Supplementary Material

Expression of the Acanthamoeba polyphaga mimivirus NDK gene product

NDKapm was cloned and expressed as previously described (20, 21). Briefly, the Mimivirus gene was inserted into the pDIGS02 expression plasmid and expressed in phase with a N-terminal His6-tag in E. coli Rosetta(DE3)pLysS at 298K. NDKapm as well as all NDKapm mutants were produced using the same system. Induction was performed when the cell culture absorbance A600 reached 1 using 0.5 mM IPTG. Pellets were resuspended in a 50 mM sodium phosphate, 300 mM NaCl buffer, pH 9.0 (buffer A), containing 0.1% Triton X-100 and 5% glycerol, and total proteins were extracted by sonication.

NDK Mutants

The N62L, R107G mutants, as well as the insertion of the Kpn sequence (+Kpn) from Dictyostelium discoideum, were produced using the QuickChange II Site-directed Mutagenesis Kit (Stratagene) using the following oligonucleotides (F: Forward, R: Reverse)

F-Kpn: GACTCCAAGGTAATACC AACCCATTAGCCTCAGCC CCAGGAACTATCAG

R-Kpn: CTGATAGTTCCTGG GGCTGAGGCTAATGGGTT GGTATTACCTTGGAGTC

F-N62L:CAA TCC TAT TTT AAT GAT CTT TGT GAT TTC ATG GTA AGT

R-N62L:ACT TAC CAT GAA ATC ACA AAG ATC ATT AAA ATA GGA TTG

F-R107G:GCA AAT GAT ATC GGA GAA AAT CTT ATT CAC GCC

R-R107G:GGC GTG AAT AAG ATT TTC TCC GAT ATC ATT TGC

Crystallization of NDKapm and Mutants complexes

Crystallization of the NDKapm and the NDK mutants nucleotide complexes were performed by vapour diffusion experiments. Due to the ATPase activity of NDK enzymes, The NTP nucleotides are spontaneously transformed into NDP (25) as evidence by the complexes structures. Consequently, all NDK complexes contain NDP even if NTP has been used for crystallization. Conditions were initially identified through a screening protocol performed on 3x96-well crystallization plates (Greiner) loaded by an 8-needle dispensing robot (Tecan, WS 100/8 workstation modified for our needs), using one 1μl sitting drop per condition (2). Crystallization conditions were improved by hanging drop vapor diffusion using 24-well culture plates (Greiner). Each hanging drop was prepared by mixing 0.5 to 1 µl of the NDK complexes with 0.5 to 1 μl of the reservoir solution. The hanging drop on the cover glass was vapor-equilibrated against 1 ml of the reservoir solution in each well of the tissue culture plate.

Structure determination of the NDKapm native form

The sequence of the Acanthamoeba polyphagamimivirus NDK shares 44% identical amino acid residues with the one of Mycobacterium tuberculosis NDK (PDB code 1K44, S1) and 38% with the one of Drosophila melanogaster (PDB code 1NDL,S2). The structure was solved by molecular replacement on the CaspR web-server (S3; using the above structures as references to generate the Acanthamoeba polyphagamimivirus NDK models (21).

Supplementary Figure Legends

Fig. S1: Structural alignment of NDKapm with other NDK structures.

The alignment was produced using the 3D-coffee web-server ( S7) based on the structural alignment of NDK 3D-structures (NDKdd: 1NDC, NDKph: 2CWK, NDKmt: 1K44, NDKbh: 1NB2, NDKec: 2HUR, NDKhsap: 1ZS6; NDKbt: 1BHN, NDKtt: 1WKJ, NDKpf: 1XIQ, NDKhsal: 2AZ3, NDKos: 1PKU, NDKat:1S59, NDKps: 1W7W, NDKdm: 1NDL, NDKpa: 1XQI, NDKapm: 2B8Q). Residues boxed in red correspond to strictly conserved residues; red coloured residues correspond to physico-chemically equivalent residues. We marked secondary structure elements of NDKdd, NDKph and NDKapm, NDKpa at the top and bottom of the alignment respectively using the ESPript program(14). Black arrows correspond to the punctual mutations on NDKapm.

