research article crystal structure of pav1-137: a protein from … · 2019. 7. 31. · archaea...

Hindawi Publishing CorporationArchaeaVolume 2013 Article ID 568053 5 pageshttpdxdoiorg1011552013568053

Research ArticleCrystal Structure of PAV1-137 A Protein from the Virus PAV1That Infects Pyrococcus abyssi

N Leulliot12 S Quevillon-Cheruel1 M Graille13 C Geslin4 D Flament4

M Le Romancer4 and H van Tilbeurgh1

1 Institut de Biochimie et de Biophysique Moleculaire et Cellulaire CNRS-UMR 8619 IFR115 Universite Paris-SudBatiment 430 91405 Orsay France

2 Laboratoire de Cristallographie et RMN Biologiques-CNRS UMR-8015 Universite Paris DescartesFaculte des Sciences Pharmaceutiques et Biologiques 4 av de lrsquoObservatoire 75270 Paris CEDEX 06 France

3 Laboratoire de Biochimie (BIOC) CNRS UMR 7654 Ecole Polytechnique Route de Saclay 91128 Palaiseau France4Universite de Brest CNRS IFREMER UMR 6197 Laboratoire de Microbiologie des Environnements Extremes OSU-IUEMTechnopole Brest-Iroise Avenue Dumont DrsquoUrville 29280 Plouzane France

Correspondence should be addressed to H van Tilbeurgh hermanvan-tilbeurghu-psudfr

Received 31 October 2012 Accepted 12 February 2013

Academic Editor Martin Lawrence

Copyright copy 2013 N Leulliot et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strainGE23) that has been isolated and characterized It is lemon shaped and is decorated with a short fibered tail PAV1 morphologicallyresembles the fusiformmembers of the family Fuselloviridae or the genus SalterprovirusThe 18 kb dsDNAgenome of PAV1 contains25 predicted genes most of them of unknown function To help assigning functions to these proteins we have initiated structuralstudies of the PAV1 proteomeWe determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of22 ATheprotein forms dimers both in solution and in the crystalThe fold of PAV1-137 is a four-120572-helical bundle analogous to thosefound in some eukaryotic adhesion proteins such as focal adhesion kinase suggesting that PAV1-137 is involved in protein-proteininteractions

1 Introduction

The archaea domain is organized into two major phyla theCrenarchaeota and the Euryarchaeota The first phylum con-tains mainly the extremely thermophilic Sulfolobales Desul-furococcales and Thermoproteales The vast majority ofhyperthermophilic viruses were isolated from the Crenar-chaeota infecting in particular the genera SulfolobusThermo-proteusAcidianus Pyrobaculum Stygiolobus andAeropyrum[1 2] Their shapes are characterized by unusual morpholo-gies very different from bacterialviruses and eukaryoticviruses Genomic sequences were determined for some ofthese archaeal viruses and revealed a very high portion ofORFan genes [3] Due to their exceptionalmorphological andgenomic properties they were assigned to eight novel viralfamilies [1]

The Euryarchaeota phylum includes extreme halophilesmethanogens and hyperthermophilic sulfur reducers (Ther-mococcales) Most of the viruses infecting this phylum areisolated frommesophilic hosts and are tailed viruses whereaspleomorphic types are relatively rare [4] The knowledgeabout archaeal viruses is still very limited and this is evenmore poignant for viruses that infect hyperthermophilicEuryarchaeota [5 6] To date PAV1 and TPV1 (Thermococcusprieurii virus 1) are the only viruses isolated from culti-vatedmarine hyperthermophilic euryarchaeaThese spindle-shaped viruses are morphologically similar to the halovirusesof the genus Salterprovirus [7 8] that infect extremehalophiles and to crenarchaeal viruses assigned to thefusiform family Fuselloviridae [9] but they do not share anygenomic properties PAV1 isolated from Pyrococcus abyssi

2 Archaea

was the first virus isolated and described in Thermococ-cales [10] PAV1 virions display a lemon-shaped morphology(120 nm long and 80 nmwide) with a short tail (15 nm) termi-nated by fibers Very recently a novel fusiform virus TPV1was isolated and characterized from the hyperthermophiliceuryarchaeal genusThermococcus [11]

