PDBsum entry 2omt

Cell invasion/cell adhesion

PDB id: 2omt

Contents

Protein chains

462 a.a. *

105 a.a. *

Metals

_CL

_CA

Waters ×602

* Residue conservation analysis

PDB id:

2omt

Name:

Cell invasion/cell adhesion

Title:

Crystal structure of inla g194s+s/hec1 complex

Structure:

Internalin-a. Chain: a. Fragment: internalin domain. Engineered: yes. Mutation: yes. Epithelial-cadherin. E-cad/ctf1. Chain: b. Fragment: n-terminal domain of human e-cadherin. Engineered: yes

Source:

Listeria monocytogenes. Organism_taxid: 169963. Strain: egd-e. Gene: inla. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Homo sapiens. Human. Organism_taxid: 9606.

Resolution:

2.00Å R-factor: 0.199 R-free: 0.283

Authors:

T.Wollert,D.W.Heinz,W.D.Schubert

Key ref:

T.Wollert et al. (2007). Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin. Proc Natl Acad Sci U S A, 104, 13960-13965. PubMed id: 17715295 DOI: 10.1073/pnas.0702199104

Date:

23-Jan-07 Release date: 28-Aug-07

Protein chain

A4GWM6  (A4GWM6_LISMN) -  InlA (Fragment) from Listeria monocytogenes

Seq: 800 a.a.

Struc: 462 a.a.*

Protein chain

P12830  (CADH1_HUMAN) -  Cadherin-1 from Homo sapiens

Seq: 882 a.a.

Struc: 105 a.a.*

* PDB and UniProt seqs differ at 7 residue positions (black crosses)

DOI no: 10.1073/pnas.0702199104

Proc Natl Acad Sci U S A 104:13960-13965 (2007)

PubMed id: 17715295

Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin.

T.Wollert, D.W.Heinz, W.D.Schubert.

ABSTRACT

Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.

Selected figure(s)

Figure 1.
Fig. 1. Overall structure of InlA/hEC1 (dark/light blue). (A) Reengineered residues are indicated by spheres. Although separated by 34 Å, combinations of substitutions from entropically (pink) and enthalpically (orange) dominated "hot spots" act synergistically by stabilizing -strand b of hEC1 (red). (B) Closeup view of the interaction interface. S192N and Y369A/S (ball and stick) stabilize opposite ends of b. G194S+S shortens the distance to residues Glu-54[hEC1] and Lys-61[hEC1] (ball and stick) in d and e, respectively. Stabilization is transmitted through -sheet bde to the N terminus of b. All structural figures were prepared by using Pymol.

Figure 2.
Fig. 2. Structural details of InlA variants. (A) Tyr-369[InlA]-induced side-chain rearrangements during complex formation. Superposition of uncomplexed InlA (pink) and InlA/hEC1 (blue). On complex formation, Ans27[hEC1] (light blue, top) causes Tyr-369[InlA] (dark blue) to flip to an alternative less-favorable conformation, displacing Asn-370 and His-392 from their stacking interaction with Phe-348 (black arrows). (B) The interface near Tyr-369[InlA] in InlA/hEC1 (blue), Y369A/hEC1 (green), and S192N-Y369S/hEC1 (orange). Molecular surface of Y369A is in gray, and that of hEC1 is in pink. Side chains changing conformation during complex formation are shown for InlA/hEC1 (blue) and S192N-Y369S/hEC1 (dark orange). Spheres represent water molecules: black, conserved in all complexes; orange, present in variant complexes; blue, present only in the wild-type complex. Y369A/S prevents the disruptive reorientation of Tyr-369 during complex formation exposing a water-filled cavity. Ser-369-O[]binds two conserved water molecules, one also coordinated by Asn-27[hEC1]-O[ 1]. (C) Ser-192[InlA] in InlA/hEC1 (blue) and S192N/hEC1 (dark red/pink). The two conformations of Ser-192 form water-bridged hydrogen bonds to Phe-17[hEC1]-O or Pro-18[hEC1]-O, respectively. The water molecules are additionally hydrogen-bonded by Asp-213[InlA] and Ser-172[InlA]. Substituting Ser-192 by asparagine displaces one of the water molecules and introduces a direct hydrogen bond to Pro-18-O. The second water molecule maintains the hydrogen-bonding pattern of the wild-type complex. (D–F) InlA/hEC1 (blue) and G194S+S/hEC1 (pink). (D) The interaction of InlA-LRR5 and -6 with hEC1. The mutation G194S and the insertion of an additional serine (+S) restore the canonical LRR-repeat geometry in LRR6, flipping Asn-195 (arrow) into the hydrophobic core of InlA (dark/light gray, wild-type/variant) and removing a large water-filled cavity. (E) Electron density (1 –contoured 2F[O] – F[C]; green, protein; red, water) of LRR6 in InlA/hEC1 (5). The 21-residue LRR6 creates a hydrophobic cavity between Gly-194[InlA], Glu-54[hEC1], and Lys-61[hEC1]. Water molecules filling the gap are poorly defined (weak electron density). (F) The equivalent view as E for G194S+S/hEC1. The gap between interaction partners is narrower, yielding a well defined yet unsaturated water cluster.

Figures were selected by the author.

Author's comment

Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), the authors analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding were quantified by isothermal titration calorimetry. All single substitutions were confirmed to significantly increase binding affinity. Structural changes were verified crystallographically at 2.0 Å resolution or better, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5000-fold. We thus demonstrate that rational re-engineering of protein complexes is possible by making use of physically distant hot spots of recognition.