and
C2(AB)-PsaDCOM
(C2(AB)-PsaECOM) is approximately 45°
(155°). In this projection, PsaDCOM and
PsaECOM are located ~22 and ~26 Å from
PsaCCOM. Together with some loops from membrane-integral
subunits, the three stromal subunits form a wide cavity, the docking
site for ferredoxin and flavodoxin, as first suggested by Fromme
et al. (25) and later confirmed by combined
cross-linking and electron microscopy studies (26, 27) (Fig.
3c).
Orientation of PsaE in the PSI Complex--
In all previously
calculated electron density maps of PSI, the precise positioning of the
subunit PsaE proved elusive. The region presumed to be occupied by PsaE
had been located, but an interpretation as in the case of PsaC was not
possible. The current electron density map (see Figs. 2d and
5a) clearly reveals the five-stranded
Initially, a representative NMR model of PsaE (12) was visually fitted
into the electron density map. The structural model of PsaE from
Synechococcus sp. strain PCC7002 was then modified to
reflect the sequence of S. elongatus (see Fig.
4). Following Falzone et al.
(12), the
Superimposing the NMR and x-ray model structures of the subunit PsaE
reveals both to be almost identical in the core region (Fig.
5b). The long loop CD, described as flexible in the NMR study (12), is observed to adopt a twisted conformation in the x-ray
structural model directed away from the PsaE core in a finger-like manner. Although changes in the main conformation of the PsaE model are
anticipated to be minimal, a shift in the amino acid positions by one
or two is possible, although this remains uncertain, since individual
amino acid side chains are not visible in the electron density map.
The position and orientation of PsaE within the PSI complex may be
described as follows. The N- and C termini face the stroma and are
directed away from the C3-axis toward the outer rim of the
PSI trimer (Figs. 3 and 5). The loop AB is located close to the loop
j'-k' (~6.5 Å based on C
The loop CD is the longest and most flexible in PsaE (12). Its tip is
directed toward FX, the shortest distance between the two
being ~11 Å. It is furthermore located near
The
The orientation of PsaE in the PSI complex described here is not
entirely compatible with that previously suggested by Rousseau et
al. (29), who on the basis of fluorescein isothiocyanate-labeled thylakoid membranes and mutational arginine replacement analyses in
Synechocystis PCC6803, had concluded that both N and C
termini are buried within the PSI complex. Interestingly, only two PsaE polypeptides with triple mutations (K7N,K11E,R12A and R4A,R9N,R12A) and
another one with a stop codon at the position of Leu65 were
not recovered in the PSI complex, while single mutations affected
neither binding nor ferredoxin reduction behavior (29). Clearly, only
drastic changes among the first 12 N-terminal amino acid residues,
possibly affecting the overall structure of PsaE, suffice to prevent
its binding to the PSI complex. Fluorescein isothiocyanate labeling of
accessible amino groups previously performed on membranes at pH 9.3 failed to probe the N-terminal Ala1 and the C-terminal
Lys73. When the same reaction is conducted at pH 9.8, a pH
where amino groups with an alkaline-shifted pK are more likely to
react, the N-terminal Ala1 and all
A strongly conserved aromatic region in the loop AB of PsaE, which
contains a Tyr-Trp-Tyr triple, is thought to be involved in binding
PsaC (12). This is supported by our structural model, where this loop
faces PsaC, the protein backbones being ~7 Å apart. Replacement of
the tyrosines by phenylalanine or of the tryptophan by alanine was not
reported to affect the binding behavior of PsaE in the PSI complex
(29). The x-ray structural model places the Tyr-Trp-Tyr triple close to
j'-k' (core subunits), thereby indicating its possible
importance in binding PsaE to the PSI core.
Apart from a suggested role in cyclic electron transfer (31, 32), PsaE
has been implicated in stabilizing the stromal ridge of PSI (33) and is
known to influence fast electron transfer between PsaC and ferredoxin
(29). PsaD-less mutants show that PsaE binds to the PSI core in the
absence of PsaD and that it appears to influence neither the binding of
PsaC nor the photoreduction of the terminal iron sulfur clusters
FA and FB (45). In fact, however, the x-ray
structural model reveals the loop CD of PsaE to be sandwiched between
PsaC and the PSI core. Removing PsaE would therefore introduce an
opening between PsaC and PsaA/PsaB, which could destabilize the correct
binding of PsaC to the core. Three aliphatic residues
(Val43, Ala52, and
Leu55/Val55 in Synechococcus sp.
