From the Department of Microbiology, Swammerdam
Institute for Life Sciences, University of Amsterdam, 1018WV,
Amsterdam, The Netherlands and ¶ Wellcome Trust Biocentre, School
of Life Sciences, University of Dundee, Dundee DD1 5EH,
Scotland
Received for publication, February 19, 2003, and in revised form, March 14, 2003
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ABSTRACT |
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PAS
(PER-ARNT-SIM) domains are a family
of sensor protein domains involved in signal transduction in a wide
range of organisms. Recent structural studies have revealed that these
domains contain a structurally conserved PAS1 domains are
structural modules that can be found in proteins in all kingdoms of
life (1, 2). The PAS module was first identified in the
Drosophila clock protein PER and the basic helix-loop-helix containing transcription factors ARNT
(aryl-hydrocarbon receptor nuclear translocator) in mammals and SIM
(single-minded protein) iinsects (3). Most PAS domains are sensory
modules, typically sensing oxygen tension, redox potential, or light
intensity (1, 4). Alternatively, they mediate protein-protein
interactions or bind small ligands (5). Although the amino acid
sequences of the different PAS domains show little similarity, their
three-dimensional structures appear to be conserved. All of the PAS
domains resemble the structure of photoactive yellow protein (PYP) (4),
a photoreceptor presumed to be involved in a phototactic response of
the bacterium Ectothiorhodospira halophila to intense blue
light (6). Its structure reveals an Here we investigate whether the dynamic properties of PAS domains are
intrinsic properties associated with their conserved fold. First, we
have mutated the PYP from E. halophila into a minimal PAS
domain by the removal of the N-terminal cap (see also Ref. 32). To be
able to tackle the dynamic properties of this minimal PAS domain, its
three-dimensional structure was refined against 1.14-Å synchrotron
diffraction data. Second, this structure was used in a comparative
computational study on the conformational flexibility of all of the PAS
domains for which crystals structures are available: HERG, the
N-terminal domain of a human potassium channel (33); LOV2, a
photoreceptor domain from plants (34); and FixL, a bacterial oxygen
sensor (35). Essential dynamics analyses on the sampled configurational
space of all of these PAS domains reveal conserved concerted motions.
This supports the hypothesis that the common structure of PAS domains
implies common flexibility and that it is this conserved property that is fundamental for PAS domain function in signal transduction.
Crystallization, Diffraction, and Refinement
Diffraction data were collected at beamline ID14-EH1 (European
Synchrotron Radiation Facility, Grenoble, France) and processed with
the HKL package (Table I) (36). The structure of Residues 113 (leucine) and 114 (serine) in one monomer and residue 116 (aspartic acid) in the other monomer were disordered, although some
evidence for several possible conformations was visible in the map. The
building of these regions was attempted, but their conformations could
not be determined with confidence. Similar observations were made in
previous crystallographic studies of PYP in the P65 space
group (30, 31, 41). At the N terminus, the well defined electron
density was present for Ala-27 at the early stages of refinement.
Subsequent maps also defined the conformation of Leu-26.
Computational Details
CONCOORD Simulations--
Sampling of conformational space by
the computer simulation method CONCOORD (42) was performed for crystal
structures of the PAS domains depicted in Fig. 2. Besides these
existing PAS domain structures, the Essential Dynamics--
Essential dynamics (43) determines
concerted motions of atoms from an ensemble of structures, for example,
a set of crystal structures (44-47) or a trajectory from a computer
simulation (43, 48-51). Here the CONCOORD ensembles were used as
input. A covariance matrix is constructed that describes the
correlation of the positional shifts of one atom with those of another
atom as shown in Equation 1,
To allow direct comparison of concerted motions for different proteins,
an equal number of atoms must be used in the essential dynamics
calculations. A first simplification is that only C Atomic Resolution Crystal Structure of
Native PYP contains two hydrophobic cores, one within the PAS domain
between the
As observed earlier (4, 34), none of the other currently known PAS
domain structures (Fig. 3) possesses extra residues similar to the N
terminus of wtPYP. Thus, this may be a unique feature that plays a
specific but, as yet, unidentified role in PYP function. Although the
PAS domains share a common fold, only few residues are conserved at the
sequence level (Figs. 3 and 4). When
these residues (mostly leucines, isoleucines, and valines) are compared
among the different PAS domains, they appear to form part of a
conserved hydrophobic core (Figs. 3 and 4). Interestingly, most
of these residues are located in a cluster near helix PAS Domain Flexibility--
Because PAS domains function as sensor
proteins in signaling pathways and share a common fold, it is possible
that they also have common dynamic properties, which would allow them
to communicate with transducer proteins through a conserved mechanism.
