(A) Reactions catalyzed by T. brucei S-adenosyl-L-methionine decarboxylase (TbAdoMetDC/prozyme heterodimer), ornithine decarboxylase (TbODC) and spermidine synthase (TbSpdSyn) are shown. AdoMet, S-adenosyl-L-methionine; dcAdoMet, decarboxylated S-adenosyl-L-methionine; MTA, methylthioadenosine. (B) CGP 40215 (CGP) is a competitive inhibitor of AdoMetDC (C) Bis-tris propane (B3P), a buffer component in the TbAdoMetDC/prozyme crystallization solution.
DOI: http://dx.doi.org/10.7554/eLife.20198.002

(A) Schematic representation of TbAdoMetDC β-(pink) and α-(beige) chains resulting from autocatalytic serinolysis and pyruvoyl (Pvl86) formation. (B) Sequence alignment of the N-termini of trypanosomatid (Tb, T. brucei; Tc, T. cruzi; and Lm, Leishmania major) and Hs, human, AdoMetDCs. For the complete sequence alignment see . (C) Ribbon diagram of TbAdoMetDC△26 (β-chain in pink and α-chain in beige). Select β-strands are numbered. Pvl86 is shown in spheres and select residues in the autoinhibitory sequence and active site as sticks. Atom colors follow standard nomenclature where carbon is the indicated color, nitrogen (blue), oxygen (red) and sulfur (yellow). (D) 2m|F_o−DF_c| electron density map of autoinhibitory residues contoured at the 1.2 σ. Dashed lines indicate distances (Å). (E) Active site comparison of TbAdoMetDC△26 and HsAdoMetDC (3DZ6) (β-chain dark green, α-chain light green). HsAdoMetDC active site surface is gray. Structures aligned with an RMSD of 2.6 Å over 277 C_α atoms. For full structural alignment see .
DOI: http://dx.doi.org/10.7554/eLife.20198.003

Alignment of TbAdoMetDCΔ26 monomer β/α (pink/beige) and HsAdoMetDC β/α (bright green/light green) (PDB access code 1i7M). The HsAdoMetDC putrescine is shown in green spheres and the HsAdoMetDC inhibitor 4-amidinoindan-1-one-2'-amidinohydrazone (AAH) is shown in transparent green spheres. TbAdoMetDCΔ26 contains no ligands. Structures aligned with an RMSD of 2.6 Å over 277 C_α atoms.
DOI: http://dx.doi.org/10.7554/eLife.20198.004

(A) Ribbon diagram of the CGP-bound heterodimer of TbAdoMetDC (teal (β) and sky blue (α)) and prozyme (yellow). The schematic depicts color codes for the various chains. Ligand colors are as follows: Pvl86 (sky blue), CGP (purple), Put (orange)(AdoMetDC site), Put’ (orange)(prozyme site), and B3P (violet) are shown as spheres. Residues and ligands in prozyme are marked (’). (B) Superposition of TbAdoMetDC and prozyme subunits from the CGP-heterodimer (RMSD = 2.4 Å over 261 C_α atoms). TbAdoMetDC active site helices/strands (residues 202–247) absent from prozyme are colored orange, Y243 (green), CGP (purple), B3P (violet), and prozyme Put' (orange). (C) Schematic representation of the TbAdoMetDC/prozyme heterodimer. Prozyme helices (rectangles) and strands (arrows) were numbered based on structural homology to TbAdoMetDC. (D) Superposition of the scaffolding sheets from TbAdoMetDC△26 and the apo-TbAdoMetDC heterodimer subunit showing main- and side-chain H-bond network (dashed lines) across the dimer interface (overall structures RMSD = 2.1 Å over 310 C_α atoms). For the tetramer structure observed in the asymmetric unit see and for the electron density supporting ligand placement see .
DOI: http://dx.doi.org/10.7554/eLife.20198.007

The asymmetric unit of CGP 40215-bound TbAdoMetDC/prozyme heterodimer (AdoMetDC β/α (teal/sky blue) and prozyme (yellow)) is shown with pyruvoyl groups (Pvl 86) and ligands as spheres (Put and Put', in orange; B3P, in violet; CGP, in purple). Select TbAdoMetDC helices (h1, h6–h8) and prozyme N-terminal β-strands (0') are marked. The tetramer interface (buried surface area of 2,100 Ų) is formed by the non-crystallographic symmetry related prozyme β-strands β0’ configured as crossed arms (domain swap), with β0’ forming additional interactions with h6 and h6/h7 loop from the opposite TbAdoMetDC. A salt bridge between prozyme R5’ and the TbAdoMetDC residue E148 of h8 within the same dimer is also formed (not shown). The tetramer is not relevant to the solution configuration since TbAdoMetDC/prozyme highest molecular weight species in AUC experiments was a heterodimer (; ). The predicted solvation energy gain of burying this surface is small (△G = −10 kcal/mol as calculated by PDBePISA ()), again consistent with the insignificance of the tetramer to the solution structure.
DOI: http://dx.doi.org/10.7554/eLife.20198.008

