Structure of C-Phycocyanin from the Thermophilic Cyanobacterium Synechococcus Vulcanus At

doi:10.1006/jmbi.2001.5030availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (2001) 313, 71±81

Structureofc-PhycocyaninfromtheThermophilic Cyanobacterium Synechococcus vulcanus at 2.5 AÊ : Structural Implications for Thermal Stability in Phycobilisome Assembly NoamAdir*,YelenaDobrovetskyandNataliaLerner

Department of Chemistry and The crystal structure of the light-harvesting phycobiliprotein, c-phycocya- Institute of Catalysis, Science ninfromthethermophiliccyanobacteriumSynechochoccusvulcanushas and Technology, Technion - been determined by molecular replacement to 2.5 AÊ resolution. The crys- Israel Institute of Technology tal belongs to space group R32 with cell parameters a b 188.43 AÊ , Technion City, Haifa c 61.28 AÊ , a b 90 , g 120 , with one (ab) monomer in the asym- 32000, Israel metric unit. The structure has been re®ned to a crystallographic R factor of 20.2 % (R-free factor is 24.4 %), for all data to 2.5 AÊ . The crystals were

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 substi- tution 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 Keywords: photosynthesis; antenna; X-ray crystallography; protein *Corresponding author structure; protein stability

Introduction IandII.1,2Theabsorbedenergyisef®cientlytrans- ferred via additional accessory antenna pigments, Oxygenic photosynthesis is initiated by the into the reaction center, where photochemistry absorption of visible light by a variety of pigment- occurs.1,3Thephotosyntheticreactioncentersofall protein antenna complexes bound to Photosystems oxygenic species are remarkably homologous on boththeproteinsequenceandco-factorlevels.4,5 There are however two types of antenna systems, Abbreviations used: APC, allophycocyanin; Cc-PC, of completely different composition, assembly and Cyanidium caldarium phycocyanin; DM, n-dodecyl-b-D maltopyranoside; Fd-PC, Fremyella diplosiphon attachment to the reaction center. All plants and phycocyanin; Hepes, 4-(2-hydroxyethyl)-1- green algae contain a collection of transmembrane piperazineethanesulfonic acid; LHC, light harvesting pigment protein light-harvesting complexes complex; Ml-PC, Mastigoclaudus laminosus phycocyanin; (LHCs), encoded by different members of the CAB Mes, 2-(N-morpolino)ethanesulfonic acid; PC, gene family, which contain non-covalently bound phycocyanin; PCB, phycocyanobilin cofactor; PE, chlorophyll a and b, and carotenoids as pigments. phycoerythrin; rms, root mean square; PEG4000, poly- These pigment/protein complexes are found in ethylene glycol M 4000; PSII, Photosystem II; S7-PC, r varying amounts bound to the reaction center com- Synechococcus sp PCC7002 phycocyanin; Se-PC, 6 Synechcoccus elongatus phycocyanin; Sv-PC, plexes. TheLHCincyanobacteria,redalgae,cryp- Synechcoccus vulcanus phycocyanin. tomonads and glaucophytes are large multi protein E-mail address of the corresponding author: structurescalledphycobilisomes,2,3,7±9whichare [email protected] bound to the cytoplasmic side of the reaction cen-