We have attempted to investigate this through computer simulation of
all of the structurally characterized PAS domains, namely PYP (7), FixL
(35), HERG (33), and LOV (34). The complete proteins were subjected to
CONCOORD simulations (42) followed by extraction of the common
C
Sets of eigenvectors can be projected onto each other yielding a
cumulative square inner product, indicating the degree of similarity of
the motions described by the eigenvectors. Here we have focused on the
first 12 eigenvectors (5% 3N = 234 total eigenvectors), because these together describe approximately
95% of the total motion in the ensembles. Table
II shows that the eigenvectors from the
different PAS domains are very similar, suggesting that the cores of
the PAS domains share common motions, which are not present in lysozyme
(the negative control). This is further confirmed by projection of the
PAS domain eigenvectors onto the first three eigenvectors calculated
from the wtPYP ensemble (Fig. 5). Whereas
the other PAS domains reproduce these largest wtPYP motions for up to
90% within the first 12 eigenvectors, they are almost absent in the
lysozyme ensemble. Thus, the PAS domains not only share a common
structure but also share a common conformational flexibility.
To understand the motions described by the eigenvectors on a molecular
level, the minimum and maximum projections onto an eigenvector can be
translated back to Cartesian space and compared as C The data presented here show that in the absence of the
N-terminal domain, PYP maintains its PAS-fold despite the exposure of
several hydrophobic residues to solvent. The /
-fold, whereas almost
no conservation is observed at the amino acid sequence level. The
photoactive yellow protein, a bacterial light sensor, has been proposed
as the PAS structural prototype yet contains an N-terminal
helix-turn-helix motif not found in other PAS domains. Here we describe
the atomic resolution structure of a photoactive yellow protein
deletion mutant lacking this motif, revealing that the PAS domain is
indeed able to fold independently and is not affected by the removal of
these residues. Computer simulations of currently known PAS domain
structures reveal that these domains are not only structurally conserved but are also similar in their conformational flexibilities. The observed motions point to a possible common mechanism for communicating ligand binding/activation to downstream transducer proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
/
-fold with the
light-sensitive chromophore p-coumaric acid bound to the
protein via a thioester linkage (7). It is the only PAS domain of which
the catalytic function, i.e. signal generation and
transduction, has been studied in great detail. The protein has been
shown to undergo a photocycle linked to isomerization of the
chromophore (8-12). The ground state (pG) has a UV-visible absorbance
maximum at 446 nm. After absorption of a blue photon, the protein
returns from the primary excited state into the first transient ground
state, a strongly red-shifted intermediate, at the picosecond
time scale (13-17). A more moderately red-shifted
intermediate absorbing maximally at 465 nm is formed on the
nanosecond time scale (18). The red-shifted intermediate spontaneously converts into a blue-shifted intermediate absorbing maximally at 355 nm at the sub-millisecond time scale (18, 19). The blue-shifted intermediate subsequently relaxes back to pG on a
sub-second time scale (15, 18-20) or faster in a
light-dependent reaction (21, 22). Several detailed
studies, including Laue diffraction and cryo-crystallography (9, 11,
12), NMR spectroscopy (23), small angle x-ray scattering (24, 25),
biochemical experiments (26), and Fourier transform infrared
spectroscopy (27, 28) and computer simulations (29, 30), have
revealed that during the PYP photocycle distinct significant
conformational changes occur. It is these conformational changes that
are thought to translate the photon signal into a cellular response via
subsequent protein/protein interactions. To study the possible protein
motions involved in the photocycle, PYP dynamics have been investigated by computer simulation (29). This study suggested that
chromophore-linked concerted motions may be present in pG and that
these motions might be amplified upon isomerization of the chromophore.
