Download Structure of c-Phycocyanin from the Thermophilic

doi:10.1006/jmbi.2001.5030 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 313, 71±81
Structure of c-Phycocyanin from the Thermophilic
Ê:
Cyanobacterium Synechococcus vulcanus at 2.5 A
Structural Implications for Thermal Stability in
Phycobilisome Assembly
Noam Adir*, Yelena Dobrovetsky and Natalia Lerner
Department of Chemistry and
Institute of Catalysis, Science
and Technology, Technion Israel Institute of Technology
Technion City, Haifa
32000, Israel
The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechochoccus vulcanus has
Ê resolution. The crysbeen determined by molecular replacement to 2.5 A
Ê,
tal belongs to space group R32 with cell parameters a ˆ b ˆ 188.43 A
Ê , a ˆ b ˆ 90 , g ˆ 120 , with one (ab) monomer in the asymc ˆ 61.28 A
metric unit. The structure has been re®ned to a crystallographic R factor
Ê . The crystals were
of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 A
grown from phycocyanin (ab)3 trimers that form (ab)6 hexamers in the
crystals, in a fashion similar to other phycocyanins. Comparison of the
primary, tertiary and quaternary structures of the S. vulcanus phycocyanin structure with phycocyanins from both the mesophilic Fremyella
diplsiphon and the thermophilic Mastigocladus laminosus were performed.
We show that each level of assembly of oligomeric phycocyanin, which
leads to the formation of the phycobilisome structure, can be stabilized in
thermophilic organisms by amino acid residue substitutions. Each substitution can form additional ionic interactions at critical positions of each
association interface. In addition, a signi®cant shift in the position of ring
D of the B155 phycocyanobilin cofactor in the S. vulcanus phycocyanin,
enables the formation of important polar interactions at both the (ab)
monomer and (ab)6 hexamer association interfaces.
# 2001 Academic Press
*Corresponding author
Keywords: photosynthesis; antenna; X-ray crystallography; protein
structure; protein stability
Introduction
Oxygenic photosynthesis is initiated by the
absorption of visible light by a variety of pigmentprotein antenna complexes bound to Photosystems
Abbreviations used: APC, allophycocyanin; Cc-PC,
Cyanidium caldarium phycocyanin; DM, n-dodecyl-b-D
maltopyranoside; Fd-PC, Fremyella diplosiphon
phycocyanin; Hepes, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; LHC, light harvesting
complex; Ml-PC, Mastigoclaudus laminosus phycocyanin;
Mes, 2-(N-morpolino)ethanesulfonic acid; PC,
phycocyanin; PCB, phycocyanobilin cofactor; PE,
phycoerythrin; rms, root mean square; PEG4000, polyethylene glycol Mrˆ4000; PSII, Photosystem II; S7-PC,
Synechococcus sp PCC7002 phycocyanin; Se-PC,
Synechcoccus elongatus phycocyanin; Sv-PC,
Synechcoccus vulcanus phycocyanin.
E-mail address of the corresponding author:
[email protected]
0022-2836/01/010071±11 $35.00/0
I and II.1,2 The absorbed energy is ef®ciently transferred via additional accessory antenna pigments,
into the reaction center, where photochemistry
occurs.1,3 The photosynthetic reaction centers of all
oxygenic species are remarkably homologous on
both the protein sequence and co-factor levels.4,5
There are however two types of antenna systems,
of completely different composition, assembly and
attachment to the reaction center. All plants and
green algae contain a collection of transmembrane
pigment protein light-harvesting complexes
(LHCs), encoded by different members of the CAB
gene family, which contain non-covalently bound
chlorophyll a and b, and carotenoids as pigments.
These pigment/protein complexes are found in
varying amounts bound to the reaction center complexes.6 The LHC in cyanobacteria, red algae, cryptomonads and glaucophytes are large multi protein
structures called phycobilisomes,2,3,7 ± 9 which are
bound to the cytoplasmic side of the reaction cen# 2001 Academic Press
72
ter. Each phycobilisome contains a core and rods,
built up of stacks of hexamers of monomers that
contain two protein subunits, a and b, which are
quite homologous. Each phycobilisome can contain
varying ratios of different members of this protein
family: phycoerythrin (PE, lmax ˆ 560 nm), phycocyanin (PC, lmax ˆ 620 nm), allophycocyanin
(APC, lmax ˆ 650 nm) and a number of other variants. The rods also contain linker proteins situated
in the central trimer cavity,10,11 however selfassembly into hexamers occurs in vitro and during
crystallization12 in the absence of linker proteins as
well.
