The crystal structure con-tains three copies of the cytochrome c6 molecule per asymmetric unit, and is characterized by an unusually high packing density, with solvent occupy-ing barely
Trang 1cytochrome c6 with an unusual sequence insertion
Wojciech Bialek1, Szymon Krzywda2, Mariusz Jaskolski2,3and Andrzej Szczepaniak1
1 Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, Poland
2 Department of Crystallography, Faculty of Chemistry, A Mickiewicz University, Poznan, Poland
3 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
Introduction
During photosynthesis, electron transfer between two
membrane-bound complexes, cytochrome b6f and
photosystem I, can be accomplished by the
copper-containing protein plastocyanin (PC) or the heme
protein cytochrome c6 [1,2] PC is found as a unique
electron carrier in higher plants Some algae and cyanobacteria express either PC or cytochrome c6, whereas others are able to produce both proteins, depending on copper availability [3] Cytochromes c6 are water-soluble, low-spin, heme-containing proteins
Keywords
cyanobacteria; cytochrome; high-resolution
structure; photosynthesis; Synechococcus
sp PCC 7002
Correspondence
A Szczepaniak, Department of Biophysics,
Faculty of Biotechnology, University of
Wroclaw, Przybyszewskiego 63 ⁄ 77, 51-148
Wroclaw, Poland
Fax: +48 71 3756234
Tel: +48 71 3756236
E-mail: andrzej.szczepaniak@ibmb.uni.wroc.pl
M Jaskolski, Department of
Crystallography, Faculty of Chemistry, A.
Mickiewicz University, Grunwaldzka 6,
60-780 Poznan, Poland
Fax: +48 61 829 1505
Tel: +48 61 829 1274
E-mail: mariuszj@amu.edu.pl
Database
Atomic coordinates and structure factors are
available from the Protein Data Bank under
the accession code 3DR0
(Received 5 May 2009, revised 9 June
2009, accepted 11 June 2009)
doi:10.1111/j.1742-4658.2009.07150.x
The structure of the reduced form of cytochrome c6 from the mesophilic cyanobacterium Synechococcus sp PCC 7002 has been determined at 1.2 A˚ and refined to an R-factor of 0.107 This protein is unique among all known cytochromes c6, owing to the presence of an unusual seven-residue insertion, KDGSKSL(44–50), which differs from the insertion found in the recently discovered plant cytochromes c6A Furthermore, the present pro-tein is unusual because of its very high content (36%) of the smallest resi-dues (glycine and alanine) The structure reveals that the overall fold of the protein is similar to that of other class I c-type cytochromes, despite the presence of the specific insertion The insertion is located within the most variable region of the cytochrome c6 sequence, i.e between helices II and III The first six residues [KDGSKS(44–49)] form a loop, whereas the last residue, Leu50, extends the N-terminal beginning of helix III Several spe-cific noncovalent interactions are found inside the insertion, as well as between the insertion and the rest of the protein The crystal structure con-tains three copies of the cytochrome c6 molecule per asymmetric unit, and
is characterized by an unusually high packing density, with solvent occupy-ing barely 17.58% of the crystal volume
Abbreviation
PC, plastocyanin.
