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PDBsum entry 1ggw
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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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Structure of cdc4p, A contractile ring protein essential for cytokinesis in schizosaccharomyces pombe.
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Authors
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C.M.Slupsky,
M.Desautels,
T.Huebert,
R.Zhao,
S.M.Hemmingsen,
L.P.Mcintosh.
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Ref.
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J Biol Chem, 2001,
276,
5943-5951.
[DOI no: ]
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PubMed id
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Abstract
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The Schizosaccharomyces pombe Cdc4 protein is required for the formation and
function of the contractile ring, presumably acting as a myosin light chain. By
using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric
protein with two structurally independent domains, each exhibiting a fold
reminiscent of the EF-hand class of calcium-binding proteins. Although Cdc4p has
one potentially functional calcium-binding site, it does not bind calcium in
vitro. Three variants of Cdc4p containing single point mutations responsible for
temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu,
Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular
dichroism spectroscopy. In each case, the amino acid substitution only leads to
small perturbations in the conformation of the protein. Furthermore, thermal
unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms
are all extremely stable, remaining completely folded at temperatures
significantly above those causing failure of cytokinesis in intact cells.
Therefore, the altered phenotype must arise directly from a disruption of the
function of Cdc4p rather than indirectly through a disruption of its overall
structure. Several mutant alleles of Cdc4p also show interallelic
complementation in diploid cells. This phenomenon can be explained if Cdcp4 has
more than one essential function or, alternatively, if two mutant proteins
assemble to form a functional complex. Based on the structure of Cdc4p, possible
models for interallelic complementation including interactions with partner
proteins and the formation of a myosin complex with Cdc4p fulfilling the role of
both an essential and regulatory light chain are proposed.
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Figure 1.
Fig. 1. Cdc4p is composed of two structurally distinct
domains connected by a flexible linker. Shown is the ensemble of
26 structures calculated for the wild-type protein, superimposed
using the backbone atoms in the  -helices of
the N-terminal domain (A, all residues; B, residues 2-66 only),
and the C-terminal domain (C, residue 77-141 only). Due to the
flexible linker, the N- and C-terminal domains do not have a
fixed orientation with respect to one another. A MOLSCRIPT
ribbon diagram of one representative structure of Cdc4p is shown
in D, with -helices
colored as in A-C and  -strands
indicated as white arrows. Helix boundaries are as follows: A
(8-14), red, B (26-35), orange, C (41-49), yellow, D (58-64),
green, E (79-86), green, F (96-105), blue, G (113-119), purple,
and H (133-137), magenta. The short anti-parallel -sheets
encompass residues 22-24 and 54-56 in the N-terminal domain and
93-95 and 127-129 in the C-terminal domain. Also indicated in D
are the positions of point mutations in the N- (F12L, G19E, and
R33K) and C-terminal domains (F79S, G82D, and G107S) causing
temperature-dependent cell growth arrest at cytokinesis. Serines
2 and 6, which are sites of phosphorylation in vivo (40), lie at
the exposed N terminus of the protein.
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Figure 2.
Fig. 2. Plot of backbone amide 15N T[1], T[2], and
heteronuclear 15N{1H}-NOE values versus residue number for
wild-type Cdc4p. The N and C termini and the linker region
connecting the two domains of the protein exhibit conformational
flexibility on a sub-nanosecond time scale as indicated by
anomalously high T[2] and low heteronuclear NOE values. Residues
within the loop regions, particularly between helices A and B
and helices E and F, also show evidence of conformational
mobility. The positions of the eight helices are indicated above
the figure with cylinders.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2001,
276,
5943-5951)
copyright 2001.
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Secondary reference #1
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Title
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Cdc4p, A contractile ring protein essential for cytokinesis in schizosaccharomyces pombe, Interacts with a phosphatidylinositol 4-Kinase.
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Authors
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M.Desautels,
J.P.Den haese,
C.M.Slupsky,
L.P.Mcintosh,
S.M.Hemmingsen.
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Ref.
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J Biol Chem, 2001,
276,
5932-5942.
[DOI no: ]
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PubMed id
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Figure 6.
Fig. 6. Interaction between the PI 4-kinase and cdc4
mutant alleles. YPB2 cells cotransformed with the C-terminal
domain of PI 4-kinase in the library vector and intact cdc4
(cdc4^+) or mutated alleles of cdc4 in the bait vector (Fig. 2)
were grown on SD-leu-trp-his + 3AT. Negative control (YPB2 cells
transformed with the PI 4-kinase in the library vector and with
bait vector lacking an insert) is shown at the top (  ). A
positive interaction is shown by growth at 25 °C in the
absence of histidine and presence of 3-AT (from HIS3 gene
expression) and by blue color development (lacZ expression).
Results indicate that a single point mutation, G107S, abolishes
the interaction between Cdc4p and the PI 4-kinase.
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Figure 7.
Fig. 7. The C-terminal domain of Cdc4p alone is
sufficient to establish an interaction with the PI 4-kinase.
Intact Cdc4p, as well as the C- and N-terminal domains of Cdc4p
(Fig. 1), were cloned in the bait vector and tested for
interaction with the PI 4-kinase, as described in Fig. 6. All
Gal4DB fusion proteins of the appropriate sizes accumulated in
YPB2 cells, as shown in immunoblots with anti-Cdc4p antibodies
(top panel). The bottom panel shows the results of the X-gal
filter colony assay. There is positive interaction in YPB2 cells
cotransformed with the library vector carrying the coding
sequence of the C-terminal domain of PI 4-kinase and with the
bait vector carrying the coding sequence of either Cdc4p
(Gal4DB-Cdc4p) or of its C-terminal domain (Gal4DB-Cdc4p(C)).
There is no interaction in cells transformed with bait vector
without insert (Gal4DB) or carrying the coding sequence of the
N-terminal domain of Cdc4p (Gal4DB-Cdc4p(N)).
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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