The evolution of the phycobiliproteins was studied in depth by Apt et al.9 By phylogenetic analysis and sequence alignment they proposed that all
rod biliproteins (PC, PE and PEC) arose from a
common ancestor which itself arose in a number of
steps from a single genetic ancestor of the whole
protein class. When PCs from different species are
compared, it can be seen that those belonging to
thermophilic organisms do not cluster together,
indicating that each thermophile arose separately.
Ward and Castenholz13 described the distribution
of thermophiles as restricted to speci®c geographical habitats for some species, while other species
(such as Mastigocladus laminosus) appear to be
widely spread out.
Crystal structures of a number of phycobiliproteins have been determined.11,12,14 ± 26 All structures
show a great deal of similarity, with some differences in trimer packing in the crystallographic unit
cell. Of the PC structures solved, there are representatives of mesophilic species (Fremyella
diplosiphon18 and Synechococcus sp. PCC700216), a
moderate thermophile (Cyanidium caldarium25) and
a thermophile (Mastigocladus laminosus17).
In the work presented here, we have determined
the crystal structure of PC from a thermophilic
organism (Tgrowth ˆ 55-60 C), Synechococcus vulcaÊ . Structural information
nus (Sv-PC) to 2.5 A
obtained by analysis of the Sv-PC crystal structure,
in comparison to other PC structures, has been
used to identify common factors that enhance the
stability of phycobilisome assemblies, at elevated
temperatures.
Results and Discussion
Amino acid sequence
The amino acid sequence of Sv-PC was determined by DNA sequencing of PCR ampli®cation
products of the aPC and bPC genes from isolated
genomic DNA. The sequences have been deposited
in the NCBI GeneBank, with accession numbers
AF333175 and AF333174 for the a and b subunits,
respectively. The degree of homology with other
phycocyanin proteins is high, as has been shown
for a large group of DNA sequences.9 The Sv-PC
sequences are very similar to the sequences determined for Synechococcus elongatus, (Se-PC) with
only a single residue change (IleA95 to ValA95 in
Ê
Structure of Phycocyanin from S. vulcanus at 2.5 A
Se-PC). Figure 1 shows a comparison between the
Sv-PC sequences and sequences of the other PC
proteins whose three-dimensional structures have
been determined: Mastigocladus laminosus (Ml-PC),
Cyanidium caldarium (Cc-PC), Synechococcus sp
PCC7002 (S7-PC, previously called Agmenellum
quadruplicatum) and Fremyella diplosiphon (Fd-PC).
Of these, Ml is a thermophile (growth temp. 5560 C), Cc grows at intermediate temperatures
(45 C) and Fd and S7 are mesophiles. Table 2
shows the respective level of identity in the primary sequence between the Sv-PC and the other
PCs, the rms difference in the Ca trace and percentage of residues that are potentially charged (C),
polar (P) or non-polar (NP). This information will
be used in our discussion of properties leading to
thermal stability.
Quality of the structure
The high degree of homology of residue
sequence is manifested in the similar three-dimensional structures. The overall chain fold shows the
typical phycobiliprotein eight a-helical, globin-like
structure (Figure 2). The re®ned Sv-PC structure is
well within all geometric criteria. The rms deviÊ and
ation for bond lengths and angles is 0.007 A
1.18 , respectively (Table 1). The backbone conformations are all within the allowed regions on the
Ramachandran plot (data not shown), except for
ThrB77, which has been shown to have an anomalous conformation ( ˆ 89.5 , ˆ 146.8 ) in all
Table 1. Data collection and re®nement statistics
Data collection
Space group
Cell dimensions
Ê)
Resolution range (A
Observations (unique)
Average I/s (last shell)
Rsymm (last shell) (%)
Completeness (last shell) (%)
Redundancy (last shell)
Refinement statistics
Ê)
Resolution range (A
Reflections in work set (test set)
Rcryst (last shell) (%)
Rfree (last shell) (%)
RMS deviations
Ê)
bond length deviation (A
angle deviation (degrees)
Ê 2)
Average B-factor (A
Final model
Total number of atoms
Protein
Non-protein atoms
Co-factor
Water
Covalent modification
Number of amino acids
Cofactor molecules
Matthews coefficient
R32
Ê,
a ˆ b ˆ 188.12 A
Ê,
c ˆ 60.947 A
g ˆ 120 50-2.5 (2.59-2.5)
112515 (14116)
17.6 (4.8)
9.0 (37.0)
97.5 (99.3)
5.6 (6.4)
500-2.5 (2.6-2.5)
12734 (1382)
20.2 (24.7)
24.4 (27.9)
0.007
1.18
26.4
2717
2499
129
89
1 (N-methyl-AsnB77)
332
3
2.71 (54 % solvent)
Structure of Phycocyanin from S. vulcanus at 2.5 AÊ
73
Figure 1. The alignment of phycocyanin amino acid sequences
from cyanobacterial species for
which crystal structures have been
determined. Sequence numbering
includes gaps according to the convention introduced in reference 41.
