PDBsum entry: 2vlq

Protein-binding

PDB id: 2vlq

Contents

Protein chains

84 a.a. *

134 a.a. *

Ligands

MLA ×3

Waters ×300

* Residue conservation analysis

PDB id:

2vlq

Name:

Protein-binding

Title:

F86a mutant of e9 dnase domain in complex with im9

Structure:

Colicin-e9 immunity protein. Chain: a. Synonym: imme9, microcin-e9 immunity protein. Engineered: yes. Colicin e9. Chain: b. Fragment: dnase domain, residues 450-582. Engineered: yes. Mutation: yes

Source:

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

Resolution:

1.60Å R-factor: 0.166 R-free: 0.199

Authors:

A.H.Keeble,L.A.Joachimiak,M.J.Mate,N.Meenan,N.Kirkpatrick, D.Baker,C.Kleanthous

Key ref:

A.H.Keeble et al. (2008). Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases. J Mol Biol, 379, 745-759. PubMed id: 18471830 DOI: 10.1016/j.jmb.2008.03.055

Date:

15-Jan-08 Release date: 20-May-08

Protein chain

P13479  (IMM9_ECOLX) -  Colicin-E9 immunity protein

Seq: 86 a.a.

Struc: 84 a.a.

Protein chain

P09883  (CEA9_ECOLX) -  Colicin-E9

Seq: 582 a.a.

Struc: 134 a.a.*

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

 Gene Ontology (GO) functional annotation 

• Biological process: cytolysis (4 terms)
• Biochemical function: protein binding (4 terms)

DOI no: 10.1016/j.jmb.2008.03.055

J Mol Biol 379:745-759 (2008)

PubMed id: 18471830

Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases.

A.H.Keeble, L.A.Joachimiak, M.J.Maté, N.Meenan, N.Kirkpatrick, D.Baker, C.Kleanthous.

ABSTRACT

Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K(d) values that vary between 10(-4) and 10(-14) M, characteristics that are explained by a 'dual-recognition' mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential DeltaDeltaGs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental DeltaDeltaG data, particularly for interactions not mediated by interfacial water molecules. DeltaDeltaG predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites.

Selected figure(s)

Figure 2.
Fig. 2. The conserved hotspot of Im9 meets the variable hotspot of the E9 DNase. Molecular surface representation of the E9 DNase, coloured according to values of ΔΔG for alanine mutants, in the context of the complex with Im9 (Table 1). Only two of the four helices of Im9 are shown for simplicity. Colour code for values of ΔΔG[kin]^obs: yellow, 2–4 kcal/mol; green, 1–2 kcal/mol; blue, < 1 kcal/mol. With the exception of Arg54 and Asn75, the surface of the IPE is largely variable in the DNase family. By contrast, the three Im9 residues of helix III (shown as ribbon), Asp51, Tyr54 and Tyr55, are all conserved (underlined in the figure). Wallis et al.^17 showed previously that mutation of these residues to alanine generates much greater values of ΔΔG (> 5 kcal/mol). The docking of helix III into the concave cleft of the DNase IPE positions the specificity helix of the Im protein for the appropriate recognition of residue 86 on the DNase and to make additional specificity contacts, such as with Lys97. The correct positioning of helix II is thought to be through rotation about the conserved hotspot of the Im protein in the encounter complex, possibly mediated by three conserved water molecules, shown as red spheres in the figure.

Figure 5.
Fig. 5. Crystal structures of E9 DNase alanine mutants in complex with Im9. See Table 3 for details of refinement statistics. (a) E9 DNase F86A. (b) E9 DNase N75A. (c) E9 DNase K97A. (d) E9 DNase R54A. See the text for details of the structural changes in the immediate vicinity of each mutation and the changes in bound water. All mutant structures (green, with additional water molecules shown in red) are overlaid with the wild-type structure (gray).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 379, 745-759) copyright 2008.

Figures were selected by an automated process.