PDBsum entry 2jb9

Transcription

PDB id: 2jb9

Contents

Protein chains

121 a.a. *

Waters ×170

* Residue conservation analysis

PDB id:

2jb9

Name:

Transcription

Title:

Phob response regulator receiver domain constitutively-active double mutant d10a and d53e.

Structure:

Phosphate regulon transcriptional regulatory protein phob. Chain: a, b. Fragment: receiver domain, residues 1-127. Synonym: phob response regulator d10a-d53e. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.70Å R-factor: 0.224 R-free: 0.256

Authors:

C.Ferrer-Orta,R.Arribas-Bosacoma,S.-K.Kim,A.G.Blanco,P.J.B.Pereira, F.X.Gomis-Ruth,B.L.Wanner,M.Coll,M.Sola

Key ref:

R.Arribas-Bosacoma et al. (2007). The X-ray crystal structures of two constitutively active mutants of the Escherichia coli PhoB receiver domain give insights into activation. J Mol Biol, 366, 626-641. PubMed id: 17182055 DOI: 10.1016/j.jmb.2006.11.038

Date:

05-Dec-06 Release date: 04-Jan-07

Protein chains

P0AFJ5  (PHOB_ECOLI) -  Phosphate regulon transcriptional regulatory protein PhoB from Escherichia coli (strain K12)

Seq: 229 a.a.

Struc: 121 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1016/j.jmb.2006.11.038

J Mol Biol 366:626-641 (2007)

PubMed id: 17182055

The X-ray crystal structures of two constitutively active mutants of the Escherichia coli PhoB receiver domain give insights into activation.

R.Arribas-Bosacoma, S.K.Kim, C.Ferrer-Orta, A.G.Blanco, P.J.Pereira, F.X.Gomis-Rüth, B.L.Wanner, M.Coll, M.Solà.

ABSTRACT

The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg(2+) bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerization of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving alpha1, loop beta5alpha5 and N terminus of alpha5 elements, also seen in the complex of PhoB RD with Mg(2+), in which helix alpha4 highly increases its flexibility. PhoB RD in complex with Mg(2+) and BeF(3) (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix alpha4 and shows another interaction involving alpha4, beta5 and alpha5 segments. We have selected a series of constitutively active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoB(CA) RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix alpha4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein.

Selected figure(s)

Figure 1.
Figure 1. (a) The structure of PhoB RD. Richardson diagram of PhoB RD. Helices are shown as blue ribbons (α1 to α5) and β-strands as yellow arrows (β1 to β5). The N and C-terminal ends are indicated (N and C, respectively). The green arrow points to the active site cavity. Key residue side-chains mentioned throughout the text are highlighted. (b) Dimers interacting through the α1-Lβ5α5 interface. Cα plot showing the superimposition of WT-RD (grey) and DAYC-Na (khaki) dimers. Secondary structure elements belonging to the interface are indicated; the distance between vicinal Lβ4α4 loops within a dimer is shown. The green arrows point to the active site cavities of each protomer within a dimer. The C and N-terminal ends of the RDs are shown (N and C, respectively). In each case, molecules A are on the left and molecules B on the right of the dimer. (c) Packing of WT (grey) and DAYC (khaki) helix α4 against the protein core. Mutated residues or side-chains that change the conformation are indicated twice, with colour coding according the molecule they belong to. α-helices and β-strands numbering is indicated. (d) Each constituting monomer within the WT RD dimer (superimposed with its solid Connolly surface) has been rotated vertically 90° from the position occupied in (b) to grant insight into the interacting surface. The residues of each molecule participating in direct contacts are shown and labelled. The colour coding reflects conservation of each position, ranging from 0% (light yellow) to 100% (intense red). The approximate location of the active site cleft is indicated by a green arrowhead. (e) DADE (green) and BeF-RD[3]^36 (coral) active sites are superimposed (strands β1, β3 and β4 where used for an optimal superposition of residues in the active site). Mutated residues or side-chains that change the conformation are indicated twice. The small spheres represent water (W) molecules, which are numbered according to the PDB. The M sphere represents the coordinated metal; the Be sphere the Beryllium and F1, F2 and F3 the fluorine atoms in the BeF[3]¯ molecule, an analogue of the activating phosphate moiety. Electrostatic and hydrogen bond interactions are represented by broken lines and, together with water oxygen atoms, are coloured according to the PDB they belong to. W3 and the interaction between the cation and Met55 carbonyl are not shown for clarity. Secondary structure elements are indicated. (f) DAYC (khaki) and BeF[3]-RD^36 (coral) are superimposed around their active sites. The criteria of representation are the same as for (e).

The above figure is reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 366, 626-641) copyright 2007.