Residues mentioned in the text
where signi®cant changes exist are
in bold. *, indicates a mesophile; **,
indicates a weak thermophile
(Tgrowth ˆ 45 C). ***, indicates a
thermophile (Tgrowth ˆ 55-60 C).
PC structures. This residue is in close contact with
a chromophore (A84) of the adjacent subunit in the
trimeric phycobilisome assembly.16
Calculated electron density is suf®cient to interpret the positions of all side-chains and chromophores (Figure 3(a)). Somewhat weaker density
(and higher B-factors) is located in three sections of
the b-subunit. The ®rst section is in the loops connecting helices X and Y (ArgB15) which forms part
of the (ab) monomer association interface and rod
formation. The second segment connects helix Y
with helix A (AsnB29-GluB33). The third section
resides in helix F between ArgB110 and GluB117,
and probably plays a role in PC-linker protein
interactions.15 While these sections are not highly
conserved within the phycobiliprotein family,9
they are quite conserved within the phycocyanin
subfamily. 88 water molecules were modeled into
the structure. A larger fraction of the bound water
molecules (65 %) were found located in the vicinity of the a-subunit. This may be due to the more
rigid structure of the a subunit in the Sv-PC crystals, as indicated by lower all around B-factors.
Chromophores
Each of the two subunits in the monomer has a
thio-linked chromophore situated at position 84
between helices E and F,16 which also interact with
adjacent monomers in the (ab)3 trimer. The b-subunit has an additional chromophore at position 155
which is on the outside of the trimeric ring and
may be important for intra and inter-rod energy
transfer.25 The chromophores at positions A84 and
B84 have the same stereochemistry as described
previously for the homologous chromophores
found in Fd-PC, Ml-PC and Cc-PC. The B155 chromophore is also similar for rings A, B and C, but
has a different conformation in the vicinity of ring
D compared to three of the four PCs. In the Sv-PC
structure, there is a 65 rotation of the D ring
towards the b subunit (Figure 3(b)) in comparison
to the Fd-PC. This conformation is similar to that
found in the Cc-PC structure.25 The change in
orientation in the case of the Cc-PC chromophore
was attributed to changes in the packing of the
interface between trimers in the process of (ab)6
hexamer formation.25 Since this is not the case for
Sv-PC, for which packing of trimers is very similar
74
Ê
Structure of Phycocyanin from S. vulcanus at 2.5 A
Figure 2. Overall structure and schematic ribbon representation of the (ab) phycocyanin monomer. Yellow
ribbon, a subunit; blue ribbon, b subunit. Phycocyanobilin cofactors are represented in red stick. Water molecules are red spheres.
to both Ml-PC and Fd-PC (see below), it can be
assumed that it is the sequence similarity between
Sv-PC and Cc-PC that induces the conformational
change. The most important of these changes are
AspA28 (PheA28 in Fd-PC) and LysA32, which
spatially replaces GlnA33 in Fd-PC. Ml-PC has a
similar sequence in the vicinity of the chromophore, however the Ml-PC D ring is rotated almost
a full 180 around the carbon link, bringing the
heterocycle nitrogen atom towards the b subunit.
This can be considered one of the major differences
between the thermophilic Sv-PC and Ml-PC.
Quaternary association
All phycobiliprotein structures are produced by
the same associative process: (ab) monomer subunit association ! (ab)3 trimer association ! (ab)6
hexamer association ! rods.27 We ascertained by
size-exclusion HPLC that the isolated Sv-PC that
was crystallized was in the form of the trimeric
(ab)3 unit (data not shown), indicating that the ®rst
two steps of the association process involve stable
interactions and do not require the presence of linker proteins. A major question, which has been
addressed in the cases of previously determined
phycobilin protein structures, is how the hexamers
and rods are formed, and whether the crystal form
of these proteins is relevant to the in vivo state.
