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PDBsum entry 1euv
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Ulp1-Sumo crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast.
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Authors
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E.Mossessova,
C.D.Lima.
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Ref.
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Mol Cell, 2000,
5,
865-876.
[DOI no: ]
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PubMed id
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Abstract
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Modification of cellular proteins by the ubiquitin-like protein SUMO is
essential for nuclear processes and cell cycle progression in yeast. The Ulp1
protease catalyzes two essential functions in the SUMO pathway: (1) processing
of full-length SUMO to its mature form and (2) deconjugation of SUMO from
targeted proteins. Selective reduction of the proteolytic reaction produced a
covalent thiohemiacetal transition state complex between a Ulp1 C-terminal
fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3
crystal structure and functional testing of elements within the conserved
interface elucidate determinants of SUMO recognition, processing, and
deconjugation. Genetic analysis guided by the structure further reveals a
regulatory element N-terminal to the proteolytic domain that is required for
cell growth in yeast.
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Figure 3.
Figure 3. Structure of the Ulp1-Smt3 ComplexUlp1 is colored
blue; Smt3 is colored red.(A) View looking into a side of the
complex.(B) A perpendicular view of the complex looking onto the
active site. β strands are numbered; α helices are lettered.
The C-terminal Smt3 Gly-Gly motif is located above Ulp1 helix
F.(C) Stereo representation of the Ulp1-Smt3 complex in an
orientation approximately 45° from (A) or (B). Cα positions
are numbered every ten residues with Smt3 denoted by a thicker
line. Graphics prepared using SETOR unless otherwise noted ([9]).
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Figure 5.
Figure 5. Ulp1 Motifs and the Smt3 InterfaceThe Ulp1
polypeptide backbone is depicted in ribbon representation, while
the Smt3 peptide is depicted in stick representation. Ulp1
color-coded motifs as in Figure 1. (A) Ulp1 motif 1 (pink); (B)
motif 2 (blue); (C) motif 3 (red); and (D) motif 4 (green).
Views approximate a close-up for each motif as it appears in
Figure 4B. Hydrogen bonds are denoted by black spheres. Waters
are depicted by red spheres. Amino acid residues discussed in
the text are numbered.
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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2000,
5,
865-876)
copyright 2000.
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Secondary reference #1
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Title
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A new protease required for cell-Cycle progression in yeast.
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Authors
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S.J.Li,
M.Hochstrasser.
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Ref.
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Nature, 1999,
398,
246-251.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1: Characterization of the Ulp1 enzyme. a, The
His[6]–ubiquitin–Smt3–HA substrate. Ub, ubiquitin. b,
Cleavage of His[6]–Ub–Smt3–HA by Ulp1 and Ubp1, analysed
by SDS–PAGE. Lane 1, no added enzyme; lane 10, mixture of
separately purified proteins. Preincubations with inhibitors
(NEM, PMSF and Ald, for Ub aldehyde) were done for 15 min at
room temperature. Ubp1 was used as a control for cleavage after
Ub in the substrate and to confirm the inhibitory activity of Ub
aldehyde. c, Cleavage of SUMO1–ranGAP1 synthesized in vitro.
Lane 1, ranGAP1-K526R mutant; lanes 2–6, human ranGAP1
incubated at 30 °C with purified GST–Ulp1 for indicated
times (min); lane 8, GST–Ulp1 pretreated with NEM. Relative
molecular mass standards are indicated (in thousands). d, In
vitro cleavage by Ulp1 of yeast Smt3–protein conjugates. A
longer exposure was needed to visualize free Smt3. Yeast
extracts (25  g)
were from ulp1-ts cells grown at 23 °C (lanes 1, 2) and at
37 °C (lanes 3–5); lane 3, GST added. Immunoblot used a
rabbit anti-Smt3 antibody raised against His[6]–Smt3. PGK,
phosphoglycerate kinase; Smt3-aty, Smt3 precursor; Smt3, mature
Smt3.
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Figure 4.
Figure 4: Ulp1 is required for cell-cycle progression. a,
Cell-cycle phenotype of ulp1-ts cells. Micrographs show cell
morphology (Nomarski) after 6 h at 37 °C and, for a
different set of samples, nuclear staining (with DAPI) and
microtubule immunostaining after 8.5 h at 37 °C. Mutant
cells are enlarged compared with wild-type cells. After 8 h in
YPD medium at 37 °C, 58.5  3.5%
of mutant cells (n = 572) had large buds, 7.2 1.8%
had small buds and 34.3 4.1%
had no buds. The corresponding percentages for wild-type cells
(n = 425) were 32 1.8,
4 1.7
and 46.6 1.9.
b, FACS analysis of cells synchronized with  -factor
and released at 37 °C. 1C and 2C refers to one and two sets
of chromosomes per cell.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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