The simulations, later supported by x-ray crystallographic studies (30), also suggest that conserved glycines were serving as hinge points, allowing substructures in the protein to fluctuate relative to
each other. In a subsequent study where the rigidity of the PYP
backbone was altered by mutation of these glycines, the role of these
hinge points in the signal transduction process was further confirmed
(31). The glycines that were investigated in this study fall within the
PAS-fold (4) and show a large degree of conservation throughout the PAS
family. This has led to the speculation that apart from a conserved
structure the PAS domains may have similar conformational freedom and
associated signal transduction mechanism (30).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
25PYP encompassing residues 26-125 of PYP was
expressed and purified as described previously (32). Crystals were
grown by equilibration of 1 µl of 30 mg/ml protein with 1 µl of
mother liquor (1.8 M ammonium sulfate, 10 mM
CoCl2, 100 mM MES, pH 6.5) against a 1-ml
reservoir of mother liquor. Crystals appeared after 2-3 days with a
largest dimension of 0.4 mm.
25PYP was solved by molecular replacement with AMoRe (37) using the native
PYP structure (Protein Data Bank code 2PHY) (7) as a search
model (excluding the chromophore) against 8-4-Å data. A solution was
found (r = 0.479, correlation coefficient = 0.282) with two molecules in the asymmetric unit. Initial refinement was
carried out with CNS (38) interspersed with model building in O (39).
The chromophore was not included in the refinement until it was well
defined by an unbiased Fo
Fc ,
calc map (Fig. 1). Further
rounds of refinement with SHELX97 (40) allowed the placement of water
molecules and the assignment of some alternate side chain
conformations. In the last stages of the refinement, hydrogen
atoms were included (Table I).
25PYP crystal
structure described here was also simulated. As a negative control for
the subsequent comparisons, a CONCOORD ensemble starting from the
crystal structure of turkey lysozyme (Protein Data Bank code
135L) bearing no structural resemblance to PAS domains was also
calculated. During the CONCOORD runs, 1000 structures were generated
and a damping factor of 0.25 was applied to avoid unreasonable side
chain geometries.
where xi and xj represent
the coordinates of atoms i and j in a
conformation, whereas xi,0 and xj,0 represent the average coordinates
of the atoms over the ensemble. The average is calculated over all
structures after they are superimposed on a reference structure to
remove overall translational and rotational motion. Diagonalizing this
matrix yields a set of eigenvectors and eigenvalues. The eigenvectors are directions in a 3N-dimensional space (where N
is the number of atoms), and motion along a single eigenvector
corresponds to concerted displacements of groups of atoms in Cartesian
space. The eigenvalues are a measure of the mean square fluctuation of the system along the corresponding eigenvectors. The eigenvectors are
sorted according to their eigenvalue, the first eigenvector having the
largest eigenvalue.
(Eq. 1)
atoms are taken into account, which sufficiently represent the large
motions of the protein backbone (48, 52). When the structures also
contain insertions and deletions such as in the PAS domains (Fig. 3),
further simplifications will need to be applied to reduce all of the
structures to a common core (44). Residues in the PAS domains that
overlapped structurally were selected by the DALI server (53), which
performs a pairwise comparison of secondary structure elements. The
results of the pairwise alignment on secondary structure were compared
to yield the common structural elements present in the PAS domains
(Fig. 3). For lysozyme, the negative control, an equal number of
residues was selected starting from the N terminus.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
25PYP--
The structure
of
25PYP was solved by molecular replacement and refined
to a 1.14-Å resolution (R-factor = 0.147, Rfree = 0.177) (Fig. 1 and Table
I). The asymmetric unit contains two protein molecules related by a non-crystallographic 2-fold rotation axis (Fig. 1). The molecules have a similar conformation with a root
mean square deviation of 0.77 Å on C
atoms. Compared with the wtPYP structure, the two molecules superimpose with root mean
square deviations of 0.99 and 0.76 Å, respectively. From these
superpositions, positional shifts of the C
atoms of the
mutant structures with respect to the positions of the C
atoms in wild type PYP are given in Fig.