PAV1 and TPV1 are released during all phases of hostgrowthwithout causing host lysis A simple procedure to spotviruses on cellular lawns and directly observe their impacthas been specially designedThis allows determination of thehost range and infectivity of viruses isolated from anaero-bic hyperthermophile sulfur-reducing microorganisms Weused this approach to prove the infectivity of PAV1 and toconfirm the host range of TPV1 both of them being genusspecific [12]

The genome of PAV1 is composed of a double strandedcircular DNA It was shown that the free viral genome existsas a multicopy plasmid in the host strain but no integratedprophage could be detected The complete genome of PAV1contains 18098 bp [13] A number of 25 ORFs (open readingframes) encoding at least 50 amino acids were identified andalmost all are located on the same strand The shape of theviruses is perturbed upon treatment with organic solventsor detergents suggesting that their envelopes contain lipidsThis observation is supported by the fact that half of thePAV1 genome has predicted transmembrane helices Sixty-five percent of the predicted proteins have no homologuesin the sequence databases Functions could only be vaguelysuggested for three proteins A 59 amino acid protein (PAV1-59) shares similarities to the CopG transcriptional regulatorsTwo other ORFs (PAV1-676 and PAV1-678) that are the onlyORFs shared between the hyperthermophilic euryarchaealviruses PAV1 and TPV1 contain one or two copies of thelaminin G-like jelly roll fold and may hence be involved inadhesion to the host Polycistronic mRNA analysis showedthat all predicted genes are transcribed in six mRNAs

In contrast with other lemon-shaped viruses isolatedeither from hypersaline waters (Salterprovirus) or fromextreme geothermal terrestrial environments (Fuselloviri-dae) PAV1 was isolated from a remote deep-sea hydrother-mal vent So the uniqueness of the PAV1 genome comparedto those of other archaeal viruses may be a consequence ofits evolutionary history [14] Since no function could be pro-posed for most of the predicted ORFs we set out to analyzethe structures of the proteins encoded by the PAV1 genomeThe assignment of function to archaeal proteins suffers fromthe absence of genetic data and sequence analogs in better-characterized organisms [15ndash18] 3D structure is better con-served than sequence and may reveal similarities that remainundiscovered by sequence analysis [19] Therefore struc-ture determination offers a valuable alternative for investi-gating protein function Indeed the determination of thecrystal structure of the AvtR protein from a hyperther-mophilic archaeal lipothrixvirusallowed us to establish a rolefor this protein in the transcriptional regulation of viral genes[20]We want in fine to find out if PAV1 protein structures arerelated to those of other archaeal virus proteins We presenthere the X-ray crystal structure of a putative ORFan proteinPAV1-137 to our knowledge the first for a euryarchaeal viralprotein

2 Results and Discussion

Wepurified the C-terminalHis-tagged protein from a geneticconstruct deleted for the 14 first residues because sequenceanalysis predicted an unstructured conformation for thisN-terminal region [21] MALDI-TOF mass spectrometryanalysis of the purified recombinant protein shows that theN-terminalmethioninewas cleaved off during the production inE coli Gel filtration analysis suggests that the protein formsdimers in solution (not shown) Crystals were obtained in35 PEG400 05M NH

4Cl 01M Na citrate at pH 4 at a

concentration of 15mgmL for the protein Details of datacollection and refinement are found in Table 1 All residuesof the construct except for the affinity tag are visible inthe electron density Two copies of PAV1-137 are present inthe asymmetric unit and their structures are almost identical(root mean square deviation = 05 A)

PAV1-137 contains four amphipathic helices that areorganized as a helical bundle The three N-terminal helicesform a parallel up and down configuration while the C-ter-minal helix is shorter and packs with an angle of about 45∘against helices 1 and 3 The core of the 3 helices is veryhydrophobic consisting mainly of branched aliphatic aminoacids The connections between helices 1 2 and 3 are shortThe connection between helices 3 and 4 is a longer stretchresulting in a less tight packing of helix 4 compared to theother helices

