Peptide analogs from E‐cadherin with different calcium‐binding affinities

Abstract

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.

Effect of linker segments on the stability of epithelial cadherin domain 2

“…Similar binding affinities were resolved in studies of calcium42 and cadmium43 binding to a synthetic peptide containing an important loop in Domain 2. These values are also similar to values for calcium binding to synthetic peptides that include the linker region 44…”

Purification and Structural Analysis of the Hepatitis B Virus PreS1 Expressed from Escherichia coli

“…4). The spectrum is similar to that of a largely unstructured polypeptide (31)(32)(33)(34). The helical content of the preS1 was estimated by comparing its mean residue ellipticity with that expected for a complete helix, as described in Materials and Methods.…”

“…Several proteins that are functionally different from the preS1 are also known to be unstructured or loosely folded (31)(32)(33)(34). For example, human p53 transcriptional activation domain (TAD) has no tertiary or secondary structure in aqueous solution, but it forms induced helices upon binding to target proteins (37)(38)(39).…”

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin

“…The K d values of proteins were calculated by fitting the metal titration curves, assuming that the changes are from both specific and non-specific binding using the following equation modified from that of single binding process [41]:…”