The electron density map, contoured at 1.0 σ, is shown for a radius of 1.8 Å. The map is color-coded as follows: blue mesh represents density around ligand and gray mesh is density around residues in the binding site within 5 Å of a ligand. The β- and α-chains of TbAdoMetDC are in teal/sky blue, prozyme is in yellow. Select residues are labeled. (A) Bis-tris propane, B3P. (B) CGP 40215, CGP. (C) Putrescine in prozyme, Put'. (D, E). Putrescine, Put, in TbAdoMetDC h1 binding pocket. The two molecules in the asymmetric unit bind Put in alternative conformations (D vs E).
DOI: http://dx.doi.org/10.7554/eLife.20198.009

(A) Ribbon diagram of superimposed inactive TbAdoMetDCΔ26 (pink/beige) with active apo-TbAdoMetDC/prozyme (teal/sky blue/yellow) (RMSD = 2.1 Å over 310 C_α atoms). The schematic depicts color codes for the various chains. Ligands are colored as follows: B3P (purple) and Put’ (orange). (B) Active site comparison of inactive TbAdoMetDCΔ26 with CGP-TbAdoMetDC/prozyme. Inhibitor CGP is shown in green. (C) Pyruvoyl β-sheet rearrangements between inactive TbAdoMetDC△26 and the apo-TbAdoMetDC subunit from the heterodimer. Representative residues on the β7 and β8 strands and nearby loops are highlighted as color-coded pairs: Y176 (purple); F163 (green); I164 (red); I168 (black); H172 (teal) and D169 (orange). Labels are positioned near the monomer for F163, I164, I168, and Y176 and the dimer for D169 and H172. Highlighted residues migrate over distances in parenthesis (C_α-to-C_α) between the inactive monomer and heterodimer structures: F163 (14.7 Å), I164 (15.2 Å), I168 (13.7 Å), D169 (14.3 Å), H172 (8.8 Å), Y176 (8.4 Å). (D) β6 to h8 connector (residues 130–145) rearrangements between the inactive monomer and the active heterodimer. Residues E29 (green) (5.0 Å), W137 (purple) (15.4 Å), R154 (orange) (3.5 Å) are shown as color coded pairs and the migration distances are in parenthesis. F20 is shown in pink and all other residues are colored the same as their chain color. For a schematic and surface representation of these conformational changes see and , respectively. See also .
DOI: http://dx.doi.org/10.7554/eLife.20198.010

β-sheets (flat arrows) and loops (lines) are respectively colored gray and orange in the monomer. The corresponding residues in the heterodimer are colored the same as in the monomer to demonstrate the change in secondary structure. The side chains of residues in the β-strands are shown as black ovals oriented towards either the outer helices (above plain) or scaffolding β-sheet (below plain). Side chains of F163, I164, D169, H172, and Y176 are colored as in . The backbone H-bonds (as defined by distances < 3.3 Å) are shown as dashed lines. The register shifts for the H-bond interactions going from the monomer to the heterodimer are depicted above the monomer.
DOI: http://dx.doi.org/10.7554/eLife.20198.011

Residues within 4 Å of alpha-helix h1 in the heterodimer structure are color-coded: N132-S134 (red), F135-W137 (orange), L144-A147 (yellow), I164 (green), I168-S170 (blue), D171-H172 (dark blue) and R298 (violet). The same labeling is reproduced in the monomer to demonstrate the structural changes. (A) Ribbon diagram of TbAdoMetDCΔ26 (β- and α-chains are pink and beige, respectively). Active site residues F28 (sticks) and Pvl 86 (spheres) are shown. (B) Ribbon diagram of heterodimeric CGP-TbAdoMetDC (β-/α-chains in teal/sky blue, CGP as purple sticks). (C) Surface representation of (A). (D) Surface representation of (B). Residues positioned N-terminal of P31 were not used in surface calculations.
DOI: http://dx.doi.org/10.7554/eLife.20198.012

(A) Surface overview of CGP-TbAdoMetDC/prozyme. Color coding is as follows TbAdoMetDC: (teal (β) and sky blue (α)), prozyme (yellow), CGP (purple) and Put (orange). (B) Top view of the h1 binding site. (C) TbAdoMetDC h1 Put-binding site showing the 4 Å shell. Dashed lines represent H-bonds as defined by distances <3.3 Å. (D) Select h1 interactions (4 Å shell) with TbAdoMetDC or prozyme residues (shown as spheres). (E) Steady-state kinetic analysis of TbAdoMetDC and prozyme mutants for data collected ±4 mM putrescine. The enzyme rates in triplicates over the range of AdoMet concentrations used in Michaelis-Menten analysis are in .
DOI: http://dx.doi.org/10.7554/eLife.20198.014
10.7554/eLife.20198.015Figure 5—source data 1.Enzyme rates in triplicates over the range of AdoMet concentrations used in Michaelis-Menten analysis.DOI: http://dx.doi.org/10.7554/eLife.20198.015