The A and B monomers in the asymmetrical unit forma two-fold symmetrical dimer and the symmetry axis runsperpendicular to the direction of the long helices (Figure 1)The helices of bothmonomers associate to form an antiparal-lel super helical bundle The dimer interface involves helices1 2 and 4 The accessible surface of each subunit buried bydimer formation is substantial The accessible surface areafor the monomer is 7300 A2 Dimerisation buries 1278 A2 permonomer corresponding to 17 of the solvent accessible sur-face areaThemajority of interactions between themonomersare conferred by helix 4 that lies against the extremities ofhelices 1 and 2The interface ismore hydrophilic than the coreof the helical bundle and is stabilized by 9 hydrogen bondsmainly between side chain and side or main chain atoms

Orthologs of PAV1-137 were recently reported in genomicsequences of new thermococcus plasmids [14] The genecoding for the ortholog of PAV1-137 is found in tandem withan ortholog of gene PAV1-375 in three of these plasmidsSurprisingly it also formed a three-gene cluster which isconserved within the provirus A3 VLP of the euryarchaealmethanogens Methanococcus voltae A3 [22] PAV1-375 likelyencodes for a P-loop ATPase but its association with PAV1-137 remains unclear PAV1-137 has presently no structuralanalogues in the Protein Data Bank that could help definingits function The lack of sequence analogues at the start ofthis study suggested that PAV1-137 might adopt a new foldHelical bundles are extremely common in protein structuresTherefore we found substantial structural similarities withproteins sharing helical bundle architecture Examination ofthe function of these proteins clearly shows that the structuralsimilarity does not indicate functional relationships Forexample significant overlaps are found between PAV1-137

Archaea 3

Cter-2

Cter-1

1205724

1205724

1205722

1205722

1205721

1205721

1205723

1205723

Nter-1

Nter-2

90∘

Figure 1 Two perpendicular views of the X-ray structure of the PAV1-137 dimer The two subunits are represented in rainbow colouringgoing from blue (N-terminal) to red (C-terminal) The N and C terminus and the helices of the two subunits are labelled The two-fold axisis indicated

and fragments of Talin and Focal adhesion kinase both fromeukaryotic origin (119885-score 5 and root mean square differenceof 32 A for 100 aligned residues) [23 24] Helices 1 2 and3 of PAV1-137 superpose well onto helices 2 3 and 4 of theeukaryotic helical bundles No equivalent is present in theseeukaryotic analogues for the fourth helix found in PAV1-137The C-terminal helix adopts a different orientation and hasno equivalent in Talin or Focal adhesion kinase

Since PAV1-137 does not seem to carry any active siteits biological function is probably connected with protein-protein or protein-nucleic acid interactions Helical bundleproteins are frequently involved in this type of interactionsexemplified by Talin Virtually nothing is known about thelife cycle of PAV1 Its genome sequencewas a first step towardsa better understanding of this new type of viruses which mayhave evolved from a recombination event between differentmobile genetic elements harbored by both hyperthermophilicand methanogenic euryarchaeota We report here on thefirst results of the investigation on structure and function ofproteins encoded by this virus

3 Materials and Methods

31 Protein Production and Purification The PAV1-137 ORFlacking the region encoding for the 14 N-terminal residueswas amplified by PCR using genomic DNA of PAV1 virusas a template An additional sequence coding for a 6-histi-dine tag was introduced at the 31015840 end of the ORF duringamplification The PCR product was then cloned into pET28vector Expression was done at 37∘C using the E coli BL21(Gold)DE3 strain The His-tagged protein was purified on aNi-NTA column (Qiagen Inc) followed by gel filtration usinga buffer composed of 20mM Tris-HCl pH 75 200mMNaCl and 10mM b-mercaptoethanol Selenomethionine-substituted PAV1-137 was produced and purified as the nativeprotein The peak fractions were concentrated to 15mgmLand used for crystallization

32 Crystallization and Data Collection Crystallization wasperformed using a Cartesian crystallization robot in 200 times200120583l sitting drops (volume for protein and liquid mother)and reproduced manually in 1 times 1120583L drops Crystals wereobtained from the following crystallization conditions 35PEG400 05M NH

