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PROT_23186_sm_SuppFig1.pdf76KFIGURE S1: Size-exclusion chromatographic profile of hCINAP (A) WT-hCINAP monomer (21 kDa) was eluted at a peak volume of 76 mL. (B) H79G-hCINAP was eluted as a non-separable mixture of oligomer-monomer at a peak volume of 70.3 mL. A lower peak, forming a right shoulder, corresponded to the monomer (Ve=76 mL). (C) Glucokinase (EC2.7.2.1), a globular protein with a molecular mass of 50 kDa, was used as a calibration marker and was eluted at a peak volume of 68.4mL. Comparison of the elution volumes indicated that the H79G mutant forms a dimer-monomer mixture where dimer is the predominant form.
PROT_23186_sm_SuppFig2.pdf579KFIGURE S2: Structural comparison between members of the Adenylate Kinase family. A. hCINAP exhibits unique structural features among the mammalian adenylate kinases. Structural comparison of hCINAP (hAK6; green) with the superimposed structures of hAK1 (blue), hAK2 (pink), bovine AK3 (yellow), hAK4 (purple) and hAK5 (grey) shows that the Walker A motif region (P-Loop; GxxxxGK(x)S/T) is structurally conserved, while the region of hCINAP linking the β3-β4 core strands consists of a long loop followed by a 310 helix, indicating a high degree of structural flexibility. Importantly, this region bears a signature for a Walker B motif (hhhDXXG), including the structurally conserved Asp77 (VIV-D77YH79G). This motif, although common to the ATpase/GTPase family, is unique for the AK family. The composition of the corresponding sequences for hAK1, hAK2, bovine AK3, hAK4 and hAK5 are FLI-D93GYP, FLL-D99GFP, WLL-D88GFP, WLL-D88GFP and FLI-D96GYP, respectively. The distinct orientation of this region in hCINAP allows entry of His79 into the ATP binding site and indicates a possible role of this residue in the catalytic mechanism of hCINAP. In the other mammalian AKs, the corresponding position is occupied by a hydrophobic residue that is oriented, instead, toward the AMP binding site. Moreover, hCINAP is the only one within the mammalian family that possesses a residue (Thr17), positioned immediately after the invariant Lysine of P-Loop (10-GTPGVGK16T-17), that is capable of coordinating a metal ion, This is another atypical feature that hCINAP also shares with the ATPase/GTPase family. The corresponding sequences for the invariant P-loop in hAK1, hAK2, bovine AK3, hAK4 and hAK5 are respectively: 15-GGPGSGK21(G)T-23, 22-GPPGAGK28(G)T-30, 12-GAPGSGK18(G)T-20, 12-GPPGSGK18(G)T-20 and 18-GGPGSGK24(G)T-26. B. (Same view as A.) hCINAP exhibits limited structural similarity with the archaeal AK from Sulfolobus acidocaldarius (AKsa; Orange). In particular, while in Sulfolobus the His93 is positioned similarly to the His 79 residue of hCINAP, the AKas lack the Walker B motif (LFI-D91THA). In the ATP binding site of AKas, Ser15 positioned immediately after the invariant Lysine of P-Loop (8-GIPGVGK14S-15), and its side chain partially overlaps with Thr17 side chain of hCINAP. However, its role in metal ion coordination is not established because AKas-ADP-AMP structure has been magnesium-free. The coordinates used for this comparison were 1Z83 (hAK1), 2C9Y (hAK2), 2AK3 (bovine AK3), 2AR7 (hAK4), 2BWJ (hAK5), 1NKS (AKas) and 3IIJ (hCINAP). Superimposition of the structures was performed with SUPERPOSE45.
PROT_23186_sm_SuppFig3.pdf794KFIGURE S3: Prediction of the AMP binding site of hCINAP A. Stereodiagram showing ligand positions of AKeco (AMPPNP and AMP; in red), AKas (ADP and AMP; in blue) and the hCINAP-SO42- complex. The crystallographic positions, occupied by the sulfate ion and the a-phosphates in the region of the AMP binding site, are essentially equivalent. The distance between the S atom of SO42- and the superimposed PA atoms of AMP from AKeco (1ANK) and AKas (1NKS) complexes is 1.48 ? and 3.26 ?, respectively. B. hCINAP-Mg2+ATP-AMP complex predictions from induced fit docking (IFD) calculations. Stereoscopic view of the three IFD receptor-ligand poses of structures IFD 1 (red), IFD 3 (blue) and IFD 4 (yellow), as outlined in the main text, shows the predicted AMP-relative orientations and the corresponding conformational change of the NMP-binding domain in the vicinity of the AMP binding site.
PROT_23186_sm_SuppFig4.pdf79KFIGURE S4: Inhibition of hCINAP by AP5A. IC50 plot of AP5A inhibition of hCINAP at constant concentrations of AMP (0.3 mM) and ATP (0.33 mM). Inhibitor concentrations were: 1 nM, 5 nM, 10 nM, 30 nM, 90 nM and 120 nM. All data are means from two experiments.
PROT_23186_sm_SuppFig5.pdf187KFIGURE S5: View of hCINAP-ADP (green) superimposed onto the hCINAP-Mg2+ADP-PO43- complex (blue). The crystallographic position of Wat11 (represented as a green sphere) in hCINAP-ADP complex, or Op2 of PO43- in hCINAP-Mg2+ADP-PO43- complex may correspond to the position of attacking β-phosphate oxygen. Lys16 is proposed to promote phosphoryl transfer by neutralizing the negative charges during the penta-coordinated transition state.