0022-2836/01/010071±11 $35.00/0 # 2001 Academic Press 72 Structure of Phycocyanin from S. vulcanus at 2.5 AÊ ter. Each phycobilisome contains a core and rods, Se-PC).Figure1showsacomparisonbetweenthe built up of stacks of hexamers of monomers that Sv-PC sequences and sequences of the other PC contain two protein subunits, a and b, which are proteins whose three-dimensional structures have quite homologous. Each phycobilisome can contain been determined: Mastigocladus laminosus (Ml-PC), varying ratios of different members of this protein Cyanidium caldarium (Cc-PC), Synechococcus sp family: phycoerythrin (PE, lmax 560 nm), phyco- PCC7002 (S7-PC, previously called Agmenellum cyanin (PC, lmax 620 nm), allophycocyanin quadruplicatum) and Fremyella diplosiphon (Fd-PC). (APC, lmax 650 nm) and a number of other var- Of these, Ml is a thermophile (growth temp. 55- iants. The rods also contain linker proteins situated 60 C), Cc grows at intermediate temperatures inthecentraltrimercavity,10,11howeverself- (45C)andFdandS7aremesophiles.Table2 assembly into hexamers occurs in vitro and during shows the respective level of identity in the pri- crystallization12intheabsenceoflinkerproteinsas mary sequence between the Sv-PC and the other well. PCs, the rms difference in the Ca trace and percen- The evolution of the phycobiliproteins was stu- tage of residues that are potentially charged (C), diedindepthbyAptetal.9Byphylogeneticanal- polar (P) or non-polar (NP). This information will ysis and sequence alignment they proposed that all be used in our discussion of properties leading to rod biliproteins (PC, PE and PEC) arose from a thermal stability. 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 Quality of the structure compared, it can be seen that those belonging to thermophilic organisms do not cluster together, The high degree of homology of residue indicating that each thermophile arose separately. sequence is manifested in the similar three-dimen- WardandCastenholz13describedthedistribution sional structures. The overall chain fold shows the of thermophiles as restricted to speci®c geographi- typical phycobiliprotein eight a-helical, globin-like cal habitats for some species, while other species structure(Figure2).There®nedSv-PCstructureis (such as Mastigocladus laminosus) appear to be well within all geometric criteria. The rms devi- widely spread out. ation for bond lengths and angles is 0.007 AÊ and Crystalstructuresofanumberofphycobilipro- 1.18,respectively(Table1).Thebackboneconfor- teinshavebeendetermined.11,12,14±26Allstructures mations are all within the allowed regions on the show a great deal of similarity, with some differ- Ramachandran plot (data not shown), except for ences in trimer packing in the crystallographic unit ThrB77, which has been shown to have an anoma- cell. Of the PC structures solved, there are repre- lous conformation (È 89.5 , É 146.8 ) in all sentatives of mesophilic species (Fremyella diplosiphon18andSynechococcussp.PCC700216),a moderatethermophile(Cyanidiumcaldarium25)and 17 athermophile(Mastigocladuslaminosus ). Table 1. Data collection and re®nement statistics In the work presented here, we have determined the crystal structure of PC from a thermophilic Data collection organism (T 55-60 C), Synechococcus vulca- Space group R32 growth Ê Cell dimensions a b 188.12 AÊ , nus (Sv-PC) to 2.5 A. Structural information c 60.947 AÊ , obtained by analysis of the Sv-PC crystal structure, g 120 in comparison to other PC structures, has been Resolution range (AÊ ) 50-2.5 (2.59-2.5) used to identify common factors that enhance the Observations (unique) 112515 (14116) stability of phycobilisome assemblies, at elevated Average I/s (last shell) 17.6 (4.8) Rsymm (last shell) (%) 9.0 (37.0) temperatures. Completeness (last shell) (%) 97.5 (99.3) Redundancy (last shell) 5.6 (6.4) Refinement statistics Results and Discussion Resolution range (AÊ ) 500-2.5 (2.6-2.5) Reflections in work set (test set) 12734 (1382)

Amino acid sequence Rcryst (last shell) (%) 20.2 (24.7) Rfree (last shell) (%) 24.4 (27.9) The amino acid sequence of Sv-PC was deter- RMS deviations mined by DNA sequencing of PCR ampli®cation bond length deviation (AÊ ) 0.007 angle deviation (degrees) 1.18 products of the aPC and bPC genes from isolated Average B-factor (AÊ 2) 26.4 genomic DNA. The sequences have been deposited Final model in the NCBI GeneBank, with accession numbers Total number of atoms 2717 AF333175 and AF333174 for the a and b subunits, Protein 2499 respectively. The degree of homology with other Non-protein atoms Co-factor 129 phycocyanin proteins is high, as has been shown Water 89 foralargegroupofDNAsequences.9TheSv-PC Covalent modification 1 (N-methyl-AsnB77) sequences are very similar to the sequences deter- Number of amino acids 332 mined for Synechococcus elongatus, (Se-PC) with Cofactor molecules 3 only a single residue change (IleA95 to ValA95 in Matthews coefficient 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 con- vention 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 45 C). ***, indicates a thermophile (Tgrowth 55-60 C).

