Peptide analogs from E-cadherin with different calcium-binding affinities.
Cadherins are a family of calcium-dependent cell-surface proteins that are fundamental in controlling the development and maintenance of tissues. Motif B of E-cadherin seems to be a crucial calcium-binding site as single point mutations (D134A and D134K) completely inactivate its adhesion activity. We analyzed peptide models corresponding to motif B (amino acids 128-144) as well as selected mutations of this motif. Our NMR studies showed that this motif B sequence is actually an active calcium-binding region, even in the absence of the rest of the cadherin molecule. We found that the binding affinity of this motif is very sensitive to mutations. For example, our peptide P128-144 with the native calcium-binding sequence has an affinity of Kd 0.4 mM, whereas the mutants P128-144/ D134A and P128-144/D134K containing the replacement of Asp134 by Ala and Lys, have Kd values of only 1.5 and 11 mM, respectively. Removing Asp at position 134, which correlates with the loss of adhesion activity, decreases calcium-binding affinity 20-fold. Ala132, along with residues Asp134, Asp136 and Asn143, is involved in calcium binding in solution. We also demonstrated that the calcium-binding affinity can be increased 3-fold when an additional Asp is introduced at position 132. In 50% organic solvent, this binding affinity of peptide P128-144/A132D (17-mer) from E-cadherin is similar to that of peptide P72-100/C73-77-91A (29-mer) from alpha-lactalbumin.
Yang
W
W
Department of Biology, Georgia State University, Atlanta, USA.
Tsai
T
T
Kats
M
M
Yang
J J
JJ
D000595:Amino Acid Sequence; D015820:Cadherins / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002942:Circular Dichroism; D007768:Lactalbumin / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012997:Solvents
D015820:Cadherins; D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D012997:Solvents; D007768:Lactalbumin; D002118:Calcium