PCC7002/S. elongatus) in the loop CD of PsaE, which are not
in contact with other amino acids within this subunit, probably
interact with a hydrophobic surface area of PsaA/PsaB. Similarly,
Tyr45 and Tyr48 (Phe48 in
Synechococcus sp. PCC7002), located in the loop CD of PsaE facing the PSI core, are possibly involved in binding PsaE to the core
subunits. As noted previously, they are conserved, but no specific
functional assignment could be made (12). Since the loop CD had to be
remodeled in order to fit our electron density map, it is uncertain
whether the orientations of these residues are similar to those in the
original NMR structural model of PsaE. Consequently, the structural
function of Tyr45 and Tyr48 presently remain open.
The Subunit PsaC--
Following the identification of
F1 and F2 in the electron
density map (7) as well as additional structural elements belonging to
the pseudosymmetrical core of PsaC, the orientation of PsaC within PSI
was established, except, that is, for a 2-fold ambiguity caused by the
inherent pseudosymmetry of the PsaC core (8, 10). EPR experiments
independently arrived at the same conclusion (34). The debate about the
two orientations has been raging since, initial experiments favoring
the orientation in which the N and C termini face the stroma and
FA is the terminal electron acceptor (35). The first
electron density map at 4 Å appeared to confirm this view, with a
closed extended loop between the two symmetry-equivalent halves of PsaC
being modeled near the interface between PsaC and the core subunits and
an extended C terminus covering the subunit stromally (9). Recently,
however, the opposite orientation (N and C termini on the interface
between PsaC and the core subunits and FB as the terminal
cluster) has been gaining ground (36, 37).
Examining the improved electron density map indicates that the previous
model cannot unreservedly be upheld. The electron density structure
located close to the stromal end of the PsaC core, which was previously
interpreted as the C-terminal extension of this subunit, now appears
more realistically to belong to PsaD (see below and Fig. 6).
Furthermore, electron density on the luminally directed side of PsaC,
which previously remained unidentified, is connected to the
pseudosymmetric core of PsaC in the improved electron density map and
is seen to be at least compatible with a linear extension of the C
terminus by 13-16 amino acid residues beyond the PsaC core. This would
be in agreement with the C-terminal extension of PsaC by 14 residues as
compared with the 2Fe4S4-ferredoxin from
P. asaccharolyticus. Thus, the orientation in which the N and C termini face the membrane-integral subunits is now seen to be at
least feasible. The only drawback to the interpretation of this
orientation is that electron density that would accommodate the
extended central loop insertion is connected to the PsaC core by weak
electron density only, preventing a complete modeling of this part of
PsaC. Overall, an orientation in which FA is the proximal
and FB the distal iron-sulfur center would now appear most likely.
The PsaC Binding Surface of the PsaA/PsaB Heterodimer--
Both
PsaA and PsaB contain a strictly conserved FX-binding motif
CDGPGRGGTCP, the two
cysteines of which have been proposed to coordinate FX
(38). The intercysteine loops of this motif have been suggested to play
a leading role in binding the extrinsic subunit PsaC (39, 40). In
particular, one of the central arginines of the FX-binding
motifs (boldface R in the sequence motif) was postulated to form a salt
bridge with an aspartate of PsaC and consequently to be crucial in
determining the orientation of this subunit (41). In the x-ray
structural model, the loop regions connecting
The current electron density map improves on this situation by
revealing a more continuous connection between the stromal ends of
Nevertheless, the structural model of the loop
j-k indicates that (i)
the central part of the conserved intercysteine FX-binding
loops of PsaA/PsaB containing the arginines, do indeed appear to
interact with PsaC, while (ii) the aspartate residue next to the first
cysteine (whose mutation to an arginine has a strong influence on the
assembly of the PSI core (40)) could feasibly also be involved in
interactions with PsaC. On the other hand, the intercysteine loops are
rather closely associated with the membrane-intrinsic regions of PSI.