4Cl 01M Na citrate at pH 4 at 18∘C

Crystals were transferred in the mother liquor containing30 glycerol prior to flash freezing in liquid nitrogen X-raydiffraction data of SeMet substituted protein crystals werecollected on the ID14-4 ESRF beamline and were processedusing MOSFLM and SCALA [25] The crystals belong to theP6122 space group with two molecules per asymmetric unit

The cell parameters and data collection statistics are reportedin Table 1

33 Structure Solution and Refinement The structure wassolved at a resolution of 22 A by single anomalous diffraction(SAD) using data collected at the Selenium peak wavelengthThe Hyss module of Phenix program [26] was used to findthe Selenium sites using the entire resolution range The siteswere refined with SHARP [27] and solvent flattening wasperformed with DM ArpWarp [28] built 95 of the visibleresiduesThemodel was fully refined and completed from thenative data using Buster and the graphics programme O [2930] Refinement was carried out using noncrystallographicsymmetry restraints and one TLS group per chain (statisticsare shown in Table 1) with the program Buster All theresidues fall in favourable regions of the Ramachandran plot

For homology search we did a Psi-BLAST analysis usingstandard procedures as provided by the NIH blastserver(httpblastncbinlmnihgovgate1inistfrBlastcgi)

Accession Number

The structure factor amplitudes and the refined coordinatesof PAV1-137 have been deposited in the Protein Data Bank asentry 4HR1

4 Archaea

Table 1 Data collection and refinement statistics

Data collectionSpace group P6122Cell dimensions a b c (A) 7355 7355 19155Wavelength (A) 09793Resolution (A) 200ndash220Outer resolution shell (A) 232ndash220Number of observed reflectionsunique 9403115911Completeness () (outer shell) 974 (853)Multiplicity (outer shell) 59 (28)119868120590(119868) (outer shell) 190 (24)119877merge ()1 (outer shell) 58 (459)

RefinementResolution (A) 200ndash220Reflections (workingtest) 15873800119877119877free ()2 215247RMSD

Bond lengths (A) 0010Bond angles (∘) 115

B-factors (A2)Protein 63

Ramachandran statistics ()Most favored 9871Allowed 129

1119877merge = sumℎ sum119894 |119868ℎ119894 minus ⟨119868ℎ⟩|sumℎ sum119894 119868ℎ119894 were 119868ℎ119894 is the ith observation of thereflection h while ⟨119868ℎ⟩ is the mean intensity of reflection h2119877 = sum ||119865119900|minus|119865119888|||119865119900|119877free was calculated with a set of randomly selectedreflections (5)

Acknowledgments

This work was supported by a grant from the AgenceNationale de Recherche (no ANR-BLAN-0408-03) Theauthors are indebted to Benjamin Dray and Nathalie Ulryckfor the technical assistance

References

[1] D Prangishvili and R A Garrett ldquoExceptionally diversemorphotypes and genomes of crenarchaeal hyperthermophilicvirusesrdquo Biochemical Society Transactions vol 32 no 2 pp204ndash208 2004

[2] T Mochizuki Y Sako and D Prangishvili ldquoProvirus inductionin hyperthermophilic archaea characterization of Aeropyrumpernix spindle-shaped virus 1 and Aeropyrum pernix ovoidvirus 1rdquo Journal of Bacteriology vol 193 no 19 pp 5412ndash54192011

[3] D Prangishvili R A Garrett and E V Koonin ldquoEvolutionarygenomics of archaeal viruses unique viral genomes in the thirddomain of liferdquo Virus Research vol 117 no 1 pp 52ndash67 2006

[4] F Eiserling A Pushkin M Gingery and G Bertani ldquoBacteri-ophage-like particles associated with the gene transfer agent ofMethanococcus voltae PSrdquo Journal of General Virology vol 80no 12 pp 3305ndash3308 1999

[5] H W Ackermann and D Prangishvili ldquoProkaryote virusesstudied by electron microscopyrdquo Archives of Virology vol 157no 10 pp 1843ndash1849 2012

[6] C Geslin M Le Romancer M Gaillard G Erauso and DPrieur ldquoObservation of virus-like particles in high tempera-ture enrichment cultures from deep-sea hydrothermal ventsrdquoResearch in Microbiology vol 154 no 4 pp 303ndash307 2003

