Control Activity of Yeast Geranylgeranyl Diphosphate Synthase from Dimer Interface through H‑Bonds and Hydrophobic Interaction

Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased <i>k</i>_cat values (200–250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS

Crystal Structure of Vaccinia Viral A27 Protein Reveals a Novel Structure Critical for Its Function and Complex Formation with A26 Protein

<div><p>Vaccinia virus envelope protein A27 has multiple functions and is conserved in the <i>Orthopoxvirus</i> genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21–84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel α-helices and one unusual antiparallel α-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner <i>in vitro</i> in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly <i>in vitro</i>. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.</p></div

Immunoblot analysis of A27 protein in infected cells and on mature virus particles.

<p>(A) HeLa cells were mock-infected or infected with WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A at an MOI of 5 PFU/cell. Lysates were harvested at 24 h post-infection and separated on 4% to 12% SDS-PAGE gels in reducing conditions (+2ME) for immunoblot analysis with anti-A26 (1∶1,000), anti-A27 (1∶5,000), and anti-D8 (1∶5,000) antibodies. (B and C) The same infections as in A were processed for immunoblot analysis in non-reducing conditions (−ME) and probed with anti-A27 (B) and anti-A26 and anti-H3 (C) antibodies. A26-monomers (A26-mono) appeared as doublets because of incomplete intramolecular disulfide bonding between C43 and C342 of the A26 protein <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#ppat.1003563-Ching1" target="_blank">[23]</a>. (D) Purified MV particles (1µg) of WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A were separated on 4%–12% SDS-PAGE gels and processed for immunoblot analysis in reducing conditions (+2ME) as described above. (E and F) Immunoblot analysis of purified MV in non-reducing conditions (−2ME) using anti-A27 (E) and anti-A26 and anti-H3 (F) antibodies. The black and white arrows mark 90-kDa and 70-kDa A26–A27 protein complexes, respectively. Arrowheads – labeled mono, -di, and -tri represent protein monomer, dimer, and trimer, respectively.</p

AUC-SV and SEC/MALS analysis of tA27 protein showing that tA27 protein formed concentration-dependent dimers.

<p>(A) AUC analyses of tA27-WT protein. Normalized <i>c</i>(<i>s</i>) distribution plots for tA27-WT and mutants. The figure shows the distribution plots for tA27-WT at pH 7.5 (<i>black</i>), tA27-TM-N (<i>green</i>), tA27-TM-C (<i>orange</i>), tA27-6A (<i>purple</i>) and tA27-WT at pH 3.0 (<i>red</i>) obtained from the fitting of SV data using a continuous <i>c</i>(<i>s</i>) distribution model. (B) SEC/MALS profiles of tA27-WT protein at concentrations of 1 mg/ml (<i>red</i>) and 9.5 mg/ml (<i>green</i>). BSA was used as control (<i>black</i>). Thin line segments represent the calculated molar masses; the numbers denote the corresponding molecular weights of each peak (left ordinate axis) in kDa. Solid lines represent normalized UV absorbance (280 nm, right ordinate axis), and dashed lines represent light scattering.</p

Overall structure of tA27.

<p>(A) A schematic representation of the vaccinia virus A27 domain structure. A27 comprises three domains, including the heparin binding domain (HBD), the coiled-coil domain (CCD) for hexameric assembly and interacting with A26, and the leucine zipper domain (LZD) for binding with A17. Expressed tA27 for crystallization studies contains residues from 21 to 84, with mutations in two adjacent cysteine residues (C71A and C72A). The magenta cylinder denotes the region of tA27 structure that corresponds to the α-helix. Black dashed lines indicate the disordered regions. The basic residues in HBD (<i>cyan</i>) and the critical segment for specific heparin binding (<i>underlined</i>) are shown. The residues in the N-terminal region (NTR) interface involved in trimer assembly are presented in blue, and residues in the C-terminal region (CTR) interface involved in hexamer assembly are in green. Purple and green dots denote the mutation sites for <i>in vitro</i> and <i>in vivo</i> studies. (B) This representation of hexameric tA27 is composed of trimer 1 (bottom), shown as ribbon diagram and surface model in blue (chain A), cyan (chain B), and magenta (chain C); and trimer 2 (top), presented as surface model and ribbon diagram in light blue (chain A′), light cyan (chain B′), and light magenta (chain C′). The disordered region, including the HBD, is indicated by black dotted lines. Residues 71 and 72 are colored in red. The filled circles indicate the C-terminus and the white circles denote the N-terminus of tA27. (C) The trimeric structure of Influenza virus HA2. A representative trimer-of-hairpins structure of the viral fusion protein of the influenza virus (Flu) HA2 (PDB code 1HTM) is shown <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#ppat.1003563-Bullough1" target="_blank">[60]</a>. For Flu HA2, the interior N-terminal coiled-coil structures are colored in similar blue and the exterior C-terminal antiparallel helices are in similar green. The right part of the figure shows the model rotated 90°.</p

Analysis of vaccinia A27 orthologs.