2. The N terminus and the loops
consisting of residues 84-88, 98-101, and 111-117 in
25PYP have a different conformation than those in wild
type PYP. The different conformation of the
25PYP N
terminus compared with the equivalent residues in wtPYP is most
probably caused by the deletion of the first 25 residues (Fig.
3). When the first two residues at the N
terminus of
25PYP are excluded from the superposition, the root mean square deviation is reduced by ~0.2 Å. From the NMR
structure and the comparison of two crystal forms of wild type PYP, the
loop around residue Met-100 is observed to be flexible (7, 30, 54).
Close contacts between the two monomers in the asymmetric unit cell
affect the conformation of the "100 loop" (Fig. 1). The distance
between the backbone atoms of the two Met-100 residues is <4.0 Å and
could influence the conformation of this loop. The large differences in
the orientation of the 111-117 loop, which is disordered in this loop
and previously reported PYP structures (30, 31, 41), and the 84-88
loop can again be explained by crystals contacts (Fig. 1).
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Fig. 1.
Structure and maps of
25PYP. Ribbon
representation of the crystal structure of
25PYP. The
asymmetric unit cell contains two proteins. Secondary structure
elements are marked on the structures. The Fo
Fc ,
calc map just before including
the chromophore is shown in magenta, contoured at 2.5
.
Hydrophobic residues that have become solvent-exposed because of the
deletion of residues 1-25 are shown as green sticks.
Details of data collection and structure refinement
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Fig. 2.
Conformational changes. Positional
shifts of equivalent C atoms after superposition of the
two
25PYP monomers in the asymmetric unit on wtPYP and
on each other.
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Fig. 3.
Comparison of the PAS cores. Ribbon
representations of the structures of wtPYP (7), the
25PYP mutant described here, FixL (35), HERG (33), LOV2
(34), and turkey lysozyme (Protein Data Bank code 135L). Residues that
align structurally (see also Fig. 4) and are used for comparisons are
highlighted in red. The cofactors, if present, are drawn in
a yellow colored stick model. Homologous residues at similar
structural positions (Fig. 4) are depicted in green.
-sheet and the
C helix and another between the
-sheet and the two small helices of the N-terminal domain (7). For
the latter domain, residues Phe-28 (at the start of
A),
Trp-199, and Phe-121 (both on
E) extend toward the N-terminal domain
and have become solvent exposed in
25PYP yet appear not to self-dimerize through crystal contacts. This finding agrees with the
observation that in
25PYP the fluorescence emission of
Trp-119 (the only tryptophan in PYP) is enhanced and blue-shifted, suggesting a more polar environment (32). It is likely that it is these
solvent-exposed hydrophobic residues that cause the reported decrease
in temperature stability observed for
25PYP (32). In
summary, the structural data show that the removal of the first 25 residues does not significantly affect the overall fold of the PAS
domain core. This is in agreement with the spectrophotometric data on
25PYP, which show that the
25PYP
absorbance maximum associated with the chemical environment of the
chromophore and very sensitive to perturbation is only minimally
blue-shifted (32). Similar photocycle intermediates as in wtPYP are
present. Only the kinetics of the recovery reaction in the photocycle
is slowed down, which again could be associated with a less stable ground state structure due to exposure of several hydrophobic residues.
A, which
appears to act as a lid on the ligand binding pockets and undergoes
conformational changes in PYP (9, 11, 23, 25, 29, 30).
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Fig. 4.
Structure-based sequence alignment.
Alignment based on superposition of PYP, HERG, FixL, and LOV2 using
DALI (53) and WHAT IF servers (56). Black arrows denote
-strands, gray bars indicate
-helices, and labels
identify the secondary structure elements. Residues selected for
essential dynamics are underlined. Homologous residues in
the alignment are colored black for at least three identical
residues and colored in gray for at least three homologous
residues.
atoms as defined by a structure-based sequence alignment (Figs. 3 and 4). This encompassed 78 residues (marked in
Figs. 3 and 4) including most of the central
-sheet, the
A/B helices, and part of the long
C helix. The resulting ensembles of
structures were analyzed by essential dynamics (43), yielding separate
sets of eigenvectors that describe concerted fluctuations of atoms for
each protein. These are sorted by their corresponding eigenvalues,
i.e. the first eigenvector being the one with the largest
eigenvalue, revealing in all cases that the majority (>95%) of the
motion is covered by the first 5% (12) of the eigenvectors. With this
condensed description of flexibility in the individual PAS domains,
comparisons are facilitated.