[7] C Bath T Cukalac K Porter andM L Dyall-Smith ldquoHis1 andHis2 are distantly related spindle-shaped haloviruses belongingto the novel virus group Salterprovirusrdquo Virology vol 350 no1 pp 228ndash239 2006

[8] C Bath and M L Dyall-smith ldquoHis1 an archaeal virus of theFuselloviridae family that infects Haloarcula hispanicardquo Journalof Virology vol 72 no 11 pp 9392ndash9395 1998

[9] P Redder X Peng K Brugger et al ldquoFour newly isolatedfuselloviruses from extreme geothermal environments revealunusual morphologies and a possible interviral recombinationmechanismrdquo Environmental Microbiology vol 11 no 11 pp2849ndash2862 2009

[10] C Geslin M Le Romancer G Erauso M Gaillard G Perrotand D Prieur ldquoPAV1 the first virus-like particle isolated froma hyperthermophilic euryarchaeote lsquoPyrococcus abyssirsquordquo Journalof Bacteriology vol 185 no 13 pp 3888ndash3894 2003

[11] A Gorlas E V Koonin N Bienvenu D Prieur and C GeslinldquoTPV1 the first virus isolated from the hyperthermophilicgenus Thermococcusrdquo Environmental Microbiology vol 14 no2 pp 503ndash516 2012

[12] A Gorlas and C Geslin ldquoA simple procedure to determine theinfectivity and host range of viruses infecting anaerobic andhyperthermophilic microorganismsrdquo Extremophiles 2013

[13] C Geslin M Gaillard D Flament et al ldquoAnalysis of thefirst genome of a hyperthermophilic marine virus-like particlePAV1 isolated from Pyrococcus abyssirdquo Journal of Bacteriologyvol 189 no 12 pp 4510ndash4519 2007

[14] M Krupovic M Gonnet W Ben Hania R Forterre and GErauso ldquoInsights into dynamics of mobile genetic elements inhyperthermophilic environments from five new thermococcusplasmidsrdquo PLoS One vol 8 no 1 p e49044 2013

[15] R Khayat L Tang E T Larson C M Lawrence M Youngand J E Johnson ldquoStructure of an archaeal virus capsid proteinreveals a common ancestry to eukaryotic and bacterial virusesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 52 pp 18944ndash18949 2005

[16] E T Larson D Reiter M Young and C M LawrenceldquoStructure of A197 from Sulfolobus turreted icosahedral virus acrenarchaeal viral glycosyltransferase exhibiting theGT-A foldrdquoJournal of Virology vol 80 no 15 pp 7636ndash7644 2006

[17] J Keller N Leulliot B Collinet et al ldquoCrystal structure ofAFV1-102 a protein from the acidianus filamentous virus 1rdquoProtein Science vol 18 no 4 pp 845ndash849 2009

[18] A Goulet M Pina P Redder et al ldquoORF157 from thearchaeal virus Acidianus filamentous virus 1 defines a new classof nucleaserdquo Journal of Virology vol 84 no 10 pp 5025ndash50312010

[19] J C Whisstock and A M Lesk ldquoPrediction of protein functionfrom protein sequence and structurerdquo Quarterly Reviews ofBiophysics vol 36 no 3 pp 307ndash340 2003

[20] N Peixeiro J Keller B Collinet et al ldquoStructure and functionof AvtR a novel transcriptional regulator from a hyperther-mophilic archaeal lipothrixvirusrdquo Journal of Virology vol 87no 1 pp 124ndash136 2013

[21] S Hirose K Shimizu S Kanai Y Kuroda and T NoguchildquoPOODLE-L a two-level SVM prediction system for reliablypredicting long disordered regionsrdquo Bioinformatics vol 23 no16 pp 2046ndash2053 2007

Archaea 5

[22] M Krupovic and D H Bamford ldquoArchaeal proviruses TKV4and MVV extend the PRD1-adenovirus lineage to the phylumEuryarchaeotardquo Virology vol 375 no 1 pp 292ndash300 2008