On their own, they therefore represent a much smaller binding surface
for PsaC than implied by the hypothetical model proposed previously
(39-41). However, in addition to the intercysteine loops, the parts of
the loops j-k (VIII-IX)
N-terminal to the conserved FX-binding motif additionally
approach PsaC to within ~7 Å (as compared with the ~6 Å of the
intercysteine loops), complementing the binding surface for PsaC (Fig.
3c).
Structural Model of PsaD--
Now that essentially all electron
density belonging to both PsaE and PsaC has been identified, a first
structural model of PsaD may be attempted. The model of PsaD presently
consists of 125 C
PsaD was previously reported to contain a single short
The loops of PsaD connecting the individual strands of the
Relative to PsaA/PsaB, the main volume of PsaD partly covers the
interhelical loops n'-o', j-k, and i-h as well as the stromal ends of the The Subunits PsaF, PsaJ, and PsaM--
The changes in the present
model of PSI do not exclusively concern the stromal subunits. Note in
particular that the poorly defined
On the other hand, the current electron density map allows a previously
unreported stromal
The exact assignment of the secondary structure elements in this region
is not entirely clear; because w and x appear to
be linked stromally (this is still the case in the present electron
density map), these were assigned to PsaF (9), the only subunit long
enough to possess two transmembrane
However, this assignment contradicts the positioning of PsaJ identified
by electron microscopy, which indicates PsaJ to be located in the
region of w and x (4). Possibly, therefore,
y1 and y2 belong to PsaJ
and u to PsaM. In this constellation, PsaM is distal to both
PsaJ and PsaF, accounting for the lack of cross-linking products
between PsaM and any other small PSI subunit (5). The position of PsaJ would match the electron microscopic conclusions, although PsaJ would
not be transmembrane as generally assumed.
A third assignment is possible if the loop w-x is assumed to
be an artifact. Then y1 and
y2 could represent the C terminus of PsaF, while
one of w and x would belong to PsaJ and the other
to PsaF. This constellation would best account for the finding that
PsaF does not contain two significantly hydrophobic stretches, although
it is not in perfect agreement with the apparent stromal connection
between w and x.
PsaA and PsaB--
Except for the luminal connection
k-l-m (Fig.
9), all other luminal interhelical loop
regions have not to date been located in their entirety. This is due
both to their length and to the lower effective resolution of these
luminal regions of the electron density. By contrast, the short stromal
connections have been identified to a much larger extent (9).
Nevertheless, the previous model could not differentiate between
a or d as the N-terminal
transmembrane
The loop e-f is positioned near the
loop n-o of the same subunit,
bringing the N terminus (e-f) of
PsaA and PsaB into close physical contact with its C-terminal region
(n-o). Interestingly, mapping
e and f to the amino acid
sequence N-terminal of the hydrophobic span I assigns charged residue
groups to both. In the case of the unprimed subunit, as part of the
ferredoxin binding site (Fig. 3b), these residues clearly
participate in binding the soluble electron acceptor proteins,
ferredoxin and flavodoxin. Similarly the C-terminal end of e
and the connection e-f would be involved in binding the
subunit PsaE, while e' binds PsaD, although to a lesser
degree. The functional asymmetry between the two core subunits in this
area is underscored by the fact that no sequence homology is observed
for the region immediately N-terminal of the hydrophobic span I
(
Recently, protease cleavage site analysis of PsaA and PsaB proposed
mapping the subunit PsaA to the primed core The current electron density map allows a detailed model of the
stromal ridge of PSI to be derived. The combination of the programs
SHARP and SOLOMON used for heavy atom model refinement and phase
improvement significantly increase the quality of the electron density
map. The known homologous structural models of PsaC and PsaE have been
employed in interpreting the corresponding regions of the electron
density map, underscoring the usefulness of such partial model
structures. The localization of the NMR structure of PsaE in the
stromal ridge demonstrates the improvements achieved by the described
procedures. The present, improved model shows all subunits of the
stromal ridge to be in close contact to each other. It gives the
structural basis for the stabilizing role of PsaD.