In the Sv-PC structure, the (ab)6 hexamer is
formed by (ab)3 trimer head to head association
(with contacts mostly between a-subunits), and
Figure 3. Electron density omit maps (Fo ÿ Fc) of SvPC chromophores. (a) Map contoured (at 2s) following
re®nement, superimposed on the PCBA84 chromophore
molecule shown in stick representation. The four pyrrole
rings are labeled A through D. (b) Stereoview of map
calculated after rigid-body re®nement step (contoured at
1.5s), superimposed on the PCBB155 chromophore molecule shown in green stick representation. The homologous chromophore from Fd-PC (red) was superimposed
on the Sv-PC to show the difference in position of ring
D between the two species. The b subunit is drawn in
blue ribbon form.
with each hexamer situated directly above the
adjacent hexamer. This has been the case for many
of the phycobiliprotein structures determined so
far. The Cc-PC structure shows a slightly altered
hexamer packing (due to a rotation of one hexamer
by about 30 relative to the adjacent hexamer.25 It
would appear from the differences in hexamer
interactions in the different species that crystalpacking in¯uences this level of assembly, and the
in vivo state might be somewhat different. However, the global quaternary association process to
the (ab)6 hexamer level does not appear to be very
different in thermophilic organisms as compared to
mesophiles. Since these associations must be stable
at the elevated growth temperatures of the thermophilic species, we probed the ®ne details of the
different levels of subunit association to try and
identify the structural basis for thermal stability.
Structure of Phycocyanin from S. vulcanus at 2.5 AÊ
75
Table 2. Comparison between phycocyanin subunits of different species
PC type
Subunit
Sv-PCc
a
b
a
b
a
b
a
b
a
b
Ml-PCd
Fd-PCc
Cc-PCc
S7-PCd
Primary sequence r.m.s. difference
homologya (%)
Ca traceb
86
72
80
83
84
72
67
0.62
0.56
0.45
0.40
0.52
0.73
-
Ê
A
Ê
A
Ê
A
Ê
A
Ê
A
Ê
A
Charged residues
(%)
Polar residues
(%)
Non-polar
residues (%)
19
19
18
21
17
19
17
20
19
22
49
45
50
49
50
45
50
47
49
53
51
55
50
51
50
55
50
53
51
47
a
NCBI BLAST.
InsightII.
PDB reference codes: Sv-PC-1I7Y; Fd-PC-1CPC; Cc-PC-1PHN.
d
Coordinates for Ml-PC and S7-PC have not been deposited in the PDB.
b
c
Basis for thermal stability
The function of the phycobilisome antenna is the
ef®cient absorption and transfer of excitation
energy into PSII. A secondary function is as a
reservoir of nutrients for the cyanobacterial cell in
cases of starvation,28 indicating that the proteinprotein associations that form the phycobilisome
are reversible. Each functional (ab) monomer can
be subdivided into seven functional zones involved
in: chromophore binding (two dissimilar domains
in the b subunit), (ab) monomer formation, (ab)3
trimer formation, (ab)6 hexamer formation, ((ab)6)n
rod formation (n signifying a varying number of
hexamers of different types within each rod) and
phycobilisome formation by rod to rod, and rod to
core association. It stands to reason that for each of
these zones some variation may occur in the thermophilic species, which could strengthen interactions and prevent disassembly. Indeed it has
been shown both in vivo and in vitro29 that phycocyanin, within the phycobilisome structure, is
stable to elevated temperatures. Absorption and
¯uorescence measurement clearly show that phycocyanin disassembly occurred only at temperatures above 70 C.
A recent comprehensive study was performed
by Szilagyi and Zavodsky30 in which all of the proteins found in the Protein Data Bank (up to 1998)
for which structures exist for both mesophilic and
thermophilic species were compared for a variety
of characteristics. These authors suggested that
thermostability could be conferred on a protein by
changing one or more of six parameters: cavities,
hydrogen bonding, ion pairs, secondary structure,
surface polarity and amino acid composition. The
conclusion of this report was that none of the parameters analyzed could be identi®ed as the most
signi®cant factor in obtaining thermostability in all
structures examined. The only characteristic that
had a statistically signi®cant change was the number of intermediate and weak ionic interactions
formed. Among the proteins examined were the a
and b subunits of Fd-PC (mesophile) and Cc-PC
(weak thermophile) and it was shown that while
the a subunits of these two species are quite similar, the b subunits show some variation in almost
every parameter examined. Ion pairing, hydrogen
bonds and cavities all appeared to increase stabilization of the Cc-PC when compared to the Fd-PC.
This report did not analyze association domains in
homo- or hetero-oligomeric proteins such as the
PCs. In a different report, Kannan and
Vishveshwara31 proposed that the number of
patches of aromatic residues could enhance thermal stability, but found no difference between CcPC and Fd-PC. If we perform a similar analysis,
comparing the higher temperature thermophiles
Sv-PC and Ml-PC with the mesophilic Fd-PC we
may see a number of changes in the interface
regions that might be indicative of the common
needs for thermal stability in the associative process (Tables 3 and 4).