[23] S T Arold M K Hoellerer and M E M Noble ldquoThe struc-tural basis of localization and signaling by the focal adhesiontargeting domainrdquo Structure vol 10 no 3 pp 319ndash327 2002

[24] E Papagrigoriou A R Gingras I L Barsukov et al ldquoActivationof a vinculin-binding site in the talin rod involves rearrange-ment of a five-helix bundlerdquo EMBO Journal vol 23 no 15 pp2942ndash2951 2004

[25] A Leslie Joint CCP4 and EACMBNewsletter Protein Crystallog-raphy Daresbury Laboratory Warrington UK 1992

[26] P D Adams K Gopal R W Grosse-Kunstleve et al ldquoRecentdevelopments in the PHENIX software for automated crys-tallographic structure determinationrdquo Journal of SynchrotronRadiation vol 11 no 1 pp 53ndash55 2004

[27] C Vonrhein E Blanc P Roversi and G Bricogne ldquoAutomatedstructure solution with autoSHARPrdquo Methods in MolecularBiology vol 364 pp 215ndash230 2007

[28] R J Morris A Perrakis and V S Lamzin ldquoARPwARP andautomatic interpretation of protein electron density mapsrdquoMethods in Enzymology vol 374 pp 229ndash244 2003

[29] E Blanc P Roversi C Vonrhein C Flensburg SM Lea andGBricogne ldquoRefinement of severely incomplete structures withmaximum likelihood in BUSTER-TNTrdquo Acta CrystallographicaSection D vol 60 no 12 pp 2210ndash2221 2004

[30] T A Jones ldquoInteractive electron-density map interpretationfrom INTER to Ordquo Acta Crystallographica Section D vol 60no 12 pp 2115ndash2125 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Archaea

was the first virus isolated and described in Thermococ-cales [10] PAV1 virions display a lemon-shaped morphology(120 nm long and 80 nmwide) with a short tail (15 nm) termi-nated by fibers Very recently a novel fusiform virus TPV1was isolated and characterized from the hyperthermophiliceuryarchaeal genusThermococcus [11]

PAV1 and TPV1 are released during all phases of hostgrowthwithout causing host lysis A simple procedure to spotviruses on cellular lawns and directly observe their impacthas been specially designedThis allows determination of thehost range and infectivity of viruses isolated from anaero-bic hyperthermophile sulfur-reducing microorganisms Weused this approach to prove the infectivity of PAV1 and toconfirm the host range of TPV1 both of them being genusspecific [12]

The genome of PAV1 is composed of a double strandedcircular DNA It was shown that the free viral genome existsas a multicopy plasmid in the host strain but no integratedprophage could be detected The complete genome of PAV1contains 18098 bp [13] A number of 25 ORFs (open readingframes) encoding at least 50 amino acids were identified andalmost all are located on the same strand The shape of theviruses is perturbed upon treatment with organic solventsor detergents suggesting that their envelopes contain lipidsThis observation is supported by the fact that half of thePAV1 genome has predicted transmembrane helices Sixty-five percent of the predicted proteins have no homologuesin the sequence databases Functions could only be vaguelysuggested for three proteins A 59 amino acid protein (PAV1-59) shares similarities to the CopG transcriptional regulatorsTwo other ORFs (PAV1-676 and PAV1-678) that are the onlyORFs shared between the hyperthermophilic euryarchaealviruses PAV1 and TPV1 contain one or two copies of thelaminin G-like jelly roll fold and may hence be involved inadhesion to the host Polycistronic mRNA analysis showedthat all predicted genes are transcribed in six mRNAs

In contrast with other lemon-shaped viruses isolatedeither from hypersaline waters (Salterprovirus) or fromextreme geothermal terrestrial environments (Fuselloviri-dae) PAV1 was isolated from a remote deep-sea hydrother-mal vent So the uniqueness of the PAV1 genome comparedto those of other archaeal viruses may be a consequence ofits evolutionary history [14] Since no function could be pro-posed for most of the predicted ORFs we set out to analyzethe structures of the proteins encoded by the PAV1 genomeThe assignment of function to archaeal proteins suffers fromthe absence of genetic data and sequence analogs in better-characterized organisms [15ndash18] 3D structure is better con-served than sequence and may reveal similarities that remainundiscovered by sequence analysis [19] Therefore struc-ture determination offers a valuable alternative for investi-gating protein function Indeed the determination of thecrystal structure of the AvtR protein from a hyperther-mophilic archaeal lipothrixvirusallowed us to establish a rolefor this protein in the transcriptional regulation of viral genes[20]We want in fine to find out if PAV1 protein structures arerelated to those of other archaeal virus proteins We presenthere the X-ray crystal structure of a putative ORFan proteinPAV1-137 to our knowledge the first for a euryarchaeal viralprotein

