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. 2021 Sep 22;12(1):5590.
doi: 10.1038/s41467-021-25849-0.

Structure of a Ty1 restriction factor reveals the molecular basis of transposition copy number control

Matthew A Cottee #  1 , Sean L Beckwith #  2 , Suzanne C Letham  1   3 , Sarah J Kim  2 , George R Young  4   5 , Jonathan P Stoye  5   6 , David J Garfinkel  7 , Ian A Taylor  8
Affiliations

Structure of a Ty1 restriction factor reveals the molecular basis of transposition copy number control

Matthew A Cottee et al. Nat Commun. .

Abstract

Excessive replication of Saccharomyces cerevisiae Ty1 retrotransposons is regulated by Copy Number Control, a process requiring the p22/p18 protein produced from a sub-genomic transcript initiated within Ty1 GAG. In retrotransposition, Gag performs the capsid functions required for replication and re-integration. To minimize genomic damage, p22/p18 interrupts virus-like particle function by interaction with Gag. Here, we present structural, biophysical and genetic analyses of p18m, a minimal fragment of Gag that restricts transposition. The 2.8 Å crystal structure of p18m reveals an all α-helical protein related to mammalian and insect ARC proteins. p18m retains the capacity to dimerise in solution and the crystal structures reveal two exclusive dimer interfaces. We probe our findings through biophysical analysis of interface mutants as well as Ty1 transposition and p18m restriction in vivo. Our data provide insight into Ty1 Gag structure and suggest how p22/p18 might function in restriction through a blocking-of-assembly mechanism.

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Conflict of interest statement

The authors declare no competing financial or non-financial interests.

