Calcium-Mediated Allostery of the EGF Fold.

The epidermal growth factor (EGF)-like domain is one of the most abundant disulfide-containing domains in nature and is involved in many cellular processes critical to life. Although many EGF-like domains participate in calcium-dependent functions by responding to the local calcium concentration, little is known about how this responsiveness is programmed at the molecular level. Here, we reveal the structural and environmental determinants underpinning the folding of a synthetic analogue of the EGF-A domain (from the low-density lipoprotein receptor). We show that calcium sensitivity is enabled by an allosteric folding pathway, in which calcium binding is connected to the peptide core through local inter-residue interactions. In the absence of calcium, the fold favors disorder because the inherently weak core is insufficient to stabilize the active form, resulting in substantial loss in activity of 2 orders of magnitude. The EGF-A fold, which can freely transition between active and disordered states, is volatile, and we found it to be intolerant of mutations, unlike other disulfide-rich peptides that have been used as stabilizing frameworks. This volatility is beneficial for modularity/plasticity and appears to have evolved for such a purpose, allowing cellular pathways to sense and respond to environmental cues.

Potent, Selective, and Cell-Penetrating Inhibitors of Kallikrein-Related Peptidase 4 Based on the Cyclic Peptide MCoTI-II.

Kallikrein-related peptidase 4 (KLK4) is a serine protease that has putative intracellular and extracellular functions in prostate cancer progression. Here we show that MCoTI-II, a 34-amino acid cyclic peptide found in the seeds of red gac (Momordica cochinchinensis), is an inhibitor of KLK4. By grafting a preferred KLK4 cleavage sequence into MCoTI-II, we produced a highly potent KLK4 inhibitor (K i = 0.1 nM) that displayed 100,000-fold selectivity over related KLKs and the ability to penetrate cells. Additionally, by substituting positively charged noncontact residues in this compound, we produced a potent and selective KLK4 inhibitor that does not penetrate cells. The inhibitors were shown to be nontoxic to human cells and stable in human serum. These KLK4 inhibitors provide useful chemical tools to further define the role(s) of both intracellular and extracellular KLK4 in prostate cancer cell lines and disease models.