2 Results and Discussion

Wepurified the C-terminalHis-tagged protein from a geneticconstruct deleted for the 14 first residues because sequenceanalysis predicted an unstructured conformation for thisN-terminal region [21] MALDI-TOF mass spectrometryanalysis of the purified recombinant protein shows that theN-terminalmethioninewas cleaved off during the production inE coli Gel filtration analysis suggests that the protein formsdimers in solution (not shown) Crystals were obtained in35 PEG400 05M NH

4Cl 01M Na citrate at pH 4 at a

concentration of 15mgmL for the protein Details of datacollection and refinement are found in Table 1 All residuesof the construct except for the affinity tag are visible inthe electron density Two copies of PAV1-137 are present inthe asymmetric unit and their structures are almost identical(root mean square deviation = 05 A)

PAV1-137 contains four amphipathic helices that areorganized as a helical bundle The three N-terminal helicesform a parallel up and down configuration while the C-ter-minal helix is shorter and packs with an angle of about 45∘against helices 1 and 3 The core of the 3 helices is veryhydrophobic consisting mainly of branched aliphatic aminoacids The connections between helices 1 2 and 3 are shortThe connection between helices 3 and 4 is a longer stretchresulting in a less tight packing of helix 4 compared to theother helices

The A and B monomers in the asymmetrical unit forma two-fold symmetrical dimer and the symmetry axis runsperpendicular to the direction of the long helices (Figure 1)The helices of bothmonomers associate to form an antiparal-lel super helical bundle The dimer interface involves helices1 2 and 4 The accessible surface of each subunit buried bydimer formation is substantial The accessible surface areafor the monomer is 7300 A2 Dimerisation buries 1278 A2 permonomer corresponding to 17 of the solvent accessible sur-face areaThemajority of interactions between themonomersare conferred by helix 4 that lies against the extremities ofhelices 1 and 2The interface ismore hydrophilic than the coreof the helical bundle and is stabilized by 9 hydrogen bondsmainly between side chain and side or main chain atoms

Orthologs of PAV1-137 were recently reported in genomicsequences of new thermococcus plasmids [14] The genecoding for the ortholog of PAV1-137 is found in tandem withan ortholog of gene PAV1-375 in three of these plasmidsSurprisingly it also formed a three-gene cluster which isconserved within the provirus A3 VLP of the euryarchaealmethanogens Methanococcus voltae A3 [22] PAV1-375 likelyencodes for a P-loop ATPase but its association with PAV1-137 remains unclear PAV1-137 has presently no structuralanalogues in the Protein Data Bank that could help definingits function The lack of sequence analogues at the start ofthis study suggested that PAV1-137 might adopt a new foldHelical bundles are extremely common in protein structuresTherefore we found substantial structural similarities withproteins sharing helical bundle architecture Examination ofthe function of these proteins clearly shows that the structuralsimilarity does not indicate functional relationships Forexample significant overlaps are found between PAV1-137

Archaea 3

Cter-2

Cter-1

1205724

1205724

1205722

1205722

1205721

1205721

1205723

1205723

Nter-1

Nter-2

90∘












Accession Number


4 Archaea








Acknowledgments


References






















Archaea 5

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 3

Cter-2

Cter-1

1205724

1205724

1205722

1205722

1205721

1205721

1205723

1205723

Nter-1

Nter-2

90∘












Accession Number


4 Archaea








Acknowledgments


References






















Archaea 5

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Archaea








Acknowledgments


References






















Archaea 5

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 5

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article crystal structure of pav1-137: a protein from … · 2019. 7. 31. · archaea...

Documents