Significance: The aggregation of the amyloid-βpeptide (Aβ) into neurotoxic oligomers is central to the development of Alzheimer’s disease. One possible source of their toxicity results from interactions of the Aβoligomers with the neuronal membrane, damaging membrane integrity and thus neurons. However, molecular details of these interactions are unclear. Here, we contrast the dimerization of Aβin solution and at the neuronal membrane. Our results clearly indicate that the sugar moieties of GM1 sequester Aβby forming key hydrogen bonds with the peptide, which diverts the configuration of the Aβdimers away from damagingβ-sheet–rich structures. These findings underline the importance of GM1 in Alzheimer’s disease progression and provide a nanoscopic basis for its reported neuroprotective effect.

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

Protegrin peptides are potent antimicrobial agents believed to act against a variety of pathogens by forming nonselective transmembrane pores in the bacterial cell membrane. We have employed 3D Poisson-Nernst-Planck (PNP) calculations to determine the steady-state ion conduction characteristics of such pores at applied voltages in the range of −100 to +100 mV in 0.1 M KCl bath solutions. We have tested a variety of pore structures extracted from molecular dynamics (MD) simulations based on an experimentally proposed octomeric pore structure. The computed single-channel conductance values were in the range of 290–680 pS. Better agreement with the experimental range of 40–360 pS was obtained using structures from the last 40 ns of the MD simulation, where conductance values range from 280 to 430 pS. We observed no significant variation of the conductance with applied voltage in any of the structures that we tested, suggesting that the voltage dependence observed experimentally is a result of voltage-dependent channel formation rather than an inherent feature of the open pore structure. We have found the pore to be highly selective for anions, with anionic to cationic current ratios (I_Cl−/I_K+) on the order of 10³. This is consistent with the highly cationic nature of the pore but surprisingly in disagreement with the experimental finding of only slight anionic selectivity. We have additionally tested the sensitivity of our PNP model to several parameters and found the ion diffusion coefficients to have a significant influence on conductance characteristics. The best agreement with experimental data was obtained using a diffusion coefficient for each ion set to 10% of the bulk literature value everywhere inside the channel, a scaling used by several other studies employing PNP calculations. Overall, this work presents a useful link between previous work focused on the structure of protegrin pores and experimental efforts aimed at investigating their conductance characteristics. Protegrins are small peptides with strong antimicrobial properties, believed to kill bacteria primarily by forming nonselective pores in the bacterial membrane. This nonspecific and highly effective mechanism of action has created significant excitement about the use of protegrins as therapeutic antibiotics. However, a lack of understanding of the fundamental processes that lead to pore formation and bacterial death has proven to be a major bottleneck in the rational design of protegrin-based antibiotics. In the present work, we have carried out computational investigations of the diffusion of ions through a protegrin pore. We have thereby provided a connection between previous experimental and simulation work aimed at elucidating the structure of the protegrin pore and earlier experimental work investigating the ion transport characteristics of protegrin pores. The ion diffusion characteristics of protegrin pores are likely to be important in their ability to kill bacteria, as the uncontrolled flow of ions through a bacterial membrane will result in membrane depolarization and the loss of vital membrane functions. The present work thus represents an important first step in modeling and quantifying the timeline of events that lead to the killing of bacteria by protegrins. Furthermore, the computational tools that we have presented herein are easily extendible to similar systems, in particular other antimicrobial peptides.

To investigate the stability and functional role of long-residence water molecules in the Q61H variant of the signaling protein K-ras, we analyzed all available Ras crystal structures and conformers derived from a series of independent explicit solvent molecular dynamics (MD) simulations totaling 1.76 µs. We show that the protein samples a different region of phase space in the presence and absence of several crystallographically conserved and buried water molecules. The dynamics of these waters is coupled with the local as well as the global motions of the protein, in contrast to less buried waters whose exchange with bulk is only loosely coupled with the motion of loops in their vicinity. Aided by two novel reaction coordinates involving the distance (d) between the C_α atoms of G60 at switch 2 and G10 at the P-loop and the N-C_α-C-O dihedral (ξ) of G60, we further show that three water molecules located in lobe1, at the interface between the lobes and at lobe2, are involved in the relative motion of residues at the two lobes of Q61H K-ras. Moreover, a d/ξ plot classifies the available Ras x-ray structures and MD-derived K-ras conformers into active GTP-, intermediate GTP-, inactive GDP-bound, and nucleotide-free conformational states. The population of these states and the transition between them is modulated by water-mediated correlated motions involving the functionally critical switch 2, P-loop and helix 3. These results suggest that water molecules act as allosteric ligands to induce a population shift among distinct switch 2 conformations that differ in effector recognition. K-ras belongs to the Ras family of G-proteins that regulate cell proliferation and development. To execute its function, K-ras adopts different conformational states when it is active and inactive. In addition to these two states, it samples many transient intermediate conformations as it makes the transition from one state to the other. Mutations that affect the population of these states can cause cancer or developmental disorder. Using simulation approaches, here we show that a number of water molecules buried within the structure of an oncogenic K-ras protein modulate the distribution of its conformational states. Moreover, a detailed analysis based on two novel structural parameters revealed the existence of long-range water-mediated interactions that facilitate a dynamic coupling between the two lobes of the protein. These findings pave the way for a dynamics-guided strategy to inhibit abnormal Ras signaling.