Fig. S2: The Mimivirus NDK hexamer.Cartoon representation of the NDKapm hexamer structure in complex with dTDP (white) each chain is coloured with a different colour.

Fig. S3: Stereo views of the NDK active site with dTDP/Mg2+. NDKapm is coloured in opaque red, NDKdd in transparent yellow and the NDK+Kpn-N62L-R107G triple mutant in transparent cyan. A] The specific interaction between R107-Nε and the dTDP base moiety is represented by a red dashed line.B] The specific interaction between N62-Nδ2 and the dTDP ribose and the pyrimidine base O2 is represented by the black dashed lines. The standard LKpn1 segment is coloured in green.

Fig. S4: Phylogenetic position of Mimivirus NDK.Homologous NDK protein sequences from all major phyla were aligned using the Musclesoftware (S5). This includes alpha- beta- delta- gamma- epsilon- proteobacteria, cyanobacteria, firmicutes, chlamydiales, magnetococcus, aquifex, euryarchea and chrenarchea, spirochete,one environmental sequence, fungi, plants, metazoans, protists including alveolata, mycetozoa, diplomonadida, Acanthamoeba castellanii, and Mimivirus. A maximun likelyhood tree was computed with PhyML (S6) using thedefault option on our phylogeny server at URL: phylogeny.fr (S4). Despite a number of low bootstrap values, there is no ambiguity on the lack of affinity between the NDK enzymes from Mimivirus and its host Acanthamoeba castellanii.

Supplementary Tables Legends:

Table 1: NDKapm X-ray data collection and refinement statistics

Table 2: NDKapm mutants X-ray data collection and refinement statistics

The estimated coordinate errors are based on maximum likelihood and are given in Å.

<I / σ I>, is the mean signal to noise ratio, where I is the integrated intensity of a measured reflection and σ is the estimated error in the measurement.

, where I is the integrated intensity of reflection h having i observations and is the mean recorded intensity of reflection h over multiple recording.

Rcryst = , where Fo are observed and Fc calculated structure factor amplitudes. Rfree is calculated from a randomly chosen 5% of reflections.

Supplementary references:

S1. Chen, Y., S. Morera, J. Mocan, I.Lascu, andJ. Janin. 2002. X-ray structure of Mycobacterium tuberculosis nucleoside diphosphate kinase. Proteins47:556-557.

S2. Chiadmi, M., S. Moréra,I. Lascu, C. Dumas, G. Le Bras, M. Véron, and J. Janin. 1993.Crystal structure of the Awd nucleotide diphosphate kinase from Drosophila. Structure1:283-293.

S3. Claude, J.B.,K. Suhre, C. Notredame, J.M. Claverie, and C. Abergel. 2004. CaspR: a web server for automated molecular replacement using homology modelling. Nucleic Acids Res.32:W606-W609.

S4. Dereeper, A., V. Guignon, G. Blanc, S. Audic, S. Buffet, F. Chevenet, J.F. Dufayard, S. Guindon, V. Lefort, M. Lescot, J-M. Claverie, and O. Gascuel.2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist.Nucleic Acids Res.36:W465-9.

S5. Edgar, R.C.2004.MUSCLE: multiple sequence alignment with high accuracy and high throughput.Nucleic Acids Res.32:1792-7.

S6. Guindon, S., and O. Gascuel.2003.A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood.Syst. Biol.52:696-704.

S7. O'Sullivan, O., K. Suhre, C. Abergel, D.G. Higgins, and C. Notredame. 2004. 3DCoffee: combining protein sequences and structures within multiple sequence alignments. J. Mol. Biol. 340:385-395.

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