|
Figure 1.
Figure 1. Structure of a Smt3p–Ubc9p complex. (a) Sequence
alignment of Smt3p from S. cerevisiae and human SUMO-1, SUMO-2,
and SUMO-3, with secondary structure elements indicated above,
and residues identical to Smt3p highlighted in yellow. Residues
in Smt3p that contact Ubc9p are denoted with cyan circles. (b)
Sequence alignment of Ubc9p from S. cerevisiae with human,
Arabidopsis thaliana, Schizosaccharomyces pombe, and Xenopus
laevis Ubc9. Secondary structure elements are indicated above,
and residues identical to S. cerevisiae Ubc9p are highlighted
in cyan. Residues in Ubc9p that contact Smt3p are denoted with
yellow circles. (c) Overall structure of the complex, with Smt3p
in yellow and Ubc9p in cyan. Secondary structures are shown
overlaid with a semi-transparent surface. Ubc9p's catalytic
cysteine (Cys 93) is represented by a green sphere. The crystals
form in C2 with a = 120.89 Å, b = 84.58 Å, c = 80.14
Å, and β = 124.31, and two complexes per asymmetric unit.
Data were collected using the mail-in program at the 22-BM
(SER-CAT, Southeast Regional Collaborative Access Team) beamline
at the Advanced Photon Source. Reflection data were indexed,
integrated and scaled using HKL2000.^50 Initial phases were
obtained by molecular replacement using the coordinates of
Ubc9p^37 as a search model in CNS.^51 Electron density for Smt3p
was readily visible in initial maps. The model was built
manually using O,^52 using the previous structures of Smt3p as
guides,^27.^ and ^36. and refined using CNS alternating with
cycles of rebuilding relying on simulated-annealing omit and
composite omit maps.^51 The structure was refined from 50.0
Å to 1.9 Å. The final model has excellent geometry,
with no Ramachandran outliers in disallowed regions. Statistics
from data collection and refinement are given in Table 1. This
and other Figures representing structures were generated with
Pymol.^53 (d) Close-up view of the interface between Smt3p and
Ubc9p, oriented as in (c). Smt3p is shown in yellow, with
specific residues labeled in black. Ubc9p is shown in cyan, with
specific residues labeled cyan. Oxygen atoms are colored red,
and nitrogen atoms blue. Hydrogen bonds and salt-bridges are
represented with dashes. (e) ubc9 bearing mutations in interface
residues with Smt3p do not complement ubc9Δ cells. In a plasmid
shuffle assay, ubc9Δ (PTY30 or PTY34) cells, transformed with a
LEU2 vector containing an intron-less cDNA sequence for
wild-type UBC9 or the indicated mutant allele expressed from the
UBC9 promoter, were spotted onto selective medium supplemented
with dextrose, as described.^[37]^ and ^54. Individual
transformants were successively replica plated onto 5-FOA (US
Biological) plates to cure cells of YCpUBC9·U and
incubated at 26 °C, 30 °C, and 36 °C.^37 Similar
results were obtained at all three temperatures, and
representative results are shown for the experiment at 30
°C. Plasmids were isolated from viable
YCpUBC9·U-cured strains and sequenced to verify the
identity of the ubc9 allele. (f) ubc9 bearing mutations in
interface residues with Smt3p do not restore resistance to
genotoxic stress. The ubc9-10 mutant strain of S. cerevisiae,
bearing a ubc9P123L conditional mutation, was shown previously
to display increased sensitivity to a wide range of DNA damaging
agents including hydroxyurea (HU) and methyl methane sulfonate
(MMS).^54 Exponentially growing cultures of ubc9-10 cells
(A[595] = 0.3) transformed with the indicated plasmids were
serially tenfold diluted and 5 μl aliquots were spotted onto
the appropriate selective media supplemented with dextrose and
incubated at 36 °C. To assay cell sensitivity to HU or MMS,
plates were supplemented with 5 mg/ml HU or 0.0125% MMS,
respectively.^37
|
