Abstract

The human TSHβ subunit proteins and their binding sites on the TSH receptor using molecular dynamics simulation Abstract: To gain further insight into the binding sites of the normal and variant human TSHβ subunits (TSHβ & TSHβv), previously characterized by us, we modeled the two monomeric proteins and studied their interaction with the TSH receptor ectodomain (TSHR-ECD) by using molecular dynamics simulation. Furthermore, analyzed their bioactivity in vitro using purified recombinant proteins to confirm that such binding was physiologically relevant.

Examining the interaction of TSHβ and TSHβv with the TSHR-ECD model using molecular dynamic simulation revealed strong binding of these proteins to the receptor ECD. The specificity of TSHβ and TSHβv binding to the TSHR-ECD was examined by analyzing the hydrogen-bonding residues of these subunits to the FSH receptor ECD indicating the inability of these molecules to bind to the ECD of the FSH receptors. Furthermore, the modelling suggests that TSHβ and TSHβv proteins clasped the concave surface of the leucine rich region (LRR) of the TSHR ECD in a similar way to the native TSH using dynamic hydrogen bonding. These mutually exclusive stable interactions between the subunits and ECD residues included some high affinity contact sites corresponding to binding-models of native TSH.

Furthermore, to extend the in-silico binding data of the TSHβ subunits, we cloned TSHβ and TSHβv proteins using the entire coding ORF of the TSHβ and TSHβv and purified the flag-tagged proteins. The expressed TSHβ subunit proteins retained bioactivity both in a co-culture system as well as with immune-purified proteins.

In summary, we showed that such interactions can result in a functional outcome.