-sheet
and an
-helix is suggested, indicating that this subunit partly
shields PsaC from the stromal side. In addition to the improvements on
the stromal subunits, the structural model of the membrane-integral
region of PSI is also extended. The current electron density map allows
the identification of the N and C termini of the subunits PsaA and
PsaB. The 11-transmembrane
-helices of these subunits can now be
assigned uniquely to the hydrophobic segments identified by
hydrophobicity analyses.
INTRODUCTION
Top
Abstract
Introduction
References
-barrel forming the core of this subunit (12).
-sheet, whereas the N- and
C-terminal regions are mobile (13).
-helices of the x-ray structural
model to the membrane-integral subunits of PSI has been suggested (8,
9). Regarding the connectivity of the subunits PsaA/PsaB, some
unobserved loops prevented an unambiguous assignment of individual
-helices to the primary structure. This was especially true of the
N-terminal, antenna-binding domains of PsaA and PsaB.
-helices of PsaA/PsaB is discussed.
EXPERIMENTAL PROCEDURES
X-ray data sets used for the calculation of the new electron density
map
atoms.
RESULTS AND DISCUSSION
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Fig. 1.
Distribution of FOM. a,
MIR(AS) statistics. Distribution of mean FOM versus
resolution for the procedures described in determining the heavy atom
model. b, distribution of mean FOM. c, estimated
errors in the phase angle versus resolution ( Phi
represents mean phase angle difference between MIRAS phases and phases
obtained after density modification).
Overview of heavy atom refinement
-helices in the former
appear more detailed but less continuous, whereas the MLPHARE maps
reveal more connected electron density stretches.
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Fig. 2.
Electron density map corresponding to the
core of PsaE including the backbone as modeled in the present electron
density map. The contouring is at 1.2 S.D. above mean density.
a, backbone trace of PsaE. The parts not included in
b-d are dashed. b and c,
electron density maps derived from VECREF/HEAVY/MLPHARE (no density
modification) and SHARP (no density modification), respectively.
d, density map SHARP-SOLOMON (density modified). The central
-sheet of PsaE is visible only in d. The MIR maps
b and c are both of comparatively low quality in
this region, although b is observed to be of superior
quality in other regions of the structure (not shown). Electron density
maps obtained after SOLOMON generally reveal more detailed features
than comparable maps calculated using DM. This figure was
produced using BobScript (47).
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Fig. 3.
The stromal ridge of PSI shown from the
stromal side onto the membrane plane. a, simulated
surface of the stromal ridge. PsaC is depicted in yellow,
PsaD in red, PsaE in blue, and membrane-integral
subunits in white. The approximate centers of mass of
subunits PsaD and PsaE are indicated by white dots;
for PsaC, it coincides with the symbol of the local 2-fold
axis C2(AB). I, terminal extension of PsaD. It
functions as a clasp for PsaC and is in close contact with PsaE.
II, crystal contact surface of PsaE with the next trimer in
the crystal lattice. III, loop CD of PsaE below PsaC.
IV, binding site for ferredoxin/flavodoxin. b,
the equivalent view as in a showing the observed secondary
structure elements of PSI. The -helices of the membrane-integral
parts are drawn as cylinders and shaded gray. The
stromal subunits PsaC, PsaD, and PsaE are depicted as coil models
showing their secondary structure elements. In addition, a large loop
region of PsaD involved in the contact between this subunit and the
core of PSI is marked. c, model of the stromal ridge of PSI
showing the secondary structure elements. The view is into the
ferredoxin/flavodoxin binding pocket with the trimeric axis on the
right. The stromal subunits PsaC, -D, and -E as well as the
iron-sulfur cluster FX are rendered in black.
The protein backbone of the membrane-integral subunits (
-helices
depicted as cylinders) are colored in gray.
a was produced using Grasp (48); b and
c were produced using BobScript (47).
-barrel of the PsaE
core as well as an extended loop region directed toward the PSI core.
-strands are referred to as
A to
E and the
connecting loops as AB to DE, Fig.
5b. An additional proline was
introduced into the loop BC and a glutamine into loop DE, slightly
elongating these loops. Except for the C-terminal end of
E
(Val68 has been substituted by an alanine), all remaining
substitutions are not located in the
-barrel, nor do they disturb
the length of the loops. The C terminus of PsaE in S. elongatus and the loops BC and DE are longer than in
Synechococcus sp. PCC7002; they have been modified as
indicated by the electron density map (Fig. 5a).