Trang 2involved in the high-potential electron transport
chain They are characterized by low molecular mass
(80–90 amino acid residues in the mature protein), and
have a covalently bound heme group Once believed
to be absent in plants, cytochrome c6-like proteins,
now designated cytochrome c6A, have recently also
been discovered in plants [4,5] and in the green alga
Chlamydomonas reinhardtii[6]
In general, cytochromes c6 are characterized by high
redox potential (Em,7 of 330–360 mV) However, a
number of low redox potential cytochrome c6-like
pro-teins have been identified These include plant and algal
cytochrome c6A(Em,7of 70 mV [7]), and
cyanobacte-rial cytochrome c6C (Em,7 of 150 mV [8]) and
cyto-chrome c6B, for which a low redox potential has been
postulated as well [8] Members of these recently
identi-fied cytochrome c6subfamilies are characterized by the
presence of Val, Ile or Leu in place of a conserved Gln
residue located inside the cytochrome c6 heme pocket
Because of its low redox potential and the presence of
two additional Cys residues, cytochrome c6A has been
postulated to play a role in the formation of disulfide
bridges in thylakoid lumen proteins, rather than
func-tioning in the photosynthetic electron transport chain
[9] Interestingly, the replacement of the conserved Gln
residue by Val resulted in a decrease of the
cyto-chrome c6redox potential by 100 mV [cytochrome c6
from Phormidium laminosum [7] and Synechococcus
sp PCC 7002 (Bialek, unpublished results)], whereas
the opposite Valfi Gln substitution in cytochrome c6A
from Arabidopsis thaliana increased the midpoint
potential by approximately 100 mV [7]
The structures of cytochromes c6 from three
cyano-bacteria (Arthrospira maxima [10],
Synechococ-cus elongatus [11], and P laminosum [7]), one from a
red alga (Porphyra yezoensis [12]), one from a brown
alga (Hizikia fusiformis [13]), and four from green
algae (Monoraphidium brauni [14], C reinhardtii [15],
Scenedesmus obliquus [16], and Cladophora glomerata
[17]) have been determined, as has the structure of
cytochrome c6A from A thaliana [18] Together, these
data represent a large volume of information about
the sequences and structures of cytochrome c6
How-ever, the cytochrome c6 molecule from the mesophilic
cyanobacterium Synechococcus sp PCC 7002 (Em,7 of
320 mV [8]) is an exceptional protein in this class, as
it contains an unusual heptapeptide insertion beginning
after Tyr43 Moreover, the petJ1 gene, which encodes
cytochrome c6 in Synechococcus sp PCC 7002, could
not be inactivated or replaced with petJ
(cyto-chrome c6), petE (PC) or cytM (cytochrome cM) from
Synechocystis [19], unlike the petJ gene from
Synecho-cystis [20] Consequently, it appears that only the
native cytochrome c6 of Synechococcus can function properly in electron transport and ensure cell viability
To elucidate the characteristics of this unusual cyto-chrome c6molecule, and in particular to shed light on the role of the heptapeptide insertion, the crystal struc-ture of the protein in its reduced form has been deter-mined at a resolution of 1.2 A˚ In addition, this study
is a contribution towards a better understanding of the molecular determinants of the spectroscopic and electron transfer properties of this class of proteins
Results and Discussion
Structure description Overall, the structure of Synechococcus sp PCC 7002 cytochrome c6 reveals the characteristic properties of other class I cytochromes c The molecule is composed
of a single polypeptide chain wrapped around the heme group, which is bound to the protein by two thioether links at Cys14 and Cys17, as well as by His18 and Met65, which are the axial ligands in the coordination sphere of the iron atom (Fig 1) The polypeptide chain consists of four a-helices (I–IV) con-nected by loops The long N-terminal helix I (Ala3– His18) has a characteristic kink at Cys14 The two Cys residues forming the covalent links to the heme group and the fifth iron-coordinating ligand, His18, are part
of this helix As in other cytochrome c6 molecules, the three consecutive amino acids Ala19-Gly-Gly21 form a
310 helix followed by an X-loop ranging from Asn22
to Lys32 The X-loop separates helix I from the short helix II (Ala33–Tyr39) Residues Leu40–Tyr43 and Gly46–Ser49 form type IV and type VIII b-turns, respectively [21] The latter turn is formed entirely within the sequence of the heptapeptide insert that is characteristic of this cytochrome c6 protein only Leu50, which is also part of the insert, belongs to helix III, formed by Leu50–Asn60 Gln62–Met65 and Gly69–Leu72 in the following region are type II¢ and type IV b-turns, respectively [21] Helix IV, which is nearly perpendicular to helix I, runs from Asp74 to Glu89 Two residues, Asp86 and Lys92, located near the C-terminus, form a salt bridge stabilizing this part