PC structures. This residue is in close contact with Chromophores a chromophore (A84) of the adjacent subunit in the Each of the two subunits in the monomer has a trimericphycobilisomeassembly.16 thio-linked chromophore situated at position 84 Calculated electron density is suf®cient to inter- betweenhelicesEandF,16whichalsointeractwith pret the positions of all side-chains and chromo- adjacent monomers in the (ab)3 trimer. The b-sub- phores(Figure3(a)).Somewhatweakerdensity unit has an additional chromophore at position 155 (and higher B-factors) is located in three sections of which is on the outside of the trimeric ring and the b-subunit. The ®rst section is in the loops con- may be important for intra and inter-rod energy necting helices X and Y (ArgB15) which forms part transfer.25ThechromophoresatpositionsA84and of the (ab) monomer association interface and rod B84 have the same stereochemistry as described formation. The second segment connects helix Y previously for the homologous chromophores with helix A (AsnB29-GluB33). The third section found in Fd-PC, Ml-PC and Cc-PC. The B155 chro- resides in helix F between ArgB110 and GluB117, mophore is also similar for rings A, B and C, but and probably plays a role in PC-linker protein has a different conformation in the vicinity of ring interactions.15Whilethesesectionsarenothighly D compared to three of the four PCs. In the Sv-PC conservedwithinthephycobiliproteinfamily,9 structure,thereisa65 rotationoftheDring towardsthebsubunit(Figure3(b))incomparison they are quite conserved within the phycocyanin to the Fd-PC. This conformation is similar to that subfamily. 88 water molecules were modeled into foundintheCc-PCstructure.25Thechangein the structure. A larger fraction of the bound water orientation in the case of the Cc-PC chromophore molecules (65 %) were found located in the vicin- was attributed to changes in the packing of the ity of the a-subunit. This may be due to the more interface between trimers in the process of (ab)6 rigid structure of the a subunit in the Sv-PC crys- hexamerformation.25Sincethisisnotthecasefor tals, as indicated by lower all around B-factors. Sv-PC, for which packing of trimers is very similar 74 Structure of Phycocyanin from S. vulcanus at 2.5 AÊ

Figure 2. Overall structure and schematic ribbon representation of the (ab) phycocyanin monomer. Yellow ribbon, a subunit; blue ribbon, b subunit. Phycocyanobi- lin cofactors are represented in red stick. Water molecules are red spheres.

Figure 3. Electron density omit maps (Fo Fc)ofSv- PC chromophores. (a) Map contoured (at 2s) following to both Ml-PC and Fd-PC (see below), it can be re®nement, superimposed on the PCBA84 chromophore assumed that it is the sequence similarity between molecule shown in stick representation. The four pyrrole Sv-PC and Cc-PC that induces the conformational rings are labeled A through D. (b) Stereoview of map change. The most important of these changes are calculated after rigid-body re®nement step (contoured at AspA28 (PheA28 in Fd-PC) and LysA32, which 1.5s), superimposed on the PCBB155 chromophore mol- spatially replaces GlnA33 in Fd-PC. Ml-PC has a ecule shown in green stick representation. The homolo- similar sequence in the vicinity of the chromo- gous chromophore from Fd-PC (red) was superimposed phore, however the Ml-PC D ring is rotated almost on the Sv-PC to show the difference in position of ring a full 180 around the carbon link, bringing the D between the two species. The b subunit is drawn in blue ribbon form. 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 with each hexamer situated directly above the All phycobiliprotein structures are produced by adjacent hexamer. This has been the case for many the same associative process: (ab) monomer sub- of the phycobiliprotein structures determined so far. The Cc-PC structure shows a slightly altered unit association ! (ab)3trimerassociation!(ab)6 hexamerassociation!rods.27Weascertainedby hexamer packing (due to a rotation of one hexamer size-exclusion HPLC that the isolated Sv-PC that byabout30relativetotheadjacenthexamer.25It was crystallized was in the form of the trimeric would appear from the differences in hexamer