Figures

Fig. 1
Fig. 1. Transposition assays define the minimal fragment for p22/p18 activity.
a Schematic of the Ty1 retrotransposon, highlighting the TY1A GAG and TY1B POL genes. The functional regions and cleavage site of Gag identified by genetic analysis are highlighted below, *PFAM domains, †CNCR domain, ‡NAC originally defined as residues 299-401 redefined by this study as residues 356–401. The Met249 and Met259 translational starts that initiate from the AUG1 and AUG2 codons are indicated. b Schematic of p22/p18 constructs and structural features of Gag. Gag-p49 and p22 undergo proteolytic maturation into Gag-p45 and p18, respectively. A restrictive fragment terminating at residue 355 (p18m) is defined in this study and all p18 constructs also contain a C-terminal hexa-histidine tag. c Schematic illustrating the two plasmids used to co-express the Ty1 transposon and the p18 restriction factor. The expression of each is driven by a galactose-inducible promoter from the GAL1 gene. Ty1 contains the his3-AI retromobility indicator gene; histidine prototrophy requires retromobility. d Western blot of p18 fragment expression. Protein extracts of galactose-induced cells were immunoblotted with an anti-hexa-histidine antibody. Pgk1 serves as a loading control. Strains used: empty (DG3739), p18 (DG4162), p18mAUG1 (DG4147), p18mAUG2 (DG4146). Migration of molecular weight standards is shown alongside the immunoblot. A representative image of at least 3 replicates is shown, images of entire gel immunoblots are provided in the Source Data file. e Qualitative mobility assay showing CNC effect of p18 constructs on Ty1 retromobility. Cells were galactose-induced; growth on selective media lacking histidine indicates a retromobility event. A representative image of at least 3 replicates is shown. f Quantitative mobility assay of galactose-induced cells. Each bar represents the mean of the four independent measurements displayed as points. The error bar centre represents the mean of the four measurements and the error bar extent ± the standard deviation. Fold-change compared with empty vector is indicated above the bars. Significance is calculated from a two-sided Student’s t-test compared with p18 (n.s not significant, ***p < 0.001. Exact p-values are provided in Supplementary Table 1, source data is provided in the Source Data file.
Fig. 2
Fig. 2. Crystal structure of p18m.
a Crystal structure of p18m monomer, the protein backbone is shown in cartoon representation. The five α-helices in the structure are labelled sequentially from the N- to C-terminus. b 3D structural alignment of all six copies of p18m present in the AUG1 and AUG2 structures. The view is the same as in a. Individual monomers are shown in cartoon representation coloured light-blue, cyan, pink, orange, grey, and green. Structures were aligned using backbone Cα atoms (RMSD 0.16 ± 0.04 Å over 82 Cα). c The trimer asymmetric unit of the p18m crystal structure. The three p18m protomers are shown in cartoon representation coloured cyan, light-blue and pink respectively. The dimer interfaces between protomers (Dimer-1 and Dimer-2) are highlighted with the grey box and ellipse respectively. d Close-up view of the Dimer-1 interface, the box-highlighted region in (c). Residues on helices α1 and α3 that make apolar and salt bridge interactions are shown in stick representation. The E265 and K307 salt bridge is indicated by the dashed lines. e Close-up view of the Dimer-2 interface, the ellipse-highlighted region in (c). Residues on helices α4 and α5 that make the hydrophobic interactions are shown in stick representation.
Fig. 3
Fig. 3. p18m self-associates in solution.
a, d SEC-MALLS analysis of p18m and Dimer-2 interface-mutant p18m-F323S. The sample loading concentrations were 400 µM (cyan), 200 µM (magenta), and 100 µM (wheat). The differential refractive index (dRI) is plotted against column retention time and the molar mass, determined at 1 s intervals throughout the elution of each peak, is plotted as points. The p18m monomer and dimer molecular mass is indicated with the grey dashed lines. b C(S) distributions were derived from sedimentation velocity data recorded from p18m at 42 µM (wheat), 82 µM (magenta), and 165 µM (cyan). The curves are the distribution of the sedimentation coefficients that best fit the sedimentation data (RMSD 0.004−0.016), see also Supplementary Table 3. c Multispeed sedimentation equilibrium profiles determined from interference data collected on p18m at 30 µM. Data were recorded at the speeds indicated. The solid lines represent the global best fit to the data using a monomer-dimer-tetramer model (KD(1-2) = 0.73 µM, KD(2-4) = 43.2 µM; reduced χ2 = 2.22). The lower panel shows the residuals to the fit, see also Supplementary Table 3. Source data for b and c are provided in the Source Data file. e, g Effect of p18mAUG1 and Gag mutations on Ty1 mobility. Growth on selective media; His+ cells indicate a retromobility event. A representative image of at least 3 replicates is shown. p18mAUG1 strains used: empty (DG3739), WT (DG4147), F323S (DG4350), F323D (DG4351). Gag strains used: WT (DG3735), F323S (DG4348), F323D (DG4349). f, h Protein extracts prepared from galactose-induced yeast cells expressing the indicated p18mAUG1 or Gag mutants were immunoblotted with hexa-histidine antibody to detect p18mAUG1 or TY-tag antibody to detect Gag. Pgk1 serves as a loading control. Migration of molecular weight standards is shown alongside the immunoblots. A representative image of at least 3 replicates is shown. Images of the whole gel immunoblots are provided in the Source Data file.
Fig. 4
Fig. 4. Ty1 retromobility and restriction tolerates conservative interface mutants.