of the protein fold
Conformation of the heptapeptide insertion
In general, high overall structural similarity is observed among all the compared cytochrome c6 molecules (Fig 2A, Table S1) The most striking structural difference between the present Synechococcus
sp PCC 7002 cytochrome c6 and all other
Trang 3cyto-chrome c6 molecules is the presence of the specific
insertion between helices II and III (Fig 2C) The
insertion consists of seven, mostly polar, amino acids
KDGSKSL(44–50) Its major part [GSKS(46–49)]
forms a type VIII b-turn Multiple hydrogen bonds
formed within this structure stabilize its conformation
These, as well as hydrogen bonds between the insert
residues and residues from other regions of the
pro-tein, are listed in Table 1 Additionally, the cationic
side chain of Lys48 is also involved in cation–p
inter-actions with Tyr56, which, in turn, is adjacent to
Gln57, the residue that is implicated in the control of
the heme redox potential Specifically, a structural
water molecule inside the heme pocket forms a
hydro-gen bond with the Gln57 side chain, and is thus
responsible for positioning of the c-amide towards the
heme group and within a van der Waals distance of a
methine carbon atom (CHA) of the porphyrin ring In
consequence, Gln57 is capable of tuning the redox
potential of the heme group [7,8]
An insert of three consecutive Asp residues has been found in the same region in cytochrome c6 from
A maxima [10], but its length, shape and chemical character are significantly different from those charac-terizing the present insertion of cytochrome c6 from Synechococcus sp PCC 7002 (Fig 2A,C) It is of note that an insertion has also been found in the sequence
of the protein from A thaliana However, the insertion
in that cytochrome c6A protein is not only longer but also located differently, namely between helices III and
IV, and is thus entirely different from the insertion dis-covered in the present protein Importantly, the cyto-chrome c6A loop contains two Cys residues that are also found in other cytochrome c6A molecules In neither case has the function of the insertions been completely elucidated
The heme prosthetic group Figure 3 depicts the hydrophobic heme pocket of the present cytochrome c6molecule By analogy with other cytochrome c prosthetic heme groups, the porphyrin moiety is slightly distorted into a saddle-shaped form
in all three molecules in the asymmetric unit The edge
of the pyrrole ring C and the propionic group D are exposed towards the solvent The heme group is cova-lently linked to the polypeptide chain through thioe-ther bonds from the Cys14 and Cys17 sulfhydryl groups to the two vinyl side chains The iron of the heme group is in the FeII oxidation state, maintained
by the presence of 20 mm dithionate in the mother liquor The iron ion is coordinated through His18 Ne2 and Met65 Sd, located at axial positions The latter residue has a gauche conformation along the Cb–Cc bond (torsion angle Ca–Cb–Cc–Sd of 50.0, 48.2 and 50.4 in molecules A, B and C, respectively) As in other cytochrome c6 structures, a hydrogen bond between the Nd1H donor of the axial His18 and the carbonyl oxygen atom of Asn22 serves to maintain the required orientation of the His ring with respect to the heme plane Interestingly, this is the only His present
in the protein The interactions of the nitrogen atoms
of the imidazole ring (Ne2–Fe and Nd1–HÆO) confirm that it is electrically neutral
The structure of reduced as well as oxidized cyto-chrome c6 from S obliquus has been described previ-ously [16] Comparison of both redox states revealed a different conformation of Pro62, which is adjacent to the Met61 axial ligand of the heme group The posi-tion of Cc of this Pro was found to change from exo
to endo upon oxidation The protein used in the pres-ent study was reduced with a 20-fold molar excess of dithionite in the crystallization buffers Pro66, which is
A
B
Fig 1 (A) Three-dimensional structure of the reduced
cyto-chrome c 6 from Synechococcus sp PCC 7002 The 3 10 helix
(yel-low) is followed by the X-loop (red) The specific insertion is shown
in magenta The N-terminus and C-terminus, as well as the two
Cys residues with covalent links to the heme group, are indicated.
(B) Electron density omit map contoured at 3.2r, showing the
heme group All structural figures were prepared in PYMOL [39].
