PDBsum entry 2erh

Hydrolase/hydrolase inhibitor

PDB id: 2erh

Contents

Protein chains

87 a.a.

127 a.a.

Waters ×149

References listed in PDB file

Key reference

Title

Computational design of a new hydrogen bond network and at least a 300-Fold specificity switch at a protein-Protein interface.

Authors

L.A.Joachimiak, T.Kortemme, B.L.Stoddard, D.Baker.

Ref.

J Mol Biol, 2006, 361, 195-208. [DOI no: 10.1016/j.jmb.2006.05.022]

PubMed id

16831445

Abstract

The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.

Figure 4.
Figure 4. The N517Q mutation in the G design induces a backbone shift in the DNase. Overlay of the design model (teal and yellow side-chains) with the experimentally determined structure (magenta and orange side-chains). The Q517 side-chain in the G design crystal structure does not displace a tightly bound water molecule (magenta, W12) resulting in a backbone shift to accommodate a different Q side-chain rotamer. The immunity protein backbones are colored in gray.

Figure 5.
Figure 5. Structure-based optimization of the G design. The DNase is colored in teal and the immunity protein in gray. Residues participating in the interaction that have been changed or were allowed to vary are shown in space-fill representation, in green and yellow, respectively. (a) In the G design crystal structure the T516 hydroxyl group makes a hydrogen bond to the backbone carbonyl of I54, but the methyl group of the threonine is sub-optimally packed. (b) In the wild-type interface N516 forms a water-mediated (magenta) hydrogen bond to the backbone carbonyl of I54. Following sequence optimization surrounding T516 using the G design structure, the two sequences with the lowest predicted binding energies contained the L19V/I68F(c) and I68W mutations (d) (named G_68F and G_68W, respectively).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 361, 195-208) copyright 2006.