Species specific differences in primary and
secondary structures
Warren & Petsko32 proposed that the amino acid
compositions of a-helices are different in proteins
of thermophilic origin as compared to homologous
Table 3. Exposed and buried surfaces in PCs of different species
PC type
Sv-PC
Ml-PC
Fd-PC
Total exposed surface
Ê 2)
area (A
(ab) Monomer buried
Ê 2)
interface surface area (A
(ab)3 Trimer buried
Ê 2)
interface surface area (A
(ab)6 Hexamer buried
Ê 2)
interface surface area (A
15470
15737
15352
3093
3001
3019
1242
1304
1203
2535
2575
2608
Ê
Structure of Phycocyanin from S. vulcanus at 2.5 A
76
Table 4. Unsatis®ed hydrogen bond donors and acceptors and cavities in different PCs
(ab) monomer interface cavity volumes
PC type
Sv-PC
Ml-PC
Fd-PC
Unsatisfied hydrogen bond
Total unsatisfied hydrogen donors or acceptors in the
bond donors or acceptors
(ab) monomer interface
29
34
27
proteins from mesophiles. The most prominent
changes in the protein from thermophilic species
(as opposed to mesophiles) are increases in Tyr,
Gly and Gln residues and decreases in Val, Glu,
His, Cys and Asp. In the Szilagyi and Zavodsky
report,30 different modi®cations of the amino acid
composition were identi®ed: an increase in charged
residues, and a decrease in the number of phenylalanine residues.
In the case of the PCs, the level of total homology is quite high (Table 2 and Apt et al.9) and
thus the amino acid compositions are quite similar.
There are no signi®cant differences in the number
of charged or polar residues in the thermophilic
PCs when compared to those from mesophiles.
Both Sv-PC and Ml-PC a subunits have a signi®cant increase in glutamine residues and a decrease
in valine residues when compared to the Fd-PC
a subunit, which is in accord with Warren and
Petsko.32
As indicated by the small rms differences
between the positions of the a-carbons in the structures of Sv-PC, Ml-PC and Fd-PC (Table 2), the secondary and tertiary structures are all very similar.
Thus thermal stability cannot be attributed to
stabilization due to an increase or conversion in
secondary structure.33 No increase in the number
of aromatic residue patches could be identi®ed,
negating this factor as a possible source of thermal
stability.31
8
11
4
Number
Ê 3)
Volume(A
9
12
6
350
440
365
Unsatisfied internal hydrogen bond donors
and acceptors
An increase in the number of hydrogen bonds
has also been proposed to promote thermal stability.33,30 The number of unsatis®ed hydrogen bond
donors and acceptors in the three PC types examined here were analyzed using the program
WHAT IF.35 The results are summarized in Table 4.
It appears from this analysis that there is actually
an increase in unsatis®ed hydrogen bond donors
and acceptors in the thermophilic PCs. Other
instances of such apparent ``destabilizing'' changes
were identi®ed in the Szilagyi report. It should
however be mentioned that such unsatis®ed
hydrogen-bond donors/acceptors could still serve
as sites for the formation of ionic/polar interactions.
Internal cavities and pockets
A decrease in the number, surface area and
volume of internal cavities and pockets has also
been indicated as providing thermal stability.30 The
three PC types were analyzed for presence of cavities, speci®cally in the interface region of the (ab)
monomer using the CASTp program,36 to try and
ascertain whether thermal stability is obtained by
excluding solvent from cavities. As summarized in
Table 4, the Ml-PC actually has a considerably
greater number and volume of cavities in the interface region, thus it would appear that thermal stability is not achieved by cavity minimization.
Exposed and buried surface areas
Charged/polar interactions
An additional parameter, which has been
suggested to increase thermal stability, is the area
of exposed and buried surfaces.33 In the case of the
PCs one could imagine that subunit interactions
could be strengthened by increasing their overlap,
thereby decreasing the solvent accessible surface.
Surface areas were calculated using the Lee and
Richards method as implemented in CNS.34 Surfaces analyzed were the exposed surfaces of the
two subunits and the buried interfaces formed
when the (ab) monomer, the (ab)3 trimer and (ab)6
hexamer are formed (Table 3). All differences
are within 5 % of the mean surface areas, and no
signi®cant trend in the thermophilic PCs was
identi®able.