(A) Overlay of CGP-TbAdoMetDC β/α (teal/sky blue, CGP 40215 (TbCGP) and B3P in purple), prozyme (yellow, TbPut’ in orange) and HsAdoMetDC (3DZ6) β/α (dark green/light green, HsPut in green) structures viewed from the dimer interface. The schematic above the figure depicts color codes for the various chains. (B–C) Limited 4 Å shell showing the B3P- and putrescine-binding sites in TbAdoMetDC (B) and prozyme (C). (D) Overlay of the TbAdoMetDC CGP-binding site with HsAdoMetDC showing select residues in the 4 Å shell. H-bond interactions (distance < 3.3 Å) are shown by dashed lines. The electron density supporting ligand placement is shown in , the complete structural alignment of Hs and Tb AdoMetDCs in and the comparison of the unliganded and liganded TbAdoMetDC structures in .
DOI: http://dx.doi.org/10.7554/eLife.20198.016

Alignment of TbAdoMetDC/prozyme CGP 40215-bound heterodimer (AdoMetDC β/α (teal/sky blue), prozyme (yellow)) with HsAdoMetDC 1i7M (β/α (bright green/light green). TbAdoMetDC pyruvoyl (TbPvl 86) is shown as dots. HsAdoMetDC ligands AAH (lines) and putrescine (spheres) are shown in green, TbAdoMetDC ligands (CGP and B3P) are shown in purple and Put is shown in tan, as is prozyme Put'. AdoMetDC subunits aligned with an RMSD of 1.6 Å over 290 C_α atoms.
DOI: http://dx.doi.org/10.7554/eLife.20198.017

Alignment of the AdoMetDC/prozyme apo structure (tan) with the CGP-bound structure (β/α in teal/sky blue) showing the active site region.
DOI: http://dx.doi.org/10.7554/eLife.20198.018

The diagram depicts eukaryotic AdoMetDC enzymes evolving from their bacterial counterparts by extension, gene duplication and fusion. Two bacterial half-enzymes (lower left, PDB 3iwc) form an αββα sandwich through dimerization of the β-sheet faces, with interacting chains depicted in cyan and green cartoon and active site pyruvates in red stick. The primary sequence diagram (same colors) is depicted below. The primary sequence diagram illustrates a presumed intermediate with extended C-terminal αββ extensions (pink). The duplicated and fused eukaryotic enzymes adopt the same αββα sandwich fold (now within a monomer), with extension (pink) dictating edge-to-edge dimerization of the β-sheets (). Trypanosomatid enzymes (represented by the T. brucei structure from this paper) undergo a second gene duplication, with one (prozyme in gray, lower right) losing catalytic activity. Prozyme activates the catalytic enzyme (colored cartoon, lower right) through dimerization and cis-trans isomerization of a conserved proline (P31, magenta sphere) with movement of N-terminal helix (purple). Additionally, protist and fungal sequences retain the conserved proline (magenta lines in the tree with primary sequence diagrams showing the location of the proline (magenta P)). Plant sequences (illustrated by PDB structure 1mhm) have lost the proline, N-terminal helix and dimerization; animal sequences (illustrated by PDB structure 1i7b) retain a dimeric structure of two active chains without proline and N-terminus (depicted in colored cartoon).
DOI: http://dx.doi.org/10.7554/eLife.20198.019

The model depicts a logical step-wise process assuming that the formation of the h1 binding site precedes insertion of the helix into the interface, but current data do not distinguish between a sequential versus a concerted activation mechanism and the ordering of events is hypothetical. (A) The inactive TbAdoMetDC monomer is composed of two β-sheets: pyruvoyl (blue) and scaffolding (light blue). The active site pyruvoyl residue (Pvl86, star) is blocked by the inhibitory sequence (S27–G30), which is oriented into the active site by the cis configuration of P31. This autoinhibitory closed confirmation is stabilized by π-π stacking between F28 (purple) and Y243 (blue). Residues N-terminal of S27, including helix h1 (purple rectangle), were not present in the monomer construct and their position in the diagram is hypothetical. (B) Binding of prozyme (yellow/light yellow) to AdoMetDC is nucleated by formation of the H-bond network between the two scaffolding sheets leading to formation of a continuous inter-subunit β-sheet that serves as a platform for the following conformational changes: (a) slipping of the interface strands β7 and β8 relative to β6, which results in flipping of their side chains from one surface to the other; (b) repositioning and elongation of the β7-β8 loop that forms the back of the h1 binding site, stabilized in this confirmation by interaction across the interface with prozyme; and (c) disordered-to-ordered transition and movement of the β6-h8 loop (H130-L144, orange) that leads to formation of the h1 binding pocket. (C) Upon the formation of the h1 binding pocket, (a) cis-to-trans isomerization of P31 displaces the autoinhibitory sequence from the active site and the open active site conformation is stabilized by (b) docking of the h1 helix at the dimer interface. (D) The active TbAdoMetDC/prozyme dimer is capable of binding ligands in the open active site, which leads to ~1000 fold increase in rates of AdoMet decarboxylation.
DOI: http://dx.doi.org/10.7554/eLife.20198.020