Limits...
Molecular design principles underlying β-strand swapping in the adhesive dimerization of cadherins.

Vendome J, Posy S, Jin X, Bahna F, Ahlsen G, Shapiro L, Honig B - Nat. Struct. Mol. Biol. (2011)

Bottom Line: We show that strand swapping in EC1 is driven by conformational strain in cadherin monomers that arises from the anchoring of their short N-terminal strand at one end by the conserved Trp2 and at the other by ligation to Ca(2+) ions.We also demonstrate that a conserved proline-proline motif functions to avoid the formation of an overly tight interface where affinity differences between different cadherins, crucial at the cellular level, are lost.We use these findings to design site-directed mutations that transform a monomeric EC2-EC3 domain cadherin construct into a strand-swapped dimer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. Center for Computational Biology and Bioinformatics, Columbia University, New York, New York, USA. Howard Hughes Medical Institute, Columbia University, New York, New York, USA.

ABSTRACT
Cell adhesion by classical cadherins is mediated by dimerization of their EC1 domains through the 'swapping' of N-terminal β-strands. We use molecular simulations, measurements of binding affinities and X-ray crystallography to provide a detailed picture of the structural and energetic factors that control the adhesive dimerization of cadherins. We show that strand swapping in EC1 is driven by conformational strain in cadherin monomers that arises from the anchoring of their short N-terminal strand at one end by the conserved Trp2 and at the other by ligation to Ca(2+) ions. We also demonstrate that a conserved proline-proline motif functions to avoid the formation of an overly tight interface where affinity differences between different cadherins, crucial at the cellular level, are lost. We use these findings to design site-directed mutations that transform a monomeric EC2-EC3 domain cadherin construct into a strand-swapped dimer.

Show MeSH

Related in: MedlinePlus

Comparison of E-cadherin EC1 and EC2 domains. (a) Sequence alignment of the mouse E-cadherin EC1 and EC2 N-terminal residues. The grey arrows represent the secondary structure elements, and the black triangle above the EC2 sequence indicates the beginning of the EC2-3 construct used. Trp2 and homologous residue Phe108 are highlighted in blue, and the deleted TQE residues in red. (b) Structural alignment of E-cadherin EC1 (in blue) and EC2 (in yellow) domains. The side chains of Trp2 and Phe108 are shown, and the deleted residues are colored in red in the EC2 structure. (c) Detail of the hydrophobic pocket. Residues Ala193 and Ala205 mutated to Ile in the pocket-filling mutants are represented.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113550&req=5

Figure 5: Comparison of E-cadherin EC1 and EC2 domains. (a) Sequence alignment of the mouse E-cadherin EC1 and EC2 N-terminal residues. The grey arrows represent the secondary structure elements, and the black triangle above the EC2 sequence indicates the beginning of the EC2-3 construct used. Trp2 and homologous residue Phe108 are highlighted in blue, and the deleted TQE residues in red. (b) Structural alignment of E-cadherin EC1 (in blue) and EC2 (in yellow) domains. The side chains of Trp2 and Phe108 are shown, and the deleted residues are colored in red in the EC2 structure. (c) Detail of the hydrophobic pocket. Residues Ala193 and Ala205 mutated to Ile in the pocket-filling mutants are represented.

Mentions: EC2 domains differ from EC1 domains in that they have a Phe instead of a Trp at position 2 and they have two additional residues in their A-strands 7. The expectation based on simulation results is that these two factors reduce strain in the A/A* strand and thus inhibit swapping. Indeed isolated EC2 domains are monomeric in solution 18. We produced mutant EC2-EC3 domain constructs which contained one or two of the swapping determinants. This was accomplished with mutants where Phe108 (equivalent to Trp2 in EC1 domains) was replaced with a Trp and/or where the A*/A strand was shortened by three residues. Specifically, Thr109, Gln110 and Glu111 that form a bulge in the A* strand (Fig. 5) were deleted. The relevant KDs are reported in Table 1. Consistent with the results for a single EC2 domain construct 18 wild-type EC2-3 does not dimerize in solution (Table 1). Similarly, both the F108W and the Δ109-111 mutants are also monomeric. However the combined F108W, Δ109-111 mutant is dimeric in solution with a KD of 46.5 μM which is lower than that of the wild type EC1-EC2 construct.


