2VLN

N75A mutant of E9 DNase domain in complex with Im9

Summary for 2VLN

• Entry DOI: 10.2210/pdb2vln/pdb
• Related: 1BXI 1E0H 1EMV 1FR2 1FSJ 1IMP 1IMQ 1V13 1V14 1V15 2IVZ 2VLO 2VLP 2VLQ
• Descriptor: COLICIN-E9 IMMUNITY PROTEIN, COLICIN E9, MALONIC ACID, ... (4 entities in total)
• Functional Keywords: protein-binding, protein-protein interaction, metal-binding, antimicrobial, bacteriocin immunity, hydrolase, antibiotic, bacteriocin, endonuclease, zinc, colicin, plasmid, nuclease, hth motif, protein binding
• Biological source: ESCHERICHIA COLI; More
• Total number of polymer chains: 2
• Total formula weight: 24981.68

Authors

Keeble, A.H.,Joachimiak, L.A.,Mate, M.J.,Meenan, N.,Kirkpatrick, N.,Baker, D.,Kleanthous, C. (deposition date: 2008-01-15, release date: 2008-05-20, Last modification date: 2023-12-13)

Primary citation

Keeble, A.H.,Joachimiak, L.A.,Mate, M.J.,Meenan, N.,Kirkpatrick, N.,Baker, D.,Kleanthous, C.
Experimental and Computational Analyses of the Energetic Basis for Dual Recognition of Immunity Proteins by Colicin Endonucleases.
J.Mol.Biol., 379:745-, 2008

PubMed 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.

PubMed: 18471830
DOI: 10.1016/J.JMB.2008.03.055

Experimental method

X-RAY DIFFRACTION (1.6 Å)