PROT_23186_sm_SuppFig6.pdf717KFIGURE S6: Models of the hCINAP-ADP complex from induced fit docking calculations. Comparison of the crystallographic hCINAP-ADP complex (blue) with the top-3 ranked receptor-ligand poses 1 (red), 2 (yellow) and 3 (green), revealed that IFD calculations can reproduce the crystallographic positions with a sufficiently high accuracy as judged by RMSD for hCINAP main-chain (∼ 0.25 Å) and side-chain (∼ 0.5 Å) atoms, and ligand RMSDs close to 1 Å. In more detail, superimposing the receptor-ligand poses with 3IIJ, the hCINAP main chain RMSDs for poses 1, 2 and 3 were 0.248 Å, 0.252 Å and 0.249 Å, respectively; side-chain RMSDs were 0.515 Å, 0.562 Å and 0.517 Å, respectively; ligand RMSDs (heavy atoms) were 1.369 Å, 0.992 Å and 1.347 Å. The difference in the ligand poses was mainly due to the positions of the ribose and adenine moieties. The latter conformations were correctly predicted for pose 2 and with that the lowest RMSD obtained (0.992 Å). The crystal structure hydrogen bonds (Sup-Table I) between the ADP β-phosphate and residues Thr17 N, Thr17 OG1, Gly13 N, Gly15 N, Lys16 NZ (all three poses 1-3) and Arg109 NH1 (pose 3) were all reproduced. Likewise, hydrogen bond contacts from Arg109 NH1, Thr18 N and Thr18 OG1 with the ADP a-phosphate were correctly predicted (poses 1-3). The ADP adenine ring adopts different conformations for poses 1-3, but all of them occupy the correct protein pocket and are close to the one observed in 3IIJ structure. Finally, it should be noted that our predictions point to flexibility of the Arg109 sidechain, with the guanidinium group likely to bind to either or both of the ADP a- and β-phosphates in a ‘dynamic’ system at any given time.
PROT_23186_sm_SuppFig7.pdf171KFIGURE S7: Expression of CINAP H79G has a toxic effect and increases apoptosis in NIH3T3 cells. Comparison of the percentage of apoptotic cells within the population of cells transfected with GFP-CINAP (wild type, WT) and cells transfected with GFP-CINAP H79G (H79G) at 24 and 36 h post transfection. Apoptosis was scored microscopically by identifying the characteristic disorganization of nuclei. Over 5000 cells were counted in each category at each time point. The percentage of apoptotic cells is very significantly higher in H79G mutant transfected samples compared to the WT control samples, both at 24 h (p=0.001) and at 36 h post transfection (p=0.001, Tukey's Multiple Comparison Test).
PROT_23186_sm_SuppFig8.pdf152KFIGURE S8: Phenotypes of nuclear organelles do not appear altered following transient transfections with mutant GFP-hCINAP-H79G, as compared with equivalent transfections with GFP-hCINAP-WT. A. Random field showing cells whose nucleus is marked with the fluorescent dye Hoechst (blue, left panels), some of which are transfected with GFP-hCINAP-WT (wild type, green top middle panel) or mutant GFP-hCINAP-H79G (green bottom, middle panel), as revealed by GFP fluorescence, and immunostained with an anti-fibrillarin mab to label nucleoli (red, right panels). B. Another field of cells marked with Hoechst (blue, left panels), transiently transfected with WT or mutant GFP-CINAP (green, middle panels) and immunostained with anti-PML mab to label the PML bodies (red, right panels). Scale bar: 20 µm.
PROT_23186_sm_SuppFig9.pdf80KFIGURE S9: Surface representation of hCINAP-Mg2+ADP-PO43- crystal structure. The P-loop, B-motif and NMP-binding loop regions are shown in yellow, orange and cyan, respectively. Mg2+ADP binds in a groove located near the protein surface with the adenine ring occupying the entrance of the Mg2+ATP binding site and its phosphate moiety wrapped by the P-loop residues and orientated toward the B-motif region. The protein's topography is also characterized by a 14 Å-long tunnel, with its wall formed by residues of the B-motif (Tyr78 and His79), the NMP-binding domain (Tyr51, Asp52, Cys53, Pro54 and Ile55) and the LID domain (Lys115, Asp118 and Asn119). Arg39 from a2 helix is part of the opening to the Mg2+ATP binding site.
PROT_23186_sm_SuppTab1.pdf61KSup-Table I: (A)Putative hydrogen-bonds and (B) van der Waals interactions between ADP and residues of the ATP binding site of hCINAP
PROT_23186_sm_SuppTab2.pdf26KSup-Table II: (A) Putative hydrogen-bonds and (B) Van der Waals interactions between dADP and residues of the ATP binding site of hCINAP
PROT_23186_sm_SuppTab3.pdf21KSup-Table III: (A) Putative Hydrogen-bonds and (B) Van der Waals interactions between SO42-(1) and residues of the ATP binding site of hCINAP and between SO42-(2) and residues of the AMP binding site of hCINAP
PROT_23186_sm_SuppTab4.pdf40KSup-Table IV: (A) Putative hydrogen-bonds and (B) Van der Waals interactions between ADP, PO43- and residues of the ATP binding site of hCINAP. (C). Coordination sphere of Mg2+ in the vicinity of ATP binding site of hCINAP
PROT_23186_sm_SuppTab5.pdf77KSup-Table V: (A) hCINAP complexes, as well as 1RKB structure, were superimposed (residues 1-172) on the hCINAP-SO42- structure (3IIK) with the program Lsqkab. Residues for which shifts are greater than 0.5Å are listed. (B) Comparison of B factors between hCINAP-ADP structure (3IIJ) and hCINAP-SO42- structures (3IIK, 1RKB).
PROT_23186_sm_SuppTab6.pdf59KSup-Table VI: ESP fit charges on the AMP and ATP α- and γ-phosphate atoms, respectively, calculated using QM/MM at varying interphosphate (P-P) distances.a

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