(ab)3 unit (data not shown), indicating that the ®rst interactions in the different species that crystal- two steps of the association process involve stable packing in¯uences this level of assembly, and the interactions and do not require the presence of lin- in vivo state might be somewhat different. How- ker proteins. A major question, which has been ever, the global quaternary association process to addressed in the cases of previously determined the (ab)6 hexamer level does not appear to be very phycobilin protein structures, is how the hexamers different in thermophilic organisms as compared to and rods are formed, and whether the crystal form mesophiles. Since these associations must be stable of these proteins is relevant to the in vivo state. at the elevated growth temperatures of the thermo-

In the Sv-PC structure, the (ab)6 hexamer is philic species, we probed the ®ne details of the formed by (ab)3 trimer head to head association different levels of subunit association to try and (with contacts mostly between a-subunits), 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

Primary sequence r.m.s. difference Charged residues Polar residues Non-polar PC type Subunit homologya(%) Catraceb (%) (%) residues (%) Sv-PCca--194951 b - - 19 45 55 Ml-PCda-0.62AÊ185050 b 86 0.56 AÊ 21 49 51 Fd-PCca720.45AÊ175050 b 80 0.40 AÊ 19 45 55 Cc-PCca830.52AÊ175050 b 84 0.73 AÊ 20 47 53 S7-PCda72- 194951 b 67- 225347 a NCBI BLAST. b InsightII. c 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.

Basis for thermal stability surface polarity and amino acid composition. The conclusion of this report was that none of the par- The function of the phycobilisome antenna is the ameters analyzed could be identi®ed as the most ef®cient absorption and transfer of excitation signi®cant factor in obtaining thermostability in all energy into PSII. A secondary function is as a structures examined. The only characteristic that reservoirofnutrientsforthecyanobacterialcellin had a statistically signi®cant change was the num- casesofstarvation,28indicatingthattheprotein- ber of intermediate and weak ionic interactions protein associations that form the phycobilisome formed. Among the proteins examined were the a are reversible. Each functional (ab) monomer can and b subunits of Fd-PC (mesophile) and Cc-PC be subdivided into seven functional zones involved (weak thermophile) and it was shown that while in: chromophore binding (two dissimilar domains the a subunits of these two species are quite similar, the b subunits show some variation in almost in the b subunit), (ab) monomer formation, (ab)3 every parameter examined. Ion pairing, hydrogen trimer formation, (ab)6 hexamer formation, ((ab)6)n rod formation (n signifying a varying number of bonds and cavities all appeared to increase stabiliz- hexamers of different types within each rod) and ation of the Cc-PC when compared to the Fd-PC. phycobilisome formation by rod to rod, and rod to This report did not analyze association domains in core association. It stands to reason that for each of homo- or hetero-oligomeric proteins such as the these zones some variation may occur in the ther- PCs. In a different report, Kannan and mophilic species, which could strengthen inter- Vishveshwara31proposedthatthenumberof actions and prevent disassembly. Indeed it has patches of aromatic residues could enhance ther- beenshownbothinvivoandinvitro29thatphyco- mal stability, but found no difference between Cc- cyanin, within the phycobilisome structure, is PC and Fd-PC. If we perform a similar analysis, stable to elevated temperatures. Absorption and comparing the higher temperature thermophiles ¯uorescence measurement clearly show that phy- Sv-PC and Ml-PC with the mesophilic Fd-PC we cocyanin disassembly occurred only at tempera- may see a number of changes in the interface tures above 70 C. regions that might be indicative of the common A recent comprehensive study was performed needs for thermal stability in the associative pro- bySzilagyiandZavodsky30inwhichallofthepro- cess(Tables3and4). teins found in the Protein Data Bank (up to 1998) for which structures exist for both mesophilic and Species specific differences in primary and thermophilic species were compared for a variety secondary structures of characteristics. These authors suggested that thermostability could be conferred on a protein by Warren&Petsko32proposedthattheaminoacid changing one or more of six parameters: cavities, compositions of a-helices are different in proteins hydrogen bonding, ion pairs, secondary structure, of thermophilic origin as compared to homologous