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Fig. 4.
Sequence alignment of subunit PsaE from
Synechococcus sp. Lines 1,
PCC7002; lines 2, S. elongatus. The
secondary structure element nomenclature ( -strands
A to
E) is
taken from Ref. 12. Amino acids not conserved in both sequences are
indicated by rectangular boxes.
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Fig. 5.
a, stereo view of PsaE including the
surrounding electron density. b, superposition of (i) NMR
model from Synechococcus sp. PCC7002 (NS,
CS; black) (12) and (ii) x-ray structural model
(gray) of PsaE from S. elongatus
(NSE, CSE) as modeled into the present electron
density map. This figure was produced using BobScript
(47).
positions), which
links the
-helices j' and k' of one of the
core subunits PsaA or PsaB (Fig. 3). It is similarly also near (~5.5
Å) the extension of a membrane-integral
-helix y1, not previously described (see below and
Figs. 3b and 8), as well as being nearest PsaD (~6.5 Å,
Fig. 3b). The loop BC stromally forms a large part of the
surface of PsaE and is involved in contacts (~4.5 Å) to the
central loop of PsaC, which links the two Fe4S4
cluster binding domains in this subunit (Ref. 1; see Figs.
3c and 6).
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Fig. 6.
a, stereo view of the structural model
of PsaC and its associated electron density map. Structural motifs of
PsaE and PsaD in close contact to PsaC are also shown. The clasp of
PsaD has been shortened to merely include the part covering PsaC.
b, Structural model of PsaC. The view is onto the
C2(AB)-axis. This figure was produced using
BobScript (47).
-helices e
and f of PsaA or PsaB, (~6 Å) as well as to the loop
n-o, which connects the "horizontal"
-helix
n and the C-terminal helix o (~5 Å) (see Fig.
3).
-strands
B and
C form part of the ferredoxin binding site
(Fig. 3c). It is therefore likely that some amino acids of these strands interact with the water-soluble electron acceptors. The
mutation of Arg39 to Gln (R39Q) at the C terminus of the
strand
C is known to partly inhibit ferredoxin reduction (28), an
observation that is corroborated by the structural model. The shortest
distance between PsaE and the loop w-x and
-helix
u (PsaF, PsaJ, and PsaM; see below) are ~12 and ~20 Å,
respectively (Fig. 3).
-amino groups of
lysines become labeled, except that of Lys11, suggesting
that Lys11 could be interacting with the PSI
core.2 This observation is in
agreement with our structural model, since Lys11 of PsaE
from Synechocystis PCC6803 corresponds to the C-terminal residue of the loop AB, which is located close to the loop
j'-k' of PsaA or PsaB. Recent biochemical studies on PSI
complexes from Synechocystis sp. PCC6802 using the
endoproteinase Glu-C reveal the residues Glu63 and
Glu67 near the C terminus of PsaE as the most likely
cleavage sites for this protease (30). These data would also confirm
our structural findings of a solvent-exposed C terminus.
-helices
j and k coordinate the
iron-sulfur cluster FX. As a result, the
-helices j and k were assigned to
the hydrophobic stretches VIII and IX of PsaA and PsaB (8, 9). Only an
incomplete model of the loop j-k
could, however, be derived previously (9).
-helices j and
k, including two stretches of electron density
pseudosymmetrically wrapped around the iron-sulfur cluster,
FX (Fig. 7). The length of
the loop j-k is in good agreement
with the hydrophilic loop VIII-IX. As is the case of the remaining
model, individual side chains cannot be identified directly in the
electron density map and have consequently not been modeled.
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Fig. 7.
Region of the electron density map showing
the structural model of the conserved FX-binding loops.
The loops obey the pseudo-2-fold symmetry observed for the other
features near C2(AB). This figure was produced
using Setor (49).
positions (as compared with the 138 amino acids determined from the corresponding gene psaD
(24)). Hence, the present backbone represents a preliminary model only.
This is especially true of the loops connecting the secondary structure
elements. The ambiguity regarding their length presently prevents the
protein sequence being assigned to the structure.