Trang 4equivalent to Pro62 of the S obliquus protein, has the exo pucker, confirming the reduced state of the mole-cule (Fig S1) Although the conformation of this Pro may serve as an indicator of the protein redox state, the mechanism of this change is currently unknown Its elucidation will require a comparison of the present structure with a model of the oxidized molecule deter-mined at comparably high resolution
In total, four water molecules are present around the two propionate substituents (at positions 6 and 7)
of each heme group (Fig 3) Two of them (water
20⁄ water 33, water 18 ⁄ water 45 and water 24 ⁄ water 30
in molecules A, B, C, respectively) are located between
K44
L50 G46
A
C
B
Fig 2 (A) Structural alignment of 10 cytochrome c 6 and cytochrome c 6A molecules: Synechococcus sp PCC 7002 (green, this work, 3DR0); P yezoensis (light pink, 1GDV); H fusiformis (black, 2ZBO); M braunii (blue, 1CTJ); S obliquus (yellow, 1C6O); C glomerata (magenta, 1LS9); C reinhardtii (cyan, 1CYI); A maxima (red, 1F1F); P laminosum (gray, 2V08); and A thaliana (orange, 2DGE) The char-acteristic insertions present in the proteins from Synechococcus sp PCC 7002 and A maxima (bottom left) and A thaliana (top middle) are shown (B) Side chains of the KDGSKSL(44–50) insertion shown in ball-and-stick representation (C) Alignment of cytochrome c6and cytochrome c6Asequences prepared in ESPRIPT [40] White letters on a red background represent strictly conserved residues; similar resi-dues are shown as purple letters on a white background in boxes Insertions of Synechococcus sp PCC 7002, A maxima and A thaliana are shown in green, red and orange, respectively Above the sequences, the secondary structure elements of the present cytochrome c 6
molecule are shown.
Table 1 Hydrogen bonding of amino acids of the insertion (bold),
as calculated by WHATIF [38] Distances are given in A ˚
Donor Acceptor Molecule A Molecule B Molecule C
Trang 5the propionate groups and create a network of
hydro-gen bonds involving the propionates Propionate-6 is
also hydrogen-bonded with Lys29 and Gln62, whereas
propionate-7 has a hydrogen bond contact with Thr30
Additionally, a hydrogen bond between water 20 and
the Gln57 side chain is observed Table 2 presents a
complete list of hydrogen bonds within the three heme
pockets
Electrostatic surface potential
The low isoelectric point of the molecule, calculated as
pI 3.8 [8], results from the ratio of acidic (nine) to
basic (seven) amino acids (Fig 4) The only Arg,
Arg71, is present in all cytochrome c6 molecules,
except for the protein from S obliquus Arg71 creates
a minor basic patch on the otherwise highly negative
protein surface The basic centers are predominantly
Lys residues Interestingly, two of the six Lys residues
in the protein sequence of Synechococcus sp PCC 7002
cytochrome c6, Lys44 and Lys48, are located within
the unique insertion Their long side chains point away
from a bulge with negative surface potential created by
the atoms between them (Fig 4) All of the negative charge centers in this region (Lys44 O, Asp45 O, Od1 and Od2, Gly46 O, Ser47 O, and Oc) point towards the surface of the protein and contribute to the overall negative surface potential The major negative patch, however, is created mainly by the side chains of Glu51, Asp86, and Glu89 The surface potential of the molecule shows an additional slightly positive region, near the heme crevice, created by Lys29 and Lys44
Fig 3 The heme pocket of superposed molecules A (red), B
(green), and C (blue) Hydrogen bonds involving the depicted
resi-dues are listed in Table 2 Structural water molecules associated
with protein molecules A, B and C are shown in the corresponding
colors.
Table 2 Hydrogen bond distances (A ˚ ) within the heme pockets of molecules A, B, and C Asterisks indicate water molecules located between the heme propionate groups.