Szilagyi and Zavodsky proposed in their extensive study of proteins from thermophilic
organisms30 that the only parameter with a consistent and signi®cant contribution towards achieving
thermal stability is the number of charged pairs. In
their analysis they distinguished between strong
Ê ), intermediate (6 A
Ê ) and weak (8 A
Ê ) inter(4.0 A
actions. We wished to try and ascertain whether
there were additional speci®c ionic interactions in
the two thermophilic PCs (as compared with the
mesophilic Fd-PC) in the interface regions that
might stabilize the different oligomeric stages of
phycobilisome assembly. Contacts were identi®ed
and measured using both the contact program in
CNS37 and CONTACT in the CCP4 suite.38
Structure of Phycocyanin from S. vulcanus at 2.5 AÊ
The (ab) monomer interface
In Figure 4(a), the (ab) monomer association
interface from Sv-PC is shown. Two major patches
of interactions arising from residues from the a
and b subunits ¯ank the interface. In addition,
there is an interaction with intermediate strength
between AspA28 and AsnB35. In ML-PC AspA28
is replaced with an asparagine that can also form a
polar contact with AsnB35. In mesophiles such as
Fd-PC, a phenylalanine is found in position A28,9
which cannot form a polar interaction with
AsnB35. As mentioned above, the B155 chromo-
77
phore is bound by many residues of the b subunit.
Ring D forms contacts with the a subunit, which
are of a more polar nature, mainly due to the
amino acid change at position A28. Thus A28, B35
and ring D of the cofactor form a polar triad
(Figure 4(b)), strengthening the monomer association domain.
The (ab)3 trimer formation interface
Following removal of the phycobiliproteins from
the thylakoid membranes by high salt treatment,
most of the oligomeric associations are lost and the
most prevalent form of PC is the (ab) monomer.
However, a signi®cant fraction of the protein
remains in the (ab)3 trimeric form, and indeed this
fraction was used in the crystallization protocol
described here. Trimer formation in all three PC
types is quite similar to that described for S7-PC.15
The overlap interface between the a subunit of one
(ab) monomer and the b subunit of an adjacent
monomer is considerably less than that found in
the (ab) monomer interface or for (ab)6 hexamer
interface (Table 3). Ionic/polar interactions are
similar to those described for S7-PC.15 In general,
there are many contacts spread out over the entire
overlap region (Figure 5(a)). In both Sv-PC and CcPC, B68 is a polar glutamine, which forms a close
contact with ArgA86 (Figure 5(b)), while in Fd-PC
and Ml-PC B68 is an alanine. An additional new
contact is formed between SerA72 and ArgB57
(Figure 5(b)). SerA72 is a proline in both Fd-PC
and Ml-PC, and thus cannot form this interaction.
Thus for this interface, no consistent change can be
found in all thermophiles that can be attributed to
increasing thermal stability. All of the hydrophobic
interactions indicated previously for the mesophilic
PCs15,18 are conserved in the thermophilic species,
and this may be the primary mode of trimer stability.
The (ab)6 hexamer formation interface
Figure 4. Monomer association interface in Sv-PC.
(a) Full monomer interface. a subunit in yellow and b
subunit in blue, residues forming polar interactions are
shown in sticks and PCBB155 is shown in thin red lines.
The large number of potential polar interactions at both
sides of the monomer interface are conserved in all phycocyanins while those at the center are found in thermophiles. (b) Close up of the additional polar interaction in
Sv-PC. AspA28, AsnB35 and PCBB155 are in stick representation with carbon colored green, oxygen in red
and nitrogen in blue. PCBB155 is overlayed with a segment of an omit electron density map (Fo ÿ Fc) calculated after re®nement completion (contoured at 2s, light
gray lines). Red dotted lines show potential polar interactions.
While the hexamer interface zone is quite extensive, with a buried surface area twice that of the
trimer interface zone (Table 3), upon their removal
from the membrane, phycobilisome rods dissociate
into trimers and monomers. This is perhaps indicative of the importance of phycobilisome linker proteins in proper assembly in vivo.10,11 However in
the Sv-PC crystals, as in most of the other previously determined phycobiliprotein structures, PC
trimers readily form hexamers (Figure 6(a)). Thus
crystal lattice packing parameters may induce the
formation of inter-trimer contacts lost when the linker proteins are removed. In addition to the many
ionic contacts described for the previously determined structures,15,16 additional contacts are found
in the Sv-PC and Ml-PC hexamer interface regions
(Figure 6(b)). AspA28 interacts with LysA32 in SvPC (and AsnA28 interacts with ArgA33 in Ml-PC)
of the adjacent monomer. These positions are
PheA28, ArgA32 and GlnA33 in Fd-PC. As indi-
78
Figure 5. (ab)3 Trimer association interface in Sv-PC.
