Novel simulation methods for characterizing conformational changes in nicotinic acetylcholine receptor analogues and beta-2-microglobulin
Authors:   , Stober Spencer T. , Abrams Cameron F. 
Published: July 2021

Molecular simulation tools and protocols were developed for characterizing conformational changes in several proteins: beta2-microglobulin (beta2M), the nicotinic acetylcholine receptor (nAChR), the acetylcholine binding protein (AChBP), and an alpha7-nicotinic acetylcholine receptor/acetylcholine binding protein chimera (alpha7-nAChR-AChBP chimera). The normal to amyloidogenic transformation of beta2M was analyzed using our implementation of on-the-y string method (OFSM) in collective variables (CVs), an algorithm for mapping free energy surfaces along a reaction coordinate. We show that the protonation state of two histidine residues is of key importance in mediating this transition. Also, our predicted free energy barriers for this transition agree with similar experimental evidence. Next, we constructed a membrane-embedded model of a complete nAChR based on the structure determined by Unwin via electron image reconstruction [Unwin, J Mol Biol 346:967, 2005]. Temperature accelerated molecular dynamics (TAMD), a method for rapid sampling of CV space, was used to examine the effects of CV selection and degree of TAMD acceleration on conformations visited during simulation. These results showed that TAMD explores conformational space roughly an order-of-magnitude faster than traditional molecular dynamics (MD) simulation, with correct choices for CVs and other parameters. Unfortunately, these experiments also revealed that the 4 A resolution of this structure is insufficient for application of extensive accelerated sampling techniques. The AChBP is often used as a surrogate for the nAChR because of its similarity to the nAChR extracellular domain (ECD) and the availability of high-resolution structures. A challenge in conformational analysis of AChBP structures is that apo and ligand-bound conformations for the multitude of available structures are nearly superimposable. To address this, we developed a new technique that uses an alignment-free statistical algorithm to assign state determining CVs. This method is successful at discriminating partial from full agonists via analysis of AChBP crystal structures and is a generally applicable method for efficient selection of CVs for use in molecular simulation. Finally we examine two recent crystal structures of an alpha7-nAChR-AChBP chimera in apo and agonist-bound conformations [Li, Nat. Neurosci. 1253:9, 2011]. We developed a new protocol using targeted molecular dynamics (TrMD) followed by restrained MD equilibration to synthesize the conformation of the ligand encounter complex (the conformation generated just after ligand binding occurs) from these structures. We show that this complex is stable on MD time scales and exhibits features of both apo and agonist-bound conformations. Ligand binding energies are computed for the ligand encounter complex and agonist-bound states that are similar to experimental values for the alpha7-nicotinic acetylcholine receptor (alpha7-nAChR). Finally, we discuss preliminary results of an OFSM simulation for the apo and ligand-bound alpha7-nAChR-AChBP chimera.