Molecule A
Molecule B
Molecule C
Heme O2D Water 34 ⁄ 496 ⁄ 137 2.61 2.87 2.64 Heme O2D Water 227 ⁄ 390 ⁄ 69 3.17 2.69 2.51 Heme O1D Water 227 ⁄ 390 ⁄ 69 3.22 3.38 3.16
Heme O1D Water 20 ⁄ 18 ⁄ 24 2.69 2.70 2.70 Heme O2A Water 33 ⁄ 45 ⁄ 30 2.85 2.80 2.83
*Water
20 ⁄ 18 ⁄ 24
*Water 33 ⁄ 45 ⁄ 30 2.58 2.61 2.61 Gln57 O e1 Water 20 ⁄ 18 ⁄ 24 2.70 2.70 2.75
Fig 4 Electrostatic surface potential distribution for PetJ1 The red zones correspond to negative potential, and blue zones correspond
to positive potential The color range spans )2 to 2 kT Side chains are shown as sticks The side chain of Glu89 assumes two confor-mations The pale blue island on the visible red face of the molecule corresponds to the NH3+ group of the N-terminus The figure was prepared in PYMOL using default settings of the APBS plugin [41].
Trang 6Interactions of aromatic residues
In total, there are seven aromatic residues in the
pro-tein sequence, not including the sole His, which acts as
a heme ligand The side chains of the three conserved
aromatic residues, Phe10, Tyr83, and Trp93, form a
triangular aromatic packing motif (Fig 5A) buried in
the hydrophobic core of the molecule In this motif,
C–H bonds at the edges of Tyr83 and Trp93 point to
the ring center of Phe10 (C–ring centroid distances
4.1–4.2 A˚) (Table S2) Similar C-H–p interactions are
also present in other cytochromes c6 with known crys-tal structures In some cytochrome c6A molecules, including that from A thaliana, whose crystal structure
is known, the Tyr of this ‘aromatic triangle’ is replaced
by Phe It is interesting to note that whereas the side chain of Trp93, owing to these interactions, is tightly wedged into a nearly ideally shaped pocket, the C-ter-minus of its main chain shows significant flexibility and has been modeled in two distinct conformations in all three molecules (Fig 5B)
Energetically significant cation–p interactions have been detected between Tyr43, Tyr56 and Phe68 and, respectively, Lys37, Lys48 and Arg71 by the capture software [22] Tyr56, although pointing with its OH group towards bulk solvent, is firmly held in place by two N-H–p interactions on both sides of the ring, from Lys48 (described above) and from the side chain amide
of Asn60 The latter interaction, which is very short (Table S2), may be especially important for stabiliza-tion of Tyr56 Similar interacstabiliza-tions stabilizing a key Tyr residue were noted previously in the ultrahigh-resolu-tion structure of bovine pancreatic trypsin inhibitor [23] In the present case, the amide N–p distance (3.29– 3.37 A˚) is even shorter than in the bovine pancreatic trypsin inhibitor structure (3.44–3.58 A˚) The stability
of Tyr56 may be important for the conformation of Gln57, and consequently for tuning the redox potential
of the protein Phe68 forms part of a conserved motif, Pro66-Ala-Phe68 This motif is adjacent to the second heme ligand, Met65, and Pro66 undergoes conforma-tional changes upon oxidation⁄ reduction [16] Whereas
in cyanobacterial, plant and red and brown algal cyto-chrome c6this Phe is conserved, it is replaced by Trp in green algae (Fig 2C) It is of note that Phe68 interacts with the single Arg found in cytochromes c6, Arg71 (Fig 5C) This Arg is required for cytochrome f oxida-tion [24] and photosystem I reducoxida-tion [25] In addioxida-tion, Phe68 forms an evident C–H–p interaction with the heme group itself (Table S2) All of the remaining aro-matic residues (Phe10, Tyr39, Tyr83, and Trp93) are involved in C–H–p interactions (Table S2)
Comparison of existing models of cytochrome c6 The three independent molecules of Synechococcus cytochrome c6 in the asymmetric unit are similar, with pairwise rmsd values of 0.30 (A⁄ C), 0.32 (B ⁄ C) and 0.36 A˚ (A⁄ B) for the Ca atoms (Fig 6) In general, the structures superpose very closely, but there are two regions with larger discrepancies The first one is the single Gly63, and the second is a stretch of six residues from Gly70 to Ala75, with the largest difference of 1.68 A˚ between Gly70 of chains B and C This large
A
B
C
Fig 5 Interactions of aromatic residues (A) The three aromatic
residues involved in a ‘triangular’ interaction in the protein core (B)
A pocket around Trp93 Note the tightly wedged aromatic side
chain of Trp93 and the dual conformation of its C-terminal group.
