PDBsum entry 1ggw

Cytokine

PDB id: 1ggw

Contents

Protein chain

140 a.a. *

* Residue conservation analysis

PDB id:

1ggw

Name:

Cytokine

Title:

Cdc4p from schizosaccharomyces pombe

Structure:

Protein (cdc4p). Chain: a. Synonym: ef-hand protein, myosin light chain. Engineered: yes

Source:

Schizosaccharomyces pombe. Fission yeast. Organism_taxid: 4896. Strain: wild-type 972. Cellular_location: cytoplasm. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

NMR struc:

26 models

Authors:

C.M.Slupsky,S.M.Hemmingsen,L.P.Mcintosh

Key ref:

C.M.Slupsky et al. (2001). Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe. J Biol Chem, 276, 5943-5951. PubMed id: 11087750 DOI: 10.1074/jbc.M008716200

Date:

25-Sep-00 Release date: 21-Mar-01

Protein chain

Q09196  (MLR4_SCHPO) -  Myosin regulatory light chain cdc4 from Schizosaccharomyces pombe (strain 972 / ATCC 24843)

Seq: 141 a.a.

Struc: 140 a.a.

DOI no: 10.1074/jbc.M008716200

J Biol Chem 276:5943-5951 (2001)

PubMed id: 11087750

Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe.

C.M.Slupsky, M.Desautels, T.Huebert, R.Zhao, S.M.Hemmingsen, L.P.McIntosh.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2001, 276, 5943-5951) copyright 2001.

Figures were selected by the author.