(a) Full trimer interface. a subunit in yellow, b subunit
in blue, b0 subunit (from adjacent (ab) monomer) in
orange. Residues forming polar interactions are shown
in sticks and PCBB155 is shown in red. (b) Close-up of
additional polar interactions (thin dotted lines) found
between a subunit residues and b0 residues in Sv-PC
(stick Figures).
cated for the (ab) monomer interface, ring D of the
B155 cofactor is also involved in formation of polar
interactions in the (ab)6 hexamer interface
(Figure 6(b)). GluA161 is closely paired with
AsnB21, which is the shorter and less polar serine
in Fd-PC. Thus, as for the (ab) monomer interface,
it appears that thermophilic PCs have evolved
similar solutions for increasing thermal stability at
the hexamer assembly level.
Conclusions
The functional and organizational requirements
of the protein components of phycobilisome antennae have lead to a high level of optimization as
indicated by the high degree of homology in both
sequence and structure. However, certain modi®cations must take place in order to preserve proper
function in extreme environments. We have shown
Ê
Structure of Phycocyanin from S. vulcanus at 2.5 A
Figure 6. (ab)6 Hexamer association interface in
Sv-PC. (a) Full hexamer interface, proteins are shown as
a-carbon traces with the top (ab) monomer in yellow
and blue and the bottom (ab) monomer in orange and
purple. Residues that form polar interactions are shown
in ball and stick (top monomer) or stick (bottom monomer) representations. (b) Close up of the hexamer interface showing additional polar interactions (dotted lines)
in the Sv-PC structure that are not present in mesophiles. Residues from the symmetry-related a subunit
(orange ribbon) are denoted A0 .
by structure comparison that in thermophiles, a
number of residue changes to either polar or
charged types may be important for the stabilization of both the (ab) monomer and (ab)6 hexamer
association domains, while only the Sv-PC structure has additional stabilizing contacts at the (ab)3
trimer association domain. These contacts should
now be studied to understand better the mechanism of phycobilisome formation.
Materials and Methods
Protein isolation and characterization
Synechococcus vulcanus cells were grown in a ten liter
temperature-controlled growth chamber on BG11 medium supplemented with 5 % CO2 in air at 55 C, with ¯uorescent lamp illumination. Cells were grown for three
to four days, collected by centrifugation, resuspended in
Structure of Phycocyanin from S. vulcanus at 2.5 AÊ
buffer A (20 mM Hepes (pH 7.5), 10 mM MgCl2, 10 mM
CaCl2) and 1 M sucrose, and treated with lysozyme
(1 mg/ml) for one hour at 50 C. The cells were then
passed through a Yeda press cell disrupter under 25
atm. N2. The cells were diluted and centrifuged for two
minutes at 2000 rpm in a Sorvall T21 centrifuge to
remove cell debris. The thylakoid membranes were then
pelleted by a ten minute centrifugation at 16,000 rpm
and the resulting pellet was resuspended in buffer A
with 0.5 M sucrose to 1.2-2 mg chlorophyll a/ml.
Bulk phycobiliproteins were removed from the thylakoid membranes by treatment with 0.5 M KCl and 0.1 %
dodecyl-b-D-maltoside (DM), followed by separation of
the thylakoids by centrifugation. We found that a fraction of PC tightly bound to the thylakoid membranes
formed superior crystals. This fraction was separated
from DM solubilized Photosystem II by DEAE chromatography with buffer B (50 mM Hepes, pH 8.0) as the
mobile phase. Phycocyanin was treated with PEG 4000
to remove impurities and then puri®ed by DEAE chromatography using a 0-300 mM NaCl gradient in buffer
C (50 mM Mes, pH 6.0). The puri®ed protein exhibited
an absorption maximum at 618 nm. The purity of the
isolated PC was determined by absorption spectroscopy
and SDS-PAGE (data not shown), which also showed
the absence of linker proteins. Size-exclusion-HPLC of
Ê column, Polymer Labthe puri®ed Sv-PC (PL-GF1000 A
oratories) with buffer B with 0.1 M NaCl as the mobile
phase af®rmed that the puri®ed PC was in trimeric form
(data not shown). PC was dialyzed against buffer D
(50 mM Tris, pH 8.0), and concentrated to 10 mg/ml.
