Calcium-Mediated Allostery of the EGF Fold

Abstract

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.

Linking molecular evolution to molecular grafting

“…The EGF(A) domain belongs to the EGF-like family, which encompasses many disulfide-rich domains found in humans (71). Unlike the cyclotide kalata B1, it has poor tolerance to Ala substitutions to non-Cys residues, with the mutations destabilising the overall fold and favouring highly disordered states despite retaining the disulfide bonds (72). The mutational analysis suggests almost all residues are coupled to structure, implying the peptide has low innovability as a scaffold.…”

EGF‐like and Other Disulfide‐rich Microdomains as Therapeutic Scaffolds

“…Thus, a combination of calcium coordination mechanisms is possible, and an individual domain may, in principle, solicit multiple mechanisms. We recently showed a synthetic analogue of the EGF‐A domain of LDLR requires calcium binding to facilitate folding as the core is insufficient to stabilize the active conformation without the presence of calcium …”

“…Thus, ac ombination of calcium coordination mechanisms is possible,a nd an individual domain may,i np rinciple,s olicit multiple mechanisms.W er ecently showed as ynthetic analogue of the EGF-A domain of LDLR requires calcium binding to facilitate folding as the core is insufficient to stabilize the active conformation without the presence of calcium. [33] Aside from calcium-binding EGF-like domains,there are some that do not bind to calcium (Figures 5e-g). Fort hese domains,compensatory interactions are employed to stabilize the structure,including having additional hydrogen bonds and side-chain interactions between segments 1and 4 (Figure 5e), an extended segment 1t hat can form additional interactions Figure 5.…”

EGF‐artige und andere disulfidreiche Mikrodomänen als therapeutische Molekülgerüste

“…Eine Kombination von Calcium‐Koordinationsmechanismen ist möglich, und eine einzelne Domäne kann im Prinzip mehrere Mechanismen verwenden. Wir haben kürzlich gezeigt, dass ein synthetisches Analogon der EGF‐A‐Domäne des LDLR eine Bindung an Calcium benötigt, um die Faltung zu erleichtern, da der Kern nicht ausreicht, um die aktive Konformation ohne die Anwesenheit von Calcium zu stabilisieren …”

“…Wirh aben kürzlich gezeigt, dass ein synthetisches Analogon der EGF-A-Domäne des LDLR eine Bindung an Calcium bençtigt, um die Faltung zu erleichtern, da der Kern nicht ausreicht, um die aktive Konformation ohne die Anwesenheit von Calcium zu stabilisieren. [33] Neben calciumbindenden EGF-artigen Domänen gibt es einige,d ie nicht an Calcium binden (Abbildung 5e-g). Für diese Domänen werden kompensatorische Wechselwirkungen eingesetzt, um die Struktur zu stabilisieren.…”