Table 3. Exposed and buried surfaces in PCs of different species

Total exposed surface (ab) Monomer buried (ab)3 Trimer buried (ab)6 Hexamer buried PC type area (AÊ 2) interface surface area (AÊ 2) interface surface area (AÊ 2) interface surface area (AÊ 2) Sv-PC 15470 3093 1242 2535 Ml-PC 15737 3001 1304 2575 Fd-PC 15352 3019 1203 2608 76 Structure of Phycocyanin from S. vulcanus at 2.5 AÊ

Table 4. Unsatis®ed hydrogen bond donors and acceptors and cavities in different PCs

(ab) monomer interface cavity volumes Unsatisfied hydrogen bond Total unsatisfied hydrogen donors or acceptors in the PC type bond donors or acceptors (ab) monomer interface Number Volume(AÊ 3) Sv-PC 29 8 9 350 Ml-PC 34 11 12 440 Fd-PC 27 4 6 365

proteins from mesophiles. The most prominent Unsatisfied internal hydrogen bond donors changes in the protein from thermophilic species and acceptors (as opposed to mesophiles) are increases in Tyr, Gly and Gln residues and decreases in Val, Glu, An increase in the number of hydrogen bonds His, Cys and Asp. In the Szilagyi and Zavodsky has also been proposed to promote thermal stabi- 33,30 report,30differentmodi®cationsoftheaminoacid lity. Thenumberofunsatis®edhydrogenbond composition were identi®ed: an increase in charged donors and acceptors in the three PC types exam- residues, and a decrease in the number of phenyl- ined here were analyzed using the program 35 alanine residues. WHATIF. TheresultsaresummarizedinTable4. In the case of the PCs, the level of total hom- It appears from this analysis that there is actually ologyisquitehigh(Table2andAptetal.9)and an increase in unsatis®ed hydrogen bond donors thus the amino acid compositions are quite similar. and acceptors in the thermophilic PCs. Other There are no signi®cant differences in the number instances of such apparent ``destabilizing'' changes of charged or polar residues in the thermophilic were identi®ed in the Szilagyi report. It should PCs when compared to those from mesophiles. however be mentioned that such unsatis®ed Both Sv-PC and Ml-PC a subunits have a signi®- hydrogen-bond donors/acceptors could still serve cant increase in glutamine residues and a decrease as sites for the formation of ionic/polar inter- in valine residues when compared to the Fd-PC actions. a subunit, which is in accord with Warren and Petsko.32 Internal cavities and pockets As indicated by the small rms differences between the positions of the a-carbons in the struc- A decrease in the number, surface area and turesofSv-PC,Ml-PCandFd-PC(Table2),thesec- volume of internal cavities and pockets has also ondary and tertiary structures are all very similar. beenindicatedasprovidingthermalstability.30The Thus thermal stability cannot be attributed to three PC types were analyzed for presence of cav- stabilization due to an increase or conversion in ities, speci®cally in the interface region of the (ab) secondarystructure.33Noincreaseinthenumber monomerusingtheCASTpprogram,36totryand of aromatic residue patches could be identi®ed, ascertain whether thermal stability is obtained by negating this factor as a possible source of thermal excluding solvent from cavities. As summarized in stability.31 Table4,theMl-PCactuallyhasaconsiderably 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 Szilagyi and Zavodsky proposed in their exten- suggested to increase thermal stability, is the area sive study of proteins from thermophilic ofexposedandburiedsurfaces.33Inthecaseofthe organisms30thattheonlyparameterwithaconsist- PCs one could imagine that subunit interactions ent and signi®cant contribution towards achieving could be strengthened by increasing their overlap, thermal stability is the number of charged pairs. In thereby decreasing the solvent accessible surface. their analysis they distinguished between strong Surface areas were calculated using the Lee and (4.0 AÊ ), intermediate (6 AÊ ) and weak (8 AÊ ) inter- RichardsmethodasimplementedinCNS.34Sur- actions. We wished to try and ascertain whether faces analyzed were the exposed surfaces of the there were additional speci®c ionic interactions in two subunits and the buried interfaces formed the two thermophilic PCs (as compared with the when the (ab) monomer, the (ab)3 trimer and (ab)6 mesophilic Fd-PC) in the interface regions that hexamerareformed(Table3).Alldifferences might stabilize the different oligomeric stages of are within 5 % of the mean surface areas, and no phycobilisome assembly. Contacts were identi®ed signi®cant trend in the thermophilic PCs was and measured using both the contact program in identi®able. CNS37andCONTACTintheCCP4suite.38 Structure of Phycocyanin from S. vulcanus at 2.5 AÊ 77