(C) N–H p interaction between the only Arg and Phe68 (A) and (C)
show 2F o F c electron density contoured at 1.2r.
Trang 7difference can be attributed to a peculiarity of the
packing arrangement involving molecules B and C, in
which the side chain of Asp74 of molecule C accepts
two strong hydrogen bonds from the main chain N–H
groups of Gly70 and Arg71 of molecule B In this
interaction, the carbonyl oxygen of Gly69 of
mole-cule B flips by 180, bringing the main chain nitrogen
atom of residue 70 much closer to the Od atom of the
adjacent Asp74 The changed geometry of this
frag-ment should be classified as reverse turn type III The
corresponding fragment in the other two molecules, as
in all known X-ray structures of c6-type cytochromes,
is classified as b-turn type II The higher lability of this
region in the otherwise very rigid and well-ordered
structure is not surprising, because, in general, the
region on the C-side of the heme moiety (i.e including
residues from the loop between helices II and III to
the C-terminus) is more flexible, and the acidic region
Asp73-Glu74-Asp75-Glu76 is a hot spot in the
struc-ture of cytochrome c6from M braunii [14]
There are 13 side chains that do not align in
molecu-lar superpositions, adopting different rotamers in one
or more of the three molecules With two exceptions,
all of these side chains are on the surface, and some of
them are clearly perturbed by crystal contacts The
first exception is Pro27, which in molecule C has the
exo pucker, whereas in the other two cases it is endo
The second exception is Ile84, which is buried inside
the molecular core but in molecule B has Cd in two
conformations Only 10 of the 261 side chains (3.8%)
in the asymmetric unit have the side chains in double
conformation Such a small number of alternative
con-formations is related to the unusual amino acid
com-position, where Ala accounts for 25.8% and Gly for
9.7% of all the residues in the amino acid sequence of
this cytochrome c6
Table S1 shows both the percentage of sequence
identity and the overall Ca rmsd values for pairs of
different cytochrome c6 structures The lowest Ca
backbone deviations are observed in the regions of helices I, III, and IV, as well as the 310helix and the X-loop consurf analysis [26] has revealed several resi-dues that are most conserved (data not shown) These residues are in the heme pocket area and are involved
in electron transfer Whereas they are located within regions of the lowest Ca discrepancies, the shortest helix II is sequentially the least conserved element among the cytochrome c6molecules The largest devia-tions are observed around the specific insertion of this cytochrome c6 molecule, i.e in the loop connecting helices II and III
Intermolecular contacts
As a consequence of the very low solvent content (17.58%) of the Synechococcus cytochrome c6 crystals, their structure is very densely packed, and a number of surface residues are involved in direct contacts with neighboring protein molecules The contacts between molecules A and B involve three hydrogen bonds from the main chain amide of Ala75 and the side chains of Thr28 and Lys29 to the main chain carbonyl groups
of, respectively, Asp45 (B), Gly70 (A), and Arg71 (A) There are only two direct hydrogen bonds between molecules A and C involving main chain atoms of molecule A, namely oxygen of Asn60 and nitrogen of Gly63, and the side chains of Lys29 and Tyr39 of mol-ecule C Three of the four direct hydrogen bonds between molecules B and C are formed by the carbox-ylic group of Asp74 of molecule C, which interacts with the N of Gly70, and both the Ng atoms of Arg71 The fourth bond is created between the Asp74 nitrogen atom (molecule B) and Gln52 Oe2
There are five direct hydrogen bonds between the three molecules in the asymmetric units and their sym-metry counterparts Three of them are created by Asn91, which uses its Nd(two bonds) and O for inter-actions with, respectively, the main chain atoms of
10
30
10 Fig 6 Stereoview of the superposition of
the main chain traces of the three indepen-dent molecules in the asymmetric unit, with residue numbering added for orientation The side chains of the two iron-coordinating residues (His18 and Met65) are shown as sticks Color code as in Fig 3.
