Table 1.
Statistics of data collection and refinement.
Fig 1.
1H,15N-correlation HSQC spectra of GtgE.
(A) GtgE(17–214), dispersion of peaks is characteristic of a well-folded protein. (B) Superposition of HSQC spectra of GtgE(17–214) (red) and GtgE(79–214) (cyan).
Fig 2.
Structure of GtgE(17–214, Δ33–40, C45S).
(A) Cartoon representation with the active site residues shown in a stick representation and marked with one-letter code. Position of Ser45 is marked with letter C to indicate that this residue is a cysteine in the wild type GtgE. The molecule is colored in rainbow, from blue at the N-terminus to red at the C-terminus. The disordered segment is shown by a dashed line. The extended termini contact symmetry-related molecules in the crystal. The strand ß1 extends ß-sheet of the neighboring molecule. The catalytic residues and the four C-terminal histidines are shown in a stick mode. Secondary structure elements are labeled. This and subsequent figures were prepared with PyMol (www.pymol.org). (B) The topology diagram of GtgE. The location of catalytic residues is marked with letters C, H and D. (C) The superposition of GtgE (colored rainbow, this work) and the GtgE(79–214) fragment (magenta, PDB code 4MI7, [22]). The ~20 swapped N-terminal residues of the symmetry related molecule are depicted here to show the structure of an intact GtgE (see Fig 3C). The N- and C-termini are marked, the active site residues, Ser45 (Cys45 in wild type GtgE), His151 and Asp169 are shown in stick mode and colored white for GtgE and cyan for GtgE fragment. The inserts on the right shows the expanded view of the active site as observed in our structure. A sulfate molecule is bound near the catalytic histidine and forms hydrogen bonds with its ND1 atom as well as with Tyr42 and Arg142 sidechains. In the presence of a substrate the His151 sidechain would flip by 180° to form hydrogen bond between its ND1 nitrogen and SG of Cys45. Green dashed line indicates the hydrogen bond between His151 and Asp169 observed in the GtgE but not in the GtgE(79–214) fragment.
Fig 3.
Interactions of the N- and C-terminal segments with neighboring molecules.
(A) Segments 17–32 are swapped between two molecules related by 2-fold symmetry and are shown as thick ribbon with residues 23–26 in ß-strand conformation. One molecule is painted rainbow, the other is cyan with the N-terminus colored magenta. (B) The extended C-terminus containing His-tag of magenta molecule contacts a symmetry-related molecule (rainbow colored, N-terminus in blue to C-terminus in red). The extended C-terminus is shown as thick ribbon. These interactions connect molecules into a long chain. (C) A model of GtgE reconstructed by re-swapping the N-terminal segment. The residues 40–214 of molecule A were connected to the swapped segment 17–32 of molecule B through a short linker.
Fig 4.
Superposition of GtgE (cyan) and papain (magenta). Papain structure used for the comparison has PDB code 3CVZ [33].
(A) The N-terminus of papain is shown as a thick green ribbon. The swapped N-terminus of GtgE from a neighboring molecule is shown as a thick blue ribbon. GtgE and papain superimpose well within the core of the fold, enclosed within the blue circle. (B) Close-up of the catalytic triads Cys-His-Asp/Asn in this superposition. The Ser45 of GtgE was replaced here by a cysteine and the sidechain of His151 was rotated by 180°. The catalytic residues are shown in stick representation, magenta carbons in papain, white carbons in GtgE.
Fig 5.
Superposition of GtgE (cyan) and papain (magenta). Papain structure used for the comparison has PDB code 3CVZ.
(A) Cleavage of GST-Rab32 by GtgE(2–228). Lane 1: protein markers, lane 2: GST-Rab32 fraction. The protein was only ~80% pure, other unidentified lower molecular weight bands were present in small amounts, lane 3: Cleavage of GST-Rab32 by GtgE(2–228) mixed in 2:1 (substrate:enzyme) molar ratio, lane 4: As lane 2 but 20:1 ratio of Rab32:GtgE(2–228). (B) Cleavage of GST-Rab32 by GtgE(14–214), lane 1: protein markers, lane 2: partially purified GST-Rab32 fraction, lane 3: Cleavage of GST-Rab32 by GtgE(14–228) in 2:1 molar ratio. (C) Cleavage of GST-Rab32 by GtgE(17–214, Δ33–40). Lane 1: protein marker,s lane 2: Cleavage of GST-Rab32 by GtgE(17–214, Δ33–40) in 2:1 molar ratio; D) GST-Rab32 in the presence fo GtgE(31–214). Lane 1: protein marker, lane 2: GST-Rab32 and GtgE(31–214) in 2:1 molar ratio. The unmarked bands were present in partially purified GST-Rab32 (see panel B lane 2), no band corresponding to the GST-Rab32 degradation product was observed.