The (ab) monomer interface phore is bound by many residues of the b subunit. Ring D forms contacts with the a subunit, which InFigure4(a),the(ab)monomerassociation are of a more polar nature, mainly due to the interface from Sv-PC is shown. Two major patches amino acid change at position A28. Thus A28, B35 of interactions arising from residues from the a and ring D of the cofactor form a polar triad and b subunits ¯ank the interface. In addition, (Figure4(b)),strengtheningthemonomerassoci- there is an interaction with intermediate strength ation domain. between AspA28 and AsnB35. In ML-PC AspA28 is replaced with an asparagine that can also form a The (ab)3 trimer formation interface polar contact with AsnB35. In mesophiles such as Fd-PC,aphenylalanineisfoundinpositionA28,9 Following removal of the phycobiliproteins from which cannot form a polar interaction with the thylakoid membranes by high salt treatment, AsnB35.Asmentionedabove,theB155chromo- 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 typesisquitesimilartothatdescribedforS7-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(Table3).Ionic/polarinteractionsare similartothosedescribedforS7-PC.15Ingeneral, there are many contacts spread out over the entire overlapregion(Figure5(a)).InbothSv-PCandCc- PC, B68 is a polar glutamine, which forms a close contactwithArgA86(Figure5(b)),whileinFd-PC and Ml-PC B68 is an alanine. An additional new contact is formed between SerA72 and ArgB57 (Figure5(b)).SerA72isaprolineinbothFd-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 PCs15,18areconservedinthethermophilicspecies, and this may be the primary mode of trimer stability.

The (ab)6 hexamer formation interface While the hexamer interface zone is quite extensive, with a buried surface area twice that of the trimerinterfacezone(Table3),upontheirremoval from the membrane, phycobilisome rods dissociate into trimers and monomers. This is perhaps indicative of the importance of phycobilisome linker pro- 10,11 Figure 4. Monomer association interface in Sv-PC. teinsinproperassemblyinvivo. Howeverin (a) Full monomer interface. a subunit in yellow and b the Sv-PC crystals, as in most of the other pre- subunit in blue, residues forming polar interactions are viously determined phycobiliprotein structures, PC shown in sticks and PCBB155 is shown in thin red lines. trimersreadilyformhexamers(Figure6(a)).Thus The large number of potential polar interactions at both crystal lattice packing parameters may induce the sides of the monomer interface are conserved in all phy- formation of inter-trimer contacts lost when the lin- cocyanins while those at the center are found in thermo- ker proteins are removed. In addition to the many philes. (b) Close up of the additional polar interaction in ionic contacts described for the previously deter- Sv-PC. AspA28, AsnB35 and PCBB155 are in stick rep- minedstructures,15,16additionalcontactsarefound resentation with carbon colored green, oxygen in red and nitrogen in blue. PCBB155 is overlayed with a seg- in the Sv-PC and Ml-PC hexamer interface regions (Figure6(b)).AspA28interactswithLysA32inSv- ment of an omit electron density map (Fo Fc) calculated after re®nement completion (contoured at 2s, light PC (and AsnA28 interacts with ArgA33 in Ml-PC) gray lines). Red dotted lines show potential polar inter- of the adjacent monomer. These positions are actions. PheA28, ArgA32 and GlnA33 in Fd-PC. As indi- 78 Structure of Phycocyanin from S. vulcanus at 2.5 AÊ