Trang 8Ala7 and Gly20, and the side chain of Tyr83 The
latter side chain is also a hydrogen bond donor to the
Gln89 O Gln87 and Asn90 are linked through an
NeÆOdhydrogen bond
Because of the low solvent content, the surface area
buried on intermolecular contacts (including
asym-metric unit and symmetry-related protein molecules) is
quite high, 41.7% (molecule A), 45.8% (molecule B),
and 47.1% (molecule C)
Crystal packing and water structure
With three independent molecules of cytochrome c6 in
the crystallographic asymmetric unit, the
volume-to-mass ratio VM is 1.49 A˚3ÆDa)1, corresponding to a
solvent content of merely 17.58% There are only four
protein structures deposited in the Protein Data Bank
that have lower reported solvent content (VS) (1XEK,
2J70, 2DUY, and 3C00) However, the number of
wrongly attributed Matthews coefficients and solvent
volumes in structures deposited in the Protein Data
Bank is astonishingly high Searching the Protein Data
Bank for protein structures with VS between 5.00%
and 17.57% gave 29 structures Calculations of the VM
and VS values (Matthews program, CCP4 [27]) for
those structures returned only four cases with VS
within the search interval Therefore, we would not be
surprised if the actual number of structures with lower
VSwas higher
The solvent molecules modeled in the electron
den-sity maps correspond to 252 H2O sites with full
occu-pancy and 272 with partial occuoccu-pancy, which are
equivalent to 404 effective water molecules Although
the number of partial water molecules may seem high,
the average water occupancy is 0.76, which is very
close to the value of 0.78 determined for a protein
structure at ultimate resolution (0.65 A˚) where solvent
occupancies were refined [28] Considering the low
sol-vent content of this crystal, one can conclude that
essentially all water molecules in the solvent region
have been accounted for in this atomic model of the
crystal structure Additionally, there are two partially
occupied sulfate ions One of them forms two
hydro-gen bonds with the main chain nitrohydro-gen atoms of
Asp74 (2.80 A˚) and Ala67 (2.86 A˚) and with a water
molecule (2.43 A˚) The other one forms two hydrogen
bonds with Ng2 (3.12 A˚) and Ne (3.03 A˚) of Arg71
and with two water molecules (2.79 and 2.77 A˚)
Conclusions
In summary, cytochrome c6 from Synechococcus
sp PCC 7002 has an overall structure that is similar
to that of other previously characterized cyto-chrome c6 molecules However, unlike other cyto-chrome c6 molecules, the Synechococcus sp PCC 7002 protein has a unique insertion of seven amino acid residues [KDGSKSL(44–50)], which is entirely differ-ent from the insertion found in type c6A cyto-chromes The presence of the insertion in the most variable region of cytochrome c6 might suggest that
it does not play any role in the cell On the other hand, the inability to inactivate or replace the gene encoding this cytochrome c6 by PC or cytochrome c6 from Synechocystis [19] is a strong indication that the presence of the insertion is essential for this organism The question of whether the Synechococcus
sp PCC 7002 insertion does indeed have a biological function is currently being studied by site-directed mutagenesis
Experimental procedures
Protein expression and purification
Escherichia coli strain DH5a was cotransformed with pUCJ1 and pEC86 plasmids The former harbors a gene encoding mature cytochrome c6 from Synechococcus
sp PCC 7002, whereas the latter harbors the heme matura-tion genes [29] Protein was expressed basically as described elsewhere [8] Briefly, 5 mL of overnight culture was used
to inoculate 1.7 L of LB medium supplemented with 1 mm FeCl3 in a 2 L flask Cultures were grown for 72–96 h at
30C with agitation at 150 r.p.m and harvested, and the periplasmic proteins were released by lysozyme treatment (1 mgÆmL)1) Subsequently, cytochrome c6 was purified as outlined in [8]
Protein crystallization
The hanging-drop vapor diffusion method was used for the crystallization experiments Cytochrome c6(1 mm in 10 mm Tris buffer, pH 7.5) was reduced by the addition of sodium dithionate to a final concentration of 20 mm Drops con-taining equal volumes (1 lL) of protein and reservoir solu-tions were equilibrated against 1 mL of reservoir solution
