PDBsum entry 1pv0

Signaling protein

PDB id: 1pv0

Contents

Protein chain

46 a.a. *

* Residue conservation analysis

PDB id:

1pv0

Name:

Signaling protein

Title:

Structure of the sda antikinase

Structure:

Sda. Chain: a. Engineered: yes

Source:

Bacillus subtilis. Organism_taxid: 1423. Gene: sda. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

25 models

Authors:

S.L.Rowland,W.F.Burkholder,M.W.Maciejewski,A.D.Grossman,G.F.King

Key ref:

S.L.Rowland et al. (2004). Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis. Mol Cell, 13, 689-701. PubMed id: 15023339 DOI: 10.1016/S1097-2765(04)00084-X

Date:

26-Jun-03 Release date: 13-Apr-04

Protein chain

Q7WY62  (SDA_BACSU) -  Sporulation inhibitor sda from Bacillus subtilis (strain 168)

Seq: 52 a.a.

Struc: 46 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/S1097-2765(04)00084-X

Mol Cell 13:689-701 (2004)

PubMed id: 15023339

Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis.

S.L.Rowland, W.F.Burkholder, K.A.Cunningham, M.W.Maciejewski, A.D.Grossman, G.F.King.

ABSTRACT

Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization/phosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

Selected figure(s)

Figure 4.
Figure 4. Identification of Key Functional Residues(A) Structure of Sda showing side chains of surface-exposed residues that are identical or conservatively substituted in all Sda orthologs available at the time of this study (the O. iheyensis and B. cereus sequences were released after completion of the mutagenesis).(B) Same schematic as in (A) but rotated vert, similar 90° clockwise around the long axis of the helical hairpin. All conserved surface residues, with the exception of Ser37 and Ser45, are on the N-terminal face of the hairpin.(C) Surface representation of Sda with location of key functional residues denoted in red. Molecular orientation is the same as (A).(D) Assays of the ability of wild-type and mutant Sda proteins to inhibit KinA autophosphorylation. Sda concentrations (in pmol) are given above each lane. Inhibition of KinA autophosphorylation is indicated by the lack of a KinA vert, similar P band(s) on the gel. Each small panel shows the result for a single protein. All mutants were correctly folded with the exception of the F25A mutant labeled “Misfold.”(E) Assays of the ability of wild-type and mutant Sda proteins to bind KinA autokinase domain. Sda proteins were incubated with Ni^2+-NTA agarose beads decorated with His[6]-KinA^383–606, then unbound (lanes labeled “S”) and bead-bound (lanes labeled “B”) fractions were recovered and analyzed as described in Experimental Procedures. In (D) and (E) the mutants are divided into three panels based on their ability to inhibit KinA autophosphorylation.

Figure 6.
Figure 6. Proposed Sda Binding Site and Mechanism of Action(A) Alignment of EnvZ with KinA, B, and C. Numbering refers to KinA. Residues that are identical or conservatively substituted in at least three of the four sequences are highlighted in yellow and orange, respectively. The experimentally determined secondary structure of the EnvZ DHp (Tomomori et al., 1999) and catalytic (Tanaka et al., 1998) domains is given below the alignment. Linker regions are demarcated by red lines. The percentage identity (I) and similarity (S) relative to KinA is indicated at the end of each sequence.(B) Modeled structure of the EnvZ autokinase domain (PDB file 1NJV) (Cai et al., 2003). Domains and linkers are color-coded to match the sequence alignment in (A). In (B)–(D) the side chain of the phosphorylatable His is colored orange.(C) Schematic of the Spo0F-Spo0B cocrystal structure (PDB file 1F51). Only the N-terminal four-helix bundle of the Spo0B dimer is shown; the C-terminal α/β domains have been omitted for clarity. The side chain of the active-site Asp residue in Spo0F is shown in red.(D) Schematic of the structure of the EnvZ DHp domain (PDB file 1JOY). Highlighted in red is the OmpR binding site determined by NMR chemical shift mapping (Tomomori et al., 1999).(E) Alignment of KinA^383–460 with Spo0B. The secondary structure of the four-helix bundle of Spo0B is indicated below the sequences. Residues in Spo0B that contact Spo0F (Zapf et al., 2000) are indicated by red circles, and the active-site His residues are denoted by an asterisk (His405 in KinA, His30 in Spo0B). The predicted Spo0F binding site on KinA and the area available for Sda binding are indicated above the sequences.(F) Schematic of the closed conformation of the KinA autokinase domain based on the EnvZ model structure. The two monomers are shown in orange and blue, the phosphorylatable His405 is depicted as a green circle, and the approximate location of the ATP binding site on the catalytic domain is indicated. The predicted Spo0F binding site and the area available for Sda binding are indicated.(G and H) Two alternative models of the mechanism of Sda action. Sda could lodge under the linker region at the top of the DHp domain (G) or bind exclusively to the linker region (H). Either orientation could explain why Sda enhances KinA dimerization (see text for details).

The above figures are reprinted by permission from Cell Press: Mol Cell (2004, 13, 689-701) copyright 2004.

Figures were selected by an automated process.