Binding of Zn(II) to Tropomyosin Receptor Kinase A in Complex with Its Cognate Nerve Growth Factor: Insights from Molecular Simulation and <i>in Vitro</i> Essays

The binding of the human nerve growth factor (NGF) protein to tropomyosin receptor kinase A (TrkA) is associated with Alzhemeir’s development. Owing to the large presence of zinc­(II) ions in the synaptic compartments, the zinc ions might be bound to the complex <i>in vivo.</i> Here, we have identified a putative zinc binding site using a combination of computations and experiments. First, we have predicted structural features of the NGF/TrkA complex in an aqueous solution by molecular simulation. Metadynamics free energy calculations suggest that these are very similar to those in the X-ray structure. Here, the “crab” structure of the NGF shape binds tightly to two TrkA “pincers”. Transient conformations of the complex include both more extended and more closed conformations. Interestingly, the latter features facial histidines (His60 and His61) among the N-terminal D1–D3 domains, each of which is a potential binding region for biometals. This suggests the presence of a four-His Zn binding site connecting the two chains. To address this issue, we investigated the binding of a D1–D3 domains’ peptide mimic by stability constant and nuclear magnetic resonance measurements, complemented by density functional theory-based calculations. Taken together, these establish unambiguously a four-His coordination of the metal ion in the model systems, supporting the presence of our postulated binding site in the NGF/TrkA complex