Given the key roles of integral membrane proteins as transporters and channels, it is necessary to understand their structures and, hence, mechanisms and regulation at the molecular level. Membrane proteins represent ~30% of all proteins of currently sequenced genomes. Paradoxically, however, only ~2% of crystal structures deposited in the protein data bank are of membrane proteins, and very few of these are at high resolution (better than 2 Å). The great disparity between our understanding of soluble proteins and our understanding of membrane proteins is because of the practical problems of working with membrane proteins – specifically, difficulties in expression, purification and crystallization. Thus, computational modeling has been utilized extensively to make crucial advances in understanding membrane protein structure and function.

Ionotropic glutamate receptors are essential for fast synaptic nerve transmission. Recent x-ray structures for the ligand-binding (S1S2) region of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive receptor have suggested how differences in protein/ligand interactions may determine whether a ligand will behave as a full agonist. We have used multiple molecular dynamics simulations of 2-5 ns duration to explore the structural dynamics of GluR2 S1S2 in the presence and absence of glutamate and in a complex with kainate. Our studies indicate that not only is the degree of domain closure dependent upon interactions with the ligand, but also that protein/ligand interactions influence the motion of the S2 domain with respect to S1. Differences in domain mobility between the three states (apo-S1S2, glutamate-bound, and kainate-bound) are surprisingly clear-cut. We discuss how these changes in dynamics may provide an explanation relating the mechanism of transmission of the agonist-binding event to channel opening. We also show here how the glutamate may adopt an alternative mode of binding not seen in the x-ray structure, which involves a key threonine (T480) side chain flipping into a new conformation. This new conformation results in an altered pattern of hydrogen bonding at the agonist-binding site.

No abstract

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to signi¢cantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of interdomain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Signi¢cantly, the in£uence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs. ß

Proteins possessing the same fold may undergo similar motions, particularly if these motions involve large conformational transitions. The increasing amounts of structural data provide a useful starting point with which to test this hypothesis. We have performed a total of 0.29 micros of molecular dynamics across a series of proteins within the same fold family (periplasmic binding proteinlike) in order to address to what extent similarity of motion exists. Analysis of the local conformational space on these timescales (10-20 ns) revealed that the behavior of the proteins could be readily distinguished between an apo-state and a ligand-bound state. Moreover, analysis of the root-mean-square fluctuations reveals that the presence of the ligand exerts a stabilizing effect on the protein, with similar motions occurring, but with reduced magnitude. Furthermore, the conformational space in the presence of the ligand appears to be dictated by sequence but not by the type of ligand present. In contrast, apo-simulations showed considerable overlap of conformational space across the fold as a result of their ability to undergo larger fluctuations. Indeed, we observed several transitions from different simulations between states corresponding to the closed-cleft and open-cleft forms of the fold, with the predominant motions being conserved across the different proteins. Thus, large-scale conformational changes do indeed appear to be conserved across this fold architecture, but smaller conformational motions appear to reflect the differences in sequence and local fold.