Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central...

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an ‘inactive’ low-affinity state (LAS) to an ‘active’ high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH’s ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Role of Water Mediated Interactions in Calcium-Coupled Allostery of Calmodulin Domains

Allosteric communication between distant parts of protein controls many cellular functions. Binding of Ca2+ to the helix-loop-helix motifs (termed EF-hands) in calmodulin (CaM) leads to large conformational changes poising it for the binding of target proteins involved in variety of cell signaling events. Despite the physiological importance, the mechanism of Ca2+-mediated allosteric transitions in CaM remains elusive. Particularly, it is still unclear how water molecules contribute to Ca2+ coordination and the coupled conformational motions. We use all-atom molecular dynamics simulations with enhanced sampling method to investigate the coupling between the Ca2+ binding, dehydration, and the conformational change of the isolated CaM domains, each containing two EF-hands. We reveal a water-bridged coordination mechanism during Ca2+ binding and dehydration, in which the bridging water molecules reduce the entropy penalty during the coordination of liganding residues, thus contributing to efficient ligand binding in CaM domains. Exposure of hydrophobic sites occurs by calcium induced rotation of the helices of EF-hands with the hydrophobic core serving as the pivot. Interestingly, we find that despite being structurally similar, the structural response in the two EF-hands upon Ca2+ binding is highly asymmetric, which is needed for allosteric communication between them. The atomically detailed picture for the allosteric transitions of the CaM EF-hands, which are the first events in mediating a variety of intracellular processes, reveal the complex interplay between the discrete water molecules, dehydration of Ca2+, and CaM structural changes.Table of Contents graphic

Interface residues that drive allosteric transitions also control the assembly of L-lactate Dehydrogenase

The allosteric enzyme, L-lactate dehydrogenase (LDH), is activated by fructose 1,6-metaphosphate (FBP) to reduce pyruvate to lactate. The molecular details of the FBP-driven transition between the low affinity T-state to the high affinity R-state in LDH, a tetramer composed of identical subunits, are not known. The dynamics of theT→R allosteric transition, investigated using Brownian dynamics (BD) simulations of the Self-Organized Polymer (SOP) model, revealed that coordinated rotations of the subunits drive the T→R transition. We used the structural perturbation method (SPM), which requires only the static structure, to identify the allostery wiring diagram (AWD), a network of residues that transmits signals across the tetramer, as LDH undergoes the T→R transition. Interestingly, the residues that play a major role in the dynamics, which are predominantly localized at the interfaces, coincide with the AWD identified using the SPM. The conformational changes in the T→R transition start from the region near the active site, comprising of helix αC, helix α1/2G, helix α3G and helix α2F, and proceed to other structural units, thus completing the global motion. Brownian dynamics simulations of the tetramer assembly, triggered by a temperature quench from the fully disrupted conformations, show that the bottleneck for assembly is the formation of the correct orientation between the subunits, requiring contacts between the interface residues. Surprisingly, these residues are part of the AWD, which was identified using the SPM. Taken together, our results show that LDH, and perhaps other multi-domain proteins, may have evolved to stabilize distinct states of allosteric enzymes using precisely the same AWD that also controls the functionally relevant allosteric transitions.