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 Figure 5. (ab)3 Trimer association interface in Sv-PC. (a) Full trimer interface. a subunit in yellow, b subunit and blue and the bottom (ab) monomer in orange and in blue, b0 subunit (from adjacent (ab) monomer) in purple. Residues that form polar interactions are shown orange. Residues forming polar interactions are shown in ball and stick (top monomer) or stick (bottom mono- in sticks and PCBB155 is shown in red. (b) Close-up of mer) representations. (b) Close up of the hexamer inter- additional polar interactions (thin dotted lines) found face showing additional polar interactions (dotted lines) between a subunit residues and b0 residues in Sv-PC in the Sv-PC structure that are not present in meso- (stick Figures). philes. Residues from the symmetry-related a subunit (orange ribbon) are denoted A0.

cated for the (ab) monomer interface, ring D of the B155 cofactor is also involved in formation of polar by structure comparison that in thermophiles, a number of residue changes to either polar or interactions in the (ab)6 hexamer interface (Figure6(b)).GluA161iscloselypairedwith charged types may be important for the stabilization of both the (ab) monomer and (ab) hexamer AsnB21, which is the shorter and less polar serine 6 in Fd-PC. Thus, as for the (ab) monomer interface, association domains, while only the Sv-PC structure has additional stabilizing contacts at the (ab) it appears that thermophilic PCs have evolved 3 similar solutions for increasing thermal stability at trimer association domain. These contacts should the hexamer assembly level. now be studied to understand better the mechan- ism of phycobilisome formation. Conclusions Materials and Methods The functional and organizational requirements of the protein components of phycobilisome anten- Protein isolation and characterization nae have lead to a high level of optimization as Synechococcus vulcanus cells were grown in a ten liter indicated by the high degree of homology in both temperature-controlled growth chamber on BG11 med- sequence and structure. However, certain modi®- ium supplemented with 5 % CO2 in air at 55 C, with ¯u- cations must take place in order to preserve proper orescent lamp illumination. Cells were grown for three function in extreme environments. We have shown to four days, collected by centrifugation, resuspended in Structure of Phycocyanin from S. vulcanus at 2.5 AÊ 79