in 24-well Linbro plates at 19C Red crystals suitable for X-ray analysis were obtained from 10 mm sodium Hepes (pH 6.2) and 2.2 m ammonium sulfate over a period of
1 week
X-ray diffraction data collection
A single crystal was scooped through a solution of 30% glycerol and 70% reservoir solution (v⁄ v) and placed in a stream of cold N2gas at 100 K Diffraction data were col-lected on the X11 synchrotron beamline (k = 0.8170 A˚) at
Trang 9the EMBL Outstation, c⁄ o DESY, Hamburg, using a
MAR 165 mm CCD detector Two passes were collected,
using different crystal-to-detector distances, oscillations (1
and 0.5) and exposure times to ensure reliable
measure-ment of high-resolution data as well as of the strong
low-angle reflections The Synechococcus sp PCC 7002
cytochrome c6crystals belong to space group P32with unit
cell dimensions a = 82.88 A˚ and c = 28.28 A˚ and diffract
to at least 1.226 A˚ The mean overall redundancy is 5.0,
and it is 2.8 in the highest-resolution shell (1.260–1.226 A˚)
The data are 98.7% complete with an overall Rintof 0.076
Post-refinement of the crystal parameters during scaling
indicated low mosaicity throughout the data collection
(0.29) The diffraction data were indexed, integrated and
scaled using the hkl2000 [30] package, and the final
statis-tics are included in Table 3
Structure solution and refinement
The structure was solved by the molecular replacement method as programmed in molrep [31], using diffraction data in the 20.0–3.5 A˚ resolution range and the cyto-chrome c6model from the green alga M brauni [14] (Protein Data Bank accession number: 1CTJ) as the molecular probe Analysis of the Matthews volume [32] indicated the pres-ence of two or three protein molecules in the asymmetric unit (2.24 or 1.49 A˚3ÆDa)1, solvent content 45.05% or 17.58%) Molecular replacement calculations confirmed that there were three independent protein chains The cor-rect space group enantiomorph, P32, was deduced from a comparison of the translation function solutions in the two alternative space groups (P32⁄ P31with CC = 0.430⁄ 0.235,
R= 0.454⁄ 0.526)
The structure was refined anisotropically using the maxi-mum-likelihood algorithm as implemented in refmac [33], including the TLS parameters [34] For free R-factor calcu-lations [35], 1274 reflections were randomly selected from the full resolution range of 50.0–1.226 A˚ After the first round of refmac refinement (isotropic, no TLS parame-ters), the model was rebuilt in quanta [36] The final refined model is characterized by an R-factor of 0.107 and
an Rfree of 0.138 for all 62 908 reflections between 50 and 1.226 A˚, and has an rmsd from ideal bond lengths of 0.020 A˚ The very high quality of the electron density maps allowed the identification of 524 (including 252 fully occu-pied) water sites and of two partially occupied sulfate ions Stereochemical analysis of the final model using procheck [37] indicated that there are no residues with generously allowed or unfavorable backbone dihedral angles, and that 87.1% of all residues are in the core region
of the Ramachandran plot The statistics of the refinement are shown in Table 3
Acknowledgements
Some of the calculations were carried out in the Poz-nan Metropolitan Supercomputing and Networking Center This work was funded in part by grants
N N204 245635 and N N303 3856 33 from the Minis-try of Science and Higher Education
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... determination of thecrystal structure of cytochrome c6 and comparison with
plastocyanin Structure 3, 1159–1169
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Yeates... Higher plants
contain a modified cytochrome c6 Trends Plant Sci 7,
244–245
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relation-ship of cytochrome c6 and plastocyanin... JA, Wastl J, Evans TJ, Bendall DS, Luisi BF & Howe CJ (2006) Structure of cytochrome c6A, a novel dithio-cyto-chrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications