Table 1.
Crystal parameters, data collection statistics and refinement statistics of g14-3-3.
Figure 1.
Classical dimer of g14-3-3 assumes an “open” conformation.
A) Two views of g14-3-3 N-terminal dimer (green, cyan). B) The superimposition of g14-3-3 and human 14-3-3ε (2BR9, violet) emphasizes the open conformation of g14-3-3. C) Close up of N-terminal dimerization interface. Residues are shown as lines if involved in hydrogen bonds, as sticks if involved in salt bridges. D) Detail of polar interactions between helices H and I. Residues not conserved in 14-3-3 family are represented as sticks. A conserved residue (Gln226 in g14-3-3 Gln222 in human 14-3-3ε) is shown as yellow sticks in B) and D) to highlight the different orientations of helix I.
Figure 2.
Sequence alignment of g14-3-3 protein.
The g14-3-3 protein has been aligned with two Ordeum vulgaris isoforms, one from epsilon and one from non-epsilon plant 14-3-3 subgroups (b14-3-3A, P29305.1; b14-3-3B, Q43470.1, respectively), the two Saccharomyces cerevisiae yeast isoforms (BMH1, P29311.4; BMH2, P34730.3) and the seven human 14-3-3 isoforms (h14-3-3β, accession number NP_647539.1; h14-3-3τ, NP_006817.1; h14-3-3η, NP_003396.1; h14-3-3ζ, NP_003397.1; h14-3-3γ, NP_036611.2; h14-3-3σ, NP_006133.1; h14-3-3ε, NP_006752.1). Alignment has been generated using ClustalW2 software and edited with BOXSHADE 3.21. Identical residues are black boxed, similar residues are gray boxed, divergent ones are left unboxed. Dashes indicate gaps. A grey line above the alignment indicates the α- helices, the thin line indicates a region of helix310, observed also in h14-3-3 γ, ε, η and σ. Residues involved in N-terminal dimerization are indicated by stars; the triad of residues contacting the phosphate moiety in the target phosphopeptide are indicated by empty triangles. Dots indicate other residues taking contact with the target phosphopeptide backbone and lateral chains. Black triangles indicate Thr214 and Glu246 of g14-3-3. White squares indicate the residues Arg200, Thr208, and Asn233 forming intermolecular hydrogen bound in the C-terminal dimer interface.
Figure 3.
C-terminal domain swapping leads to fibrils formation in the crystal.
A) Two adjacent dimers within the crystal (green, orange), corresponding to one asymmetric unit, give rise to non-canonical C-terminal dimerization through the swapping of helix I. B) Detail of the interface showing the residues involved in hydrogen bonds. C) The superimposition of g14-3-3 and human 14-3-3ε (violet) emphasizes the swapping of helix I (g14-3-3 αI' vs h14-3-3ε αI) as indicated by the arrow. D) Crystal packing (36 unit cells, 6x6x1). The crystal is formed by layers (blue, red, yellow) of endless parallel filaments.
Figure 4.
Phosphopeptide binding site is preserved upon helix I swapping.
A) A sulfate ion bound in correspondence of the phosphate binding site in g14-3-3. 2mFo-dFc electron density (contoured at 1σ) is shown for sulfate ion and the residues involved in the interaction. B) Superimposition of g14-3-3 (green, orange) and 14-3-3ε-phosphopeptide (2BR9, violet-pink) complex showing that the swapped helix I' can participate to peptide binding. C) Detail of the peptide-binding site. The peptide (from the 2BR9 structure) and the residues involved in the interaction, all conserved in g14-3-3, are represented as sticks. D) Orientation of the phosphopeptides modeled into a g14-3-3 filament. The phosphopeptides in the binding cleft are represented by arrows blue to red(N-terminus to C-terminus).
Figure 5.
A) The evolution of the RMSD (Root Mean Square Deviation) of WT-g14-3-3 (in red) and Pho-g14-3-3 (in black) during the simulation. In order to allow the system to equilibrate, the first 20 ns of simulation were discarded from the analyses (region shaded in grey). B) The per residue RMSF (Root Mean Square Fluctuation). Helices H and I are enclosed in dashed boxes. The position of Thr214 is indicated with an arrow. The bottom plot (grey) shows the per residue RMSF difference between WT-g14-3-3 and Pho-g14-3-3. A schematic secondary structure diagram of the g14-3-3 is reported on the top. C) Eigenvectors cumulative weight on total motion for WT-g14-3-3 (in red) and Pho-g14-3-3 (in black). Projection of motion along the first eigenvector for Pho-g14-3-3 D) and the WT-g14-3-3 E) protein structures. The amplitude of motion follows the color scale from red to blue. The phosphorylated residue is represented in grey and indicated with an arrow.
Figure 6.
A) Snapshots at 20, 30, 40, and 50 ns of the protein structures during the simulation. The proteins are superimposed to each other, WT-g14-3-3 is in green and Pho-g14-3-3 is in violet. B) Magnification of the loop between helices H and I. The phosphorylated Thr214 and the surrounding polar residues are represented in sticks colored by atom. C) Superimposition Pho-g14-3-3 at 20 ns of MD (violet), WT-g14-3-3 (green) and h14-3-3ε (grey) in complex with phosphopeptide (represented as sticks in pink-blue) showing that upon phosphorylation g14-3-3 helix I repositions closest to helix H but still doesn't overlap with h14-3-3ε helix I. The h14-3-3ε residues involved in the interaction with the peptide are represented as stick (grey).
Figure 7.
Assessment of g14-3-3 multimerization in vitro.
A) Far-UV CD spectra of 14-3-3 proteins. WT (black), T214E mutant (green), T208A mutant (cyan), R200K mutant (violet), polyglycinated 14-3-3 (red). CD spectra were measured with a Jasco J-715 spectropolarimeter (Jasco Ltd, Hachioji City, Tokyo, Japan) in 1.0 cm quartz cuvettes between 260 and 195 nm. Samples were 0.1 μg/μl in 10 mM Tris/HCl buffer (pH 7.0). B) Cross-linking assay. Proteins, pre-incubated with Raf1p phosphopeptide, were incubated with DMP (upper panels), in presence or absence of DTT, or without DMP (lower panel), separated on 4–12% reducing SDS-PAGE and immunoblotted with the indicated antibodies. Asterisks indicate: protein monomer (*), dimer (**), trimer (***) and tetramer (****). Molecular size markers (kDa) are on the left. C) Coomassie staining of 12% basic native PAGE. Proteins were either pre-incubated with Raf1p phosphopeptide or the unphosphorylated peptide Raf1. NativeMark (Invitrogen) size markers (kDa) are on the left. Asterisks indicate g14-3-3 and mutants: dimer (**) and tetramer (****). Empty dots indicate h14-3-3ζ dimer (°°). It is to note that, despite the theoretical molecular weight of g14-3-3 and h14-3-3ζ are comparable (28.9 and 28.1 kDa, respectively) their theoretical isoelectric point (Ip) are much divergent (5.09 and 4.73, respectively) thus contributing to the faster migration of h14-3-3ζ homodimer in native PAGE. Similarly, the presence of either phosphorylated T214 in the endogenous g14-3-3 or the glutamic acid at the same position in the T214E mutant decrease the theoretical Ip from 5.09 to 5.03 and 5.01, respectively, thus accounting for the increased migration of the proteins in the gel. D) Densitometric analysis of the native PAGE presented in (C) performed with ImageJ software. In the graphs: the optic density (OD) of each protein band for each lane is reported as relative percentage (%) of the total of optic density (OD) per lane and expressed as relative distribution of g14-3-3 dimer vs tetramer.
Figure 8.
Recombinant g14-3-3 form filaments in a concentration-dependent manner.
A) Recombinant g14-3-3 (0.1 μg/μl) forms numerous filaments with moderate length (between 0,8–1,6 μm) and an average diameter of 5,2±0,7 nm, as measured by iTEM soft imaging system (OLYMPUS). B) At the same concentration (0.1 μg/μl), the presence of the polyglycine stretch in the polyG20 mutant prevents filaments formation and the protein is found only as amorphous aggregates. C) Longer filaments (more than 3–4 μm) are formed by recombinant g14-3-3 at higher concentration (1 μg/μl). D) and E) are sections of panel C at high magnification (100.000X). Representative fields on the grids are depicted. F) Structure-based views of a g14-3-3 filament of concatenated 10 dimers. Dimension (nm) of the filaments are reported.