buffer A (20 mM Hepes (pH 7.5), 10 mM MgCl2,10mM scaled and merged using the DENZO/SCALEPACK 39 Ê CaCl2) and 1 M sucrose, and treated with lysozyme suite. The®naldatawere97.5%completeto2.5A and (1 mg/ml) for one hour at 50 C. The cells were then were used for structure determination by molecular passed through a Yeda press cell disrupter under 25 replacementwithAmoRe.38,40Thesearchmodelwas atm. N2. The cells were diluted and centrifuged for two based on a single (ab) monomer from the Fremyella minutes at 2000 rpm in a Sorvall T21 centrifuge to diplosphonPCstructure(PDBcode1CPC,18),including remove cell debris. The thylakoid membranes were then the chromophores. In the search model, residues in the pelleted by a ten minute centrifugation at 16,000 rpm Sv-PC sequence that were different from that of Fd-PC and the resulting pellet was resuspended in buffer A were substituted accordingly. Model parameters for with 0.5 M sucrose to 1.2-2 mg chlorophyll a/ml. molecular replacement were a b c 80 AÊ , Bulk phycobiliproteins were removed from the thyla- a b g 90 with an integration radius of 25 AÊ , koid membranes by treatment with 0.5 M KCl and 0.1 % using all data between 8 to 4 AÊ . The rotation function dodecyl-b-D-maltoside (DM), followed by separation of calculation resulted in a solution with a high correlation the thylakoids by centrifugation. We found that a frac- coef®cient (Cc 23.3). The position of one (ab) monomer tion of PC tightly bound to the thylakoid membranes in the asymmetric was determined using the translation formed superior crystals. This fraction was separated function (Cc 72.4, R-factor 32.3 %). Following rigid- from DM solubilized Photosystem II by DEAE chroma- body re®nement, the R-factor was 26.1 % in the range of tography with buffer B (50 mM Hepes, pH 8.0) as the 8to4AÊ , and correlation coef®cient increased to 81.5. mobile phase. Phycocyanin was treated with PEG 4000 The solution in the R32 space group had better packing to remove impurities and then puri®ed by DEAE chro- properties than for R3, and re®nement was performed in matography using a 0-300 mM NaCl gradient in buffer this space group. 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 Refinement and SDS-PAGE (data not shown), which also showed 37 the absence of linker proteins. Size-exclusion-HPLC of Thestructurewasre®nedusingCNS. Cross- the puri®ed Sv-PC (PL-GF1000 AÊ column, Polymer Lab- validation was performed by omitting 10 % of the data oratories) with buffer B with 0.1 M NaCl as the mobile for calculation of Rfree. The starting R-factor was 26.3 for Ê phase af®rmed that the puri®ed PC was in trimeric form all data between 50 and 2.5 A. Following simulated (data not shown). PC was dialyzed against buffer D annealing, B-factor re®nement and water molecule (50 mM Tris, pH 8.0), and concentrated to 10 mg/ml. 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 Sequence determination omit maps were used to manually adjust and con®rm the positions of all residues and co-factors. The ®nal S. vulcanus total DNA was isolated using a DNA iso- model had a crystallographic R-factor of 20.2 % and an lation kit (Biological Industries, Kibbutz Beit Haemek). R of 24.4 %. The genes encoding for the a and b phycocyanin free subunits were ampli®ed by PCR using the following oligonucleotide primers: for the a subunit, Analysis of parameters affecting protein 50-ATGAAAACGCCGATTACTACTGAAGCT-30 and thermal stability 50-TTAGCTGAGGCGGCGTAGTCGATG-30; for the b subunit, 50-ATGCTAGATGCATTTGCCAAA-30 and 50- The Sv-PC structure was analyzed in comparison to TTAGGCAACGCGGCAGCGGC-30. Ampli®cation was the mesophilic Fd-PC structure (PDB code 1CPC) and the carried out for 30 cycles of one minute, using Pfu DNA thermophilic Ml-PC structure. The coordinates of the polymerase. The PCR products were puri®ed using the later structure were kindly provided by Drs Huber and GFX PCR DNA puri®cation kit (Amersham Phamacia Schneider of the Max-Planck-Institut fur Biochemie, Mar- Biotec Inc.). DNA sequencing was performed at the Lab- tinsried. Internal cavities were identi®ed and quanti®ed oratory of DNA Analysis, Hebrew University Jerusalem. usingtheprogramCASTp.36Unsatis®edhydrogenbond Gene analysis and comparisons were performed using donors and acceptors were identi®ed using the program Gene Runner (Hastings Software Inc., version 3.05). WHATIF.35Possibleionpairswereidenti®edby distancecriterionusingCNS37andCCP4.38 Crystallization

Sv-PC was crystallized in the presence of 5 % Atomic co-ordinates PEG4000, 50 mM Tris (pH 8.0) and 2.5-5 mg protein/ml The structural coordinates for the Synechococcus vulca- hanging drop vapor diffusion at 22 C. Crystals typically nus C-phycocyanin structure have been deposited in the grew within 3-14 days. Protein Data Bank under ID code 1I7Y.

Data collection and structure determination Sv-PC crystallized in the R3orR32 space group with cell dimensions of a b 188.432 AÊ , c 61.276 AÊ and Acknowledgments g 120 and diffracted maximally to 2.1 AÊ . A data set was collected (at 293 K) using two crystals on a Rigaku This work was supported by the Israel Science Foun- R-AxisIIcsystem(Table1).TheX-raysourcewasaRU- dation founded by the Israel Academy of Sciences and 200 rotating anode generator (Rigaku, Japan) operating Humanities (366/99) and the Fund for the Promotion of at 9 kW. A graphite monochromator was used to ®lter Research at the Technion. We thank Itzhak Ohad for his Ê the CuKa radiation of 1.54 A wavelength. The data were meaningful suggestions. 80 Structure of Phycocyanin from S. vulcanus at 2.5 AÊ

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Edited by R. Huber

(Received 10 May 2001; received in revised form 20 August 2001; accepted 20 August 2001)