Continuum approaches to understanding ion and peptide interactions with the membrane.

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts

Gi- and Gs-coupled GPCRs show different modes of G-protein binding.

More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants

The Journal of Membrane Biology Continuum Approaches to Understanding Ion and Peptide Interactions with the Membrane Continuum Approaches to Understanding Ion and Peptide Interactions with the Membrane

Abstract Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts

Simulation schematics for case studies.

<p>Key perturbations to the experimental structure are indicated in red text. (A) Binding and cooperativity of a negative allosteric modulator (NAM) in the M2 muscarinic receptor (M2R). The binding pose of the NAM and its cooperativity with the orthosteric ligand are probed by performing unguided binding simulations with an unliganded receptor and a receptor with bound orthosteric ligand. Negative cooperativity leads to stronger binding of the NAM to the unliganded M2R. Simulations indicate that cooperativity in this system is due both to direct electrostatic repulsion between cationic ligands and coupled conformational changes of the two binding sites. (B) Activation mechanism of the β₂-adrenergic receptor (β₂AR). Removal of the bound G protein from the active-state, agonist-bound crystal structure of β₂AR leads to a spontaneous transition to the inactive state in simulation, capturing the activation process in reverse. (C) Mechanism of GPCR-catalyzed nucleotide release from a heterotrimeric G protein. Simulations of a G protein with and without bound guanosine diphosphate (GDP) suggested that nucleotide release from a G protein—which leads to G protein activation—takes place via a previously unexpected mechanism. (D) Force-induced uncoupling of a bacterial adhesin from a fibronectin fragment. MD simulations of a fibronectin–adhesin complex led to the discovery that application of stretching forces to fibronectin reduces its affinity to adhesin.</p

Bidirectional symmetry in allosteric systems.

<p>Suppose that a protein can bind two different ligands, A and B, each at their respective sites. The difference in free energy Δ<i>G</i>_0→AB between a state with both ligands bound and one with neither ligand bound is independent of the order in which the ligands bind, so we can write it both as Δ<i>G</i>_0→AB = Δ<i>G</i>_0→B + Δ<i>G</i>_B→AB and as Δ<i>G</i>_0→AB = Δ<i>G</i>_0→A + Δ<i>G</i>_A→AB, implying that Δ<i>G</i>_0→B + Δ<i>G</i>_B→AB = Δ<i>G</i>_0→A + Δ<i>G</i>_A→AB. Rearranging this equation yields Δ<i>G</i>_A→AB − Δ<i>G</i>_0→B = Δ<i>G</i>_B→AB − Δ<i>G</i>_0→A. That is, the difference in the binding energy of ligand A with or without ligand B bound is the same as the difference in the binding energy of ligand B with or without ligand A bound. If one side of this equation is negative, the other must also be negative, and if one side is positive, the other must also be positive. Thus, if binding of ligand B is more favorable in the presence of A, then binding of ligand A is more favorable in the presence of ligand B (positive cooperativity). Likewise, if the presence of ligand A disfavors the binding of ligand B, then the presence of ligand B disfavors the binding of ligand A (negative cooperativity).</p

Corrole-protein interactions in H-NOX and HasA.

Replacing the native porphyrin cofactor in haem proteins has led to the development of novel designer proteins for a variety of applications. In most cases, haem analogues bind in a way that is comparable to the iron porphyrin, but this is not necessarily the case for complexes bearing non-exchangeable ligands. This study probes how a P[double bond, length as m-dash]O corrole binds to functionally disparate hemoproteins: a haem-dependent oxygen sensor (H-NOX) and a haem-scavenging protein (HasA). The results demonstrate that the protein-cofactor interactions are distinct from the native, haem-bound holoprotein. In H-NOX, the P[double bond, length as m-dash]O unit primarily hydrogen bonds with the haem-ligating histidine (H102), rather than the hydrogen-bonding network that stabilises the Fe(ii)-O2 complex in the native protein. In the absence of H102, the protein is still able to bind the corrole, albeit at reduced levels. Molecular dynamics simulations were utilised to determine the flexibility of apo H-NOX and revealed the coupled motion of key residues necessary for corrole binding. In the case of HasA, the P[double bond, length as m-dash]O unit does not primarily interact with either the haem-ligating histidine (H32) or tyrosine (Y75). Instead, histidine 83, the hydrogen-bonding partner for Y75, is critical for P[double bond, length as m-dash]O corrole binding. The conformation of HasA is interrogated by site-specifically labelling the protein and exploiting Förster resonance energy transfer (FRET) to determine the dye-cofactor distance. HasA reconstituted with the P[double bond, length as m-dash]O corrole exhibits an extended, apo-like conformation. Together, these results demonstrate that non-natural cofactors can bind to proteins in unexpected ways and highlight the need to uncover these interactions for the further development of designer haem proteins