Sequence determination
S. vulcanus total DNA was isolated using a DNA isolation kit (Biological Industries, Kibbutz Beit Haemek).
The genes encoding for the a and b phycocyanin
subunits were ampli®ed by PCR using the following
oligonucleotide
primers:
for
the
a
subunit,
50 -ATGAAAACGCCGATTACTACTGAAGCT-30
and
50 -TTAGCTGAGGCGGCGTAGTCGATG-30 ; for the b
subunit, 50 -ATGCTAGATGCATTTGCCAAA-30 and 50 TTAGGCAACGCGGCAGCGGC-30 . Ampli®cation was
carried out for 30 cycles of one minute, using Pfu DNA
polymerase. The PCR products were puri®ed using the
GFX PCR DNA puri®cation kit (Amersham Phamacia
Biotec Inc.). DNA sequencing was performed at the Laboratory of DNA Analysis, Hebrew University Jerusalem.
Gene analysis and comparisons were performed using
Gene Runner (Hastings Software Inc., version 3.05).
79
scaled and merged using the DENZO/SCALEPACK
Ê and
suite.39 The ®nal data were 97.5 % complete to 2.5 A
were used for structure determination by molecular
replacement with AmoRe.38,40 The search model was
based on a single (ab) monomer from the Fremyella
diplosphon PC structure (PDB code 1CPC,18), including
the chromophores. In the search model, residues in the
Sv-PC sequence that were different from that of Fd-PC
were substituted accordingly. Model parameters for
Ê,
molecular
replacement
were
a ˆ b ˆ c ˆ 80 A
Ê,
a ˆ b ˆ g ˆ 90 with an integration radius of 25 A
Ê . The rotation function
using all data between 8 to 4 A
calculation resulted in a solution with a high correlation
coef®cient (Cc ˆ 23.3). The position of one (ab) monomer
in the asymmetric was determined using the translation
function (Cc ˆ 72.4, R-factor ˆ 32.3 %). Following rigidbody re®nement, the R-factor was 26.1 % in the range of
Ê , and correlation coef®cient increased to 81.5.
8 to 4 A
The solution in the R32 space group had better packing
properties than for R3, and re®nement was performed in
this space group.
Refinement
The structure was re®ned using CNS.37 Crossvalidation was performed by omitting 10 % of the data
for calculation of Rfree. The starting R-factor was 26.3 for
Ê . Following simulated
all data between 50 and 2.5 A
annealing, B-factor re®nement and water molecule
addition, the structure was inspected against electron
density maps calculated in X®t in the Xsight/InsightII
program suite (MSI Inc.). Extensive use of calculated
omit maps were used to manually adjust and con®rm
the positions of all residues and co-factors. The ®nal
model had a crystallographic R-factor of 20.2 % and an
Rfree of 24.4 %.
Analysis of parameters affecting protein
thermal stability
The Sv-PC structure was analyzed in comparison to
the mesophilic Fd-PC structure (PDB code 1CPC) and the
thermophilic Ml-PC structure. The coordinates of the
later structure were kindly provided by Drs Huber and
Schneider of the Max-Planck-Institut fur Biochemie, Martinsried. Internal cavities were identi®ed and quanti®ed
using the program CASTp.36 Unsatis®ed hydrogen bond
donors and acceptors were identi®ed using the program
WHAT IF.35 Possible ion pairs were identi®ed by
distance criterion using CNS37 and CCP4.38
Crystallization
Sv-PC was crystallized in the presence of 5 %
PEG4000, 50 mM Tris (pH 8.0) and 2.5-5 mg protein/ml
hanging drop vapor diffusion at 22 C. Crystals typically
grew within 3-14 days.
Atomic co-ordinates
The structural coordinates for the Synechococcus vulcanus C-phycocyanin structure have been deposited in the
Protein Data Bank under ID code 1I7Y.
Data collection and structure determination
Sv-PC crystallized in the R3 or R32 space group with
Ê , c ˆ 61.276 A
Ê and
cell dimensions of a ˆ b ˆ 188.432 A
Ê . A data set
g ˆ 120 and diffracted maximally to 2.1 A
was collected (at 293 K) using two crystals on a Rigaku
R-Axis IIc system (Table 1). The X-ray source was a RU200 rotating anode generator (Rigaku, Japan) operating
at 9 kW. A graphite monochromator was used to ®lter
Ê wavelength. The data were
the CuKa radiation of 1.54 A
Acknowledgments
This work was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and
Humanities (366/99) and the Fund for the Promotion of
Research at the Technion. We thank Itzhak Ohad for his
meaningful suggestions.
Ê
Structure of Phycocyanin from S. vulcanus at 2.5 A
80
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Edited by R. Huber
(Received 10 May 2001; received in revised form 20 August 2001; accepted 20 August 2001)