-helix, Da,
adjacent to PsaC (9). The current improved electron density map shows
that this
-helix is surrounded by a
-sheet (Fig. 3), consisting
of at least three relatively long
-strands, which is in agreement
with the observation that the structure of PsaD in solution contains a
small amount of
-sheet (13). The electron density of the PsaD core
is clearly connected to a stromal strand of electron density previously
assigned to PsaC, indicating that this strand is actually part of PsaD.
This means that one terminus of PsaD partly covers PsaC, wrapping
around PsaC in a clasplike manner (Figs. 3, a and
c, and 6a). The distal end of the clasp is
located above the transmembrane
-helices h',
j', and k', thereby partly shielding PsaE, as
well as the
-helix n (Fig. 3, b and
c). The closest distance between the backbones of PsaE and
PsaD is ~6.5 Å, supplying the structural explanation for the
observed cross-linking between PsaD and PsaE (5). The other terminus of
PsaD similarly appears to be located on the stromal surface. It is,
however, not in contact with any other subunit. The current model
thereby reveals the structural basis for the stabilizing role of PsaD
for PsaC suggested previously for both cyanobacterial and higher plant
PsaD (42), although this finding was later disputed (43). Since higher plant PsaD has an extended N terminus, possibly involved in the binding
of other stromal subunits specific for higher plants, this may explain
the more important role of PsaD in stabilizing the stromal ridge in
barley (44). In cyanobacterial PSI complexes, functionally bound PsaC
is retained under mild extraction procedures (43), whereas the
stabilizing role of PsaD is increasingly noticeable when more
aggressive detergents are used (45).
-sheet
appear largely directed toward the core subunits, thereby establishing
the interface between the core and PsaD (Fig. 3c). In the
PSI-complex, they are therefore buried and not accessible to attack by
proteases. An extended loop forms a compact domain without recognizable
secondary structure (Fig. 3c). It is located within a cavity
formed by the central subunits PsaA/PsaB and is involved in the
shortest contacts to the latter subunits.
-helices i and
j. The backbone-to-backbone distance between PsaD and other
structural elements are currently modeled to be as follows: ~15 Å to
e', ~7.5 Å to n, and ~7.5 Å to
j-k. Note that the shortest distance of ~10 Å between
PsaD and PsaL involves the surface
-helix pS
of PsaL. This newly introduced stromal surface
-helix is connected
to the transmembrane
-helix p and located on the
monomer-monomer interface (see Fig. 3, b and c).
The distance between PsaD and
-helix r, probably part of
PsaI, is ~15 Å. The current structural model indicates that PsaC and
PsaD share multiple contacts to each other as well as each being in
intimate contact with the core subunits.
-helix y previously
tentatively identified with PsaM (9) is no longer discernible and has
now been removed from the structural model of PSI. Thus, PsaM would not
possess the trimer-stabilizing role proposed (9). Similarly, the
luminal surface
-helix v has not been included in the
present structural model of PSI, since the corresponding electron
density could also be interpreted as belonging to an elongated loop structure.
-helical region distal to the C3-axis
near the
-helices w and x to be included in
the model. This
-helical structure has an unusual overall conformation (Fig. 8). After a short
stromal loop region near PsaE (~5.5 Å), the backbone forms a small
-helix (y1), inclined by about 40-50° to
the membrane plane, which, after reaching a third of the total membrane
depth, bends back toward the stromal surface as a second
-helix
(y2), ending just short of the electron density
assigned to the stromal loop connecting
-helices w and
x.
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Fig. 8.
View parallel to the membrane plane of the
region assigned to PsaF, PsaJ, and PsaM. The previously unreported
-helices y1 and y2 are
membrane-integral. They are, however, inclined by ~45° to the
membrane normal and do not span the thylakoid membrane. The stromal end
of y1 is located close to PsaE. This
figure was produced using BobScript (47).