<p>(A) Multiple sequence alignment of A27 orthologs. The alignment contains group A from Parapoxvirus, including Bovine papular stomatitis virus (BPSV) and Orf virus (ORFV) group B from Orthopoxvirus including Vaccinia virus (VV), Variola virus (VARV), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Ectromelia virus (ECTV) group C from Capripoxvirus, including Lumpy skin disease virus (LSDV), Goatpox virus (GTPV), Sheeppox virus (SPPV), and Suipoxvirus with Swinepox virus (SWPV); and group D from Leporipoxvirus, including Rabbit fibroma virus (RFV) and Myxoma virus (MYXV). Note the heparin binding domain (HBD, <i>orange</i>), coiled-coil domain (CCD, <i>purple</i>), and leucine zipper domain (LZD, <i>grey</i>) regions. The conserved residues surrounding the CCD are marked with orange boxes for NTR and green boxes for CTR. The two cysteine residues in the CCD are denoted by red boxes and dots. Identical and similar amino acid residues are shaded in black and gray, respectively. Dashes denote the sequence gaps introduced to optimize the amino acid sequence alignment. The accession numbers are: BPSV (NP958013); ORFV (AAR98199); VV (YP233032); VARV (ABF23908); MPXV (NP536566); ECTV (NP671648); CPXV (NP619946); LSDV (NP150551); GTPV (ABS72324); SPPV (NP659689); SWPV (NP570274); RFV (NP052004); and MYXV (NP051829). (B) The protein evolution of A27 orthologs. The respective A27 protein lengths of Groups A, B, C, and D are represented by black, cyan, orange and green lines, respectively. The inserted residues are marked with triangles, and the corresponding conserved regions of CCD are boxed in red dashes. (C) Computer modeling of vaccinia A27 orthologues with the X-ray structures of vaccinia virus tA27 show that the CCD regions are structurally conserved, as predicted by molecular modeling, in group A (BPSV, <i>black</i>), group C (LSDV, <i>orange</i>), and group D (RFV, <i>green</i>). The predicted modeling structures were produced by the Phyre server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#ppat.1003563-Kelley1" target="_blank">[61]</a>.</p

WRΔA27L, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A induce cell-to-cell membrane fusion on L cells at neutral pH.

<p>(A) L cells expressing GFP or RFP (1∶1 mixture) were either mock-infected or infected with WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A viruses at an MOI of 50 PFU/cell at 37°C for 30 min and monitored for cell-to-cell fusion at a neutral pH, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003563#s4" target="_blank">Materials and Methods</a>. Cell images were photographed at 2 h post-infection. (B) Quantification of cell-to-cell fusion at neutral pH. The percentage of cells containing both GFP and RFP fluorescence was quantified as cell-cell fusion using Axio Vision Rel. 4.8 with a Zeiss Axiovert fluorescence microscope.</p

WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A exhibited a small plaque phenotype on BSC40 cells.

<p>(A) Schematic representation of vaccinia virus genomes containing A26–A29 and J2R ORFs. Wild type WR virus is shown at the top, with arrows indicating the direction of transcription. In WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A, the viral A27L ORF was replaced with a dual expression cassette, Luc-Gpt, containing a luciferase (Luc) gene driven by a viral early promoter and the Eco-gpt (Gpt) gene driven by the viral p7.5 promoter. The J2 locus of the WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A genome was inserted with another dual expression cassette containing an A27 ORF driven by a viral late promoter and a <i>lacZ</i> gene driven by the viral p7.5k promoter. (B) BSC40-A27 cells express A27 protein for function complementation in <i>trans</i>. Immunoblot analysis of A27 protein expressed in BSC40-A27 cells. Beta-actin was used as a control. WRΔA27L virus, which formed small plaques on BSC40 cells, produced large plaques on BSC40-A27 cells after cell staining with 1% crystal violet in 20% ethanol. (C) Virus plaque morphology on BSC40 and BSC40-A27 cells. WR-BSC40-A27 and BSC40 cells were infected with 50–100 plaques of WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A, fixed at 2 days post-infection and subsequently stained with X-gal and photographed.</p