Comparison of essential subspaces
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Fig. 5.
Comparison of PAS CONCOORD eigenvectors.
Cumulative mean squared inner products (57) of the first, second, and
third eigenvectors calculated from CONCOORD ensembles of FixL, HERG,
LOV2, 25PYP, and lysozyme against the first 12 eigenvectors calculated from the wtPYP CONCOORD ensemble.
traces. In Fig. 6, the minimum and
maximum projections of the first 3 eigenvectors of
25PYP
are compared. The central
-sheet appears to be relatively static,
whereas the loops, most notably the
A/
B segment, show the largest
fluctuations. In the PAS domains, this segment is generally important
for the binding of the ligand (7, 34, 35). For instance, in PYP,
residue Arg-52 on this segment is known to undergo a conformational
change (9, 11, 29, 30) upon isomerization of the chromophore. Glu-46,
which shares a proton with the chromophore, is also located in this
region (Fig. 4). Similarly, the
A/
B segment is involved in
binding the heme in FixL (35) and the FMN in LOV (34) both via
interaction with a phenylalanine, which lies at the equivalent position
of Glu-46 in PYP. In addition, a recent analysis of LOV domains has
revealed that the
A/
B region participates in a conserved salt
bridge, which is also observed in FixL and HERG and has been proposed
to be involved in signal transduction (55). It is noteworthy that
despite these similar interactions and conservation of conformational flexibility, there is almost no sequence conservation in the
A/
B segment.
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Fig. 6.
PYP conformational changes. Atomic
positional shifts as described by the first three eigenvectors of
25PYP are depicted as structures in Cartesian space. Two
structures are depicted corresponding to
2 nm (colored)
and +2 nm (transparent) along the eigenvectors. Relative
degrees of positional shifts are indicated from blue
(smallest fluctuations) to red (largest fluctuations). The
transparent structures indicate the direction of the motion.
CONCLUSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
25PYP
structure together with the recently determined of the LOV domain in
complex with FMN (34) allowed further structural comparisons of
the PAS family. Although these proteins have almost entirely dissimilar sequences, their structures are remarkably similar with the conserved parts, the
-sheet and the
A/B helices, making up the PAS core. This finding suggests that although these proteins bind different ligands, their signaling states are reached through similar
conformational changes. We investigated this by simulating the complete
PAS domain proteins that have been structurally defined to date and
extracting from that the structurally conserved core. An analysis of
the data shows that in particular the
A/B segment moves in a
concerted fashion. Thus, we propose that despite the absence of any
sequence conservation, the PAS domains are not only structurally
conserved but also share a common conformational flexibility that may
have evolved to (i) accommodate the various input signals from
different ligands/co-factors located at different positions in the
domain and (ii) transmit the sensing event to downstream transducer proteins.
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ACKNOWLEDGEMENTS |
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We thank Raafat Hamze for contributions in the early stages of the project. We thank the European Synchrotron Radiation Facility (Grenoble, France) for the time at beamline ID14-EH1.
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FOOTNOTES |
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* 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.
The atomic coordinates and the structure factors (code 1ODV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ To whom correspondence may be addressed. E-mail: dava@davapc1.bioch.dundee.ac.uk (D. M. F. vA.) or E-mail: W.Crielaard@science.uva.nl (W. C.).
Supported by a Wellcome Trust Career Development
Research Fellowship.
Published, JBC Papers in Press, March 14, 2003, DOI 10.1074/jbc.M301701200
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ABBREVIATIONS |
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The abbreviations used are: PAS, PER-ARNT-SIM; PER, periodic clock pattern; LOV, light oxygen voltage; PYP, photoactive yellow protein; pG, ground state; MES, 4-morpholineethanesulfonic acid; wt, wild type.
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