-helices. As a result,
u was assigned to PsaJ. If this assignment is maintained,
y1 and y2 would belong to
PsaM, supporting the notion that PsaM is involved in cyclic electron
transfer.3
-helix due to the ambiguity in the stromal
(N-terminal) connection of
-helices g. The
electron density, hereby described, clearly reveals that
g is stromally connected to
a in both subunits. Thus,
a may now be identified with the fourth
hydrophobic span (IV) in both PsaA and PsaB. On condition that pairs of
neighboring transmembrane
-helices
(a,b and
c,d) are connected luminally, b then maps to III,
c to II (c and
b are connected stromally (9)), and
d to the hydrophobic span I (Fig. 9). The latter
is in turn stromally linked to e, a surface
-helix, which is again connected to f (the
N-terminal
-helix of the core subunit).
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Fig. 9.
A schematic correlation of the secondary
structures of the x-ray structural model to the amino acid sequence for
the central subunits PsaA and PsaB. The N terminus is at -helix
f, the C terminus at
-helix
o. Binding sites for the phylloquinone cofactors
(QK) and the iron sulfur cluster FX
are indicated. An assignment of primed or unprimed
-helices to PsaA
or PsaB is still not possible on purely structural grounds (however,
see Ref. 46).
-helix e). A conserved motif AHDF(D/E) about
10 amino acids from the N-terminal end of d
(span I) would by contrast correspond to the connection e-f. It is involved in interactions
with n-o, a function common to both subunits.
-helices and PsaB to the
nonprimed (46). This assignment is based on the finding that the
absence of PsaE opens additional protease cleavage sites on PsaA, while
the absence of PsaD leads to new cleavage products in the N-terminal
region of PsaB. On the basis of the electron density map, an
independent assignment of the primed and nonprimed
-helices to the
subunits PsaA and PsaB, respectively, is not feasible. Two clear
differences between PsaA and PsaB ((i) the longer N terminus (~25
amino acids) of PsaA and (ii) an 11 amino acid insertion N-terminal of
the surface
-helix l in PsaA) cannot at
present be unambiguously matched to either the primed or unprimed subunit.
CONCLUSION
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ACKNOWLEDGEMENTS |
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We thank B. Lagoutte (Commissariat à l'Energie Atomique Saclay) and Y. N. Utkin (Russian Academy of Sciences, Moscow) for sharing unpublished data and biochemical discussions. Synchrotron beam time provided by the EMBL outstation at ESRF (Grenoble, beamline ID2), LURE (Orsay, beamline DW32), SRS (Daresbury, beamline 9.6), EMBL outstation at DESY (Hamburg, beamline BW7B), and MPG-ASMB at DESY (Hamburg, beamline BW6) is gratefully acknowledged. Facilities for crystallization under microgravity were provided by the European Space Agency during the Second United States Microgravity Laboratory and the Life and Microgravity Spacelab missions of the National Aeronautics and Space Administration.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 312), the Bundesministerium für Bildung und Forschung, the Fonds der Chemischen Industrie, and the Deutsche Agentur für Raumfahrt-Angelegenheiten.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
¶ To whom correspondence should be addressed. Tel.: 49 30 838 6326; Fax: 49 30 838 6702; E-mail: nkrauss{at}chemie.fu-berlin.de.
2 B. Lagoutte, personal communication.
3 D. A. Bryant, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
PSI, photosystem I;
a to o and a' to o', x-ray
structural -helix nomenclature of the core subunits PsaA/PsaB;
a to o, pertaining to
both primed and unprimed a to o;
p to
y, x-ray structural model nomenclature of
-helices of the
smaller membrane-integral subunits PsaF to PsaM;
c-axis, crystallographic c-axis, parallel to the membrane normal;
C2(AB), 2-fold pseudosymmetry axis relating the subunits
PsaA and PsaB to each other;
Da, sole
-helix of PsaD;
C2(C), 2-fold pseudosymmetry axis inherent to PsaC;
C3-axis, crystallographic 3-fold axis coinciding with the
trimer axis of PSI;
F1 and
F2, preliminary x-ray structural model names for
FA and FB (FB and FA);
FOM, figure(s) of merit;
MIRAS, multiple isomorphous replacement and
anomalous scattering;
psaA to psaF and
psaI to psaM, genes coding for the subunits of
cyanobacterial PSI;
PsaA to PsaF and PsaI to PsaM, subunits of
cyanobacterial PSI;
PsaCCOM, PsaDCOM, and
PsaECOM, approximate centers of mass of the stromal
subunits.
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REFERENCES |
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