Molecular design principles underlying β-strand swapping in the adhesive dimerization of cadherins.

Vendome J, Posy S, Jin X, Bahna F, Ahlsen G, Shapiro L, Honig B - Nat. Struct. Mol. Biol. (2011)

Comparison of E-cadherin EC1 and EC2 domains. (a) Sequence alignment of the mouse E-cadherin EC1 and EC2 N-terminal residues. The grey arrows represent the secondary structure elements, and the black triangle above the EC2 sequence indicates the beginning of the EC2-3 construct used. Trp2 and homologous residue Phe108 are highlighted in blue, and the deleted TQE residues in red. (b) Structural alignment of E-cadherin EC1 (in blue) and EC2 (in yellow) domains. The side chains of Trp2 and Phe108 are shown, and the deleted residues are colored in red in the EC2 structure. (c) Detail of the hydrophobic pocket. Residues Ala193 and Ala205 mutated to Ile in the pocket-filling mutants are represented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113550&req=5

Figure 5: Comparison of E-cadherin EC1 and EC2 domains. (a) Sequence alignment of the mouse E-cadherin EC1 and EC2 N-terminal residues. The grey arrows represent the secondary structure elements, and the black triangle above the EC2 sequence indicates the beginning of the EC2-3 construct used. Trp2 and homologous residue Phe108 are highlighted in blue, and the deleted TQE residues in red. (b) Structural alignment of E-cadherin EC1 (in blue) and EC2 (in yellow) domains. The side chains of Trp2 and Phe108 are shown, and the deleted residues are colored in red in the EC2 structure. (c) Detail of the hydrophobic pocket. Residues Ala193 and Ala205 mutated to Ile in the pocket-filling mutants are represented.
Mentions: EC2 domains differ from EC1 domains in that they have a Phe instead of a Trp at position 2 and they have two additional residues in their A-strands 7. The expectation based on simulation results is that these two factors reduce strain in the A/A* strand and thus inhibit swapping. Indeed isolated EC2 domains are monomeric in solution 18. We produced mutant EC2-EC3 domain constructs which contained one or two of the swapping determinants. This was accomplished with mutants where Phe108 (equivalent to Trp2 in EC1 domains) was replaced with a Trp and/or where the A*/A strand was shortened by three residues. Specifically, Thr109, Gln110 and Glu111 that form a bulge in the A* strand (Fig. 5) were deleted. The relevant KDs are reported in Table 1. Consistent with the results for a single EC2 domain construct 18 wild-type EC2-3 does not dimerize in solution (Table 1). Similarly, both the F108W and the Δ109-111 mutants are also monomeric. However the combined F108W, Δ109-111 mutant is dimeric in solution with a KD of 46.5 μM which is lower than that of the wild type EC1-EC2 construct.

Bottom Line: We show that strand swapping in EC1 is driven by conformational strain in cadherin monomers that arises from the anchoring of their short N-terminal strand at one end by the conserved Trp2 and at the other by ligation to Ca(2+) ions.We also demonstrate that a conserved proline-proline motif functions to avoid the formation of an overly tight interface where affinity differences between different cadherins, crucial at the cellular level, are lost.We use these findings to design site-directed mutations that transform a monomeric EC2-EC3 domain cadherin construct into a strand-swapped dimer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA. Center for Computational Biology and Bioinformatics, Columbia University, New York, New York, USA. Howard Hughes Medical Institute, Columbia University, New York, New York, USA.

ABSTRACT
Cell adhesion by classical cadherins is mediated by dimerization of their EC1 domains through the 'swapping' of N-terminal β-strands. We use molecular simulations, measurements of binding affinities and X-ray crystallography to provide a detailed picture of the structural and energetic factors that control the adhesive dimerization of cadherins. We show that strand swapping in EC1 is driven by conformational strain in cadherin monomers that arises from the anchoring of their short N-terminal strand at one end by the conserved Trp2 and at the other by ligation to Ca(2+) ions. We also demonstrate that a conserved proline-proline motif functions to avoid the formation of an overly tight interface where affinity differences between different cadherins, crucial at the cellular level, are lost. We use these findings to design site-directed mutations that transform a monomeric EC2-EC3 domain cadherin construct into a strand-swapped dimer.

Show MeSH
Related in: MedlinePlus