a, d Effect of Gag and p18mAUG1 mutations on Ty1 mobility. Growth on media lacking histidine indicates a retromobility event. A representative image of at least 3 replicates is shown. Gag strains used: WT (DG3735), A273V (DG4342), I269F (DG4341). p18mAUG1 strains used: empty (DG3739), WT (DG4147), A273V (DG4165), I269F (DG4340). b, e Protein extracts prepared from galactose-induced yeast cells expressing the indicated Gag or p18mAUG1 mutants were immunoblotted with TY-tag antibody to detect Gag or hexa-histidine antibody to detect p18mAUG1. Pgk1 serves as a loading control. Migration of molecular weight standards is shown alongside the immunoblots. A representative image of at least 3 replicates is shown, Images of the whole gel immunoblots are provided in the Source Data file. c, f Quantitative mobility assay of galactose-induced cells. Each bar represents the mean of at least four independent measurements displayed as points. The error bar centre represents the mean of the measurements and the error bar extent ± the standard deviation. Fold-change compared with WT is indicated above the bars. Significance is calculated from a two-sided Student’s t-test compared with WT (n.s not significant, **p < 0.01, ***p < 0.001. Exact p-values are provided in Supplementary Table 1, See also Supplementary Table 4. Source data is provided in the Source Data file.
Fig. 5
Fig. 5. p18m Dimer-1 interface mutants assembly and structure.
a, b SEC-MALLS analysis of p18m-A273V and p18m-I269F, sample loading concentrations; 400 µM (cyan), 200 µM (magenta), and 100 µM (wheat). dRI is plotted against retention time. The molar mass, determined at 1 s intervals throughout peak elution, is plotted as points. Monomer and dimer molar masses are indicated with the dashed lines. c C(S) distributions were derived from sedimentation velocity data recorded from p18m-A273V at 40 µM (wheat), 86 µM (magenta), and 181 µM (cyan). Curves are the distribution of the sedimentation coefficients that best fit the sedimentation data (RMSD 0.009-0.025), see also Supplementary Table 3. (d) Thermal denaturation of p18m, p18m-A273V, and p18m-I269F monitored by CD at 222 nm upon heating from 10–80 °C. Curves are the best fit spline function to the data points, dashed lines indicate the Tm of transitions determined from the 1st derivative of the fitted curves. Error bars are standard deviations from three independent measurements. Data shown are from one representative experiment, the lower panel shows the residuals to each fit. Source data for c and d is provided in the Source Data file (e) Asymmetric unit of the p18m-A273V crystal structure. The backbone of the three p18m protomers are shown in the cartoon, coloured lime, slate and pink. The Fo-Fc map contoured at 3σ (orange mesh), produced after molecular replacement, contains residual positive density for the valine γ-methyl groups at residue 273. f 3D superimposition of the 3 chains shown in (e). Structures were aligned using backbone Cα atoms (rmsd = 0.16 ± 0.04 Å over 78 ± 3.6 Cα). g Comparison of p18m (grey) and p18m-A273V (green) dimer interfaces. Structures are shown in cartoons with A273 or V273 sidechains as sticks. The PISA-calculated average solvation energy contribution to each dimer from either A273 (p18m) or V273 (p18m-A273V) are shown below. h Close-up view of the dimer interface, boxed in g. Local shifts in the backbone conformation at the C-terminus of α1 prevent steric clashes and allow the packing of the additional γ-methyl groups of V273 preserving a favourable 3.6 Å Van der Waals distance.
Fig. 6
Fig. 6. p18m-Gag interaction is critical for restriction activity.
a–f Protein extracts from galactose-induced yeast cells (Input) were fractionated over a 7–47% continuous sucrose gradient and immunoblotted. The bars below anti-TY blots denote peak Gag fractions containing more than 1/9 of the Gag signal across the gradient, as determined by densitometric analysis. A representative image of at least 3 replicates is shown. Strains used: DG4292 (a), DG3739 (b), DG4162 (c), DG4147 (d), DG4165 (e), DG4340 (f). Migration of molecular weight standards is shown alongside the immunoblots. Images of the whole gel immunoblots are provided in the Source Data file. g (Left) Schematic illustrating endogenously expressed, chromosomal Ty1 and galactose-inducible, plasmid-borne p18mAUG1. Ty1 is tagged with the his3-AI retromobility indicator gene; histidine prototrophy requires retromobility. (Right) Relative restriction by p18mAUG1 and p18mAUG1-A273V of Ty1 and Ty1-A273V for homotypic and heterotypic pairings. The relative restriction is calculated as the percentage of fold-restriction by the homotypic pairing. Quantitative mobility data is mean from four replicates of galactose-induced cells. Each bar represents the mean of the four independent measurements displayed as points. The error bar centre represents the mean of the four measurements and the error bar extent ± the standard deviation. Significance is calculated from a two-sided Student’s t-test compared with homotypic relative restriction (*p < 0.05, exact p-values provided in Supplementary Table 1). The cartoons below illustrate homotypic or heterotypic p18m-Gag interactions. See also Supplementary Table 1 and Supplementary Fig. 7. Source data is provided in the Source Data file.
Fig. 7
Fig. 7. Model for p22/p18-dependent CNC.
a Transcription of Ty1 results in either a full-length Ty1 transcript (teal), or a short, inhibitory transcript Ty1i (red). The long transcript encodes full-length Ty1 Gag and Pol proteins, shown in green/cyan cartoon. The shorter Ty1i transcript encodes p22/p18, a C-terminal portion of Gag equivalent to a CA-protein CTD, lacking an NTD (red cartoon), as well as Pol proteins, which are not required for restriction. b Full-length Gag monomers assemble into oligomers through NTD-NTD, and CTD-CTD interactions to construct an icosahedral shell, constituting the VLP, which is required for transposition. c In the presence of p22/p18, Gag monomers are also able to oligomerise with p22/p18 through CTD-CTD interactions. Two examples of a Gag-p22/p18 hetero-interaction through the CTD interface are highlighted by the trapezoidal boxes. While Gag proteins may be able to homo-oligomerise through their NTDs, Gag oligomers that associate with p22/p18 become assembly dead ends resulting in partially assembled particles that cannot support transposition.
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