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The SecYEG translocon is a channel in bacteria, which provides a passage for secretory proteins across as well as integration of membrane proteins into the plasma membrane. The molecular mechanism, by which SecYEG manages protein transport while preventing water and ion leakage through the membrane, is still controversial. We employed molecular dynamics simulations to assess the contribution of the major structural elements – the plug and the pore ring (PR) – to the sealing of SecYEG in the active state, i.e., with a signal sequence helix occupying the lateral gate. We found, that the PR alone can provide a very tight seal for the wild-type translocon in the active state for both water and ions. Simulations of the mutant I403N, in which one of the PR-defining isoleucine residues is replaced with asparagine, suggest that hydrophobic interactions within the PR and between the PR and the plug are important for maintaining a tight conformation of the wild-type channel around the PR. Disruption of these interactions results in strong fluctuations of helix TM7 and water leakage of the translocon., The hydrophobic interactions between helix TM7, the plug and the pore ring of the translocon determine the barrier to water and ion permission.

The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Graphic abstract Legend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

Volumetric properties of proteins bear directly on their biological functions in hyperbaric environments and are useful in general as a biophysical probe. To gain insight into conformation-dependent protein volume, we developed an implicit-solvent atomic chain model that transparently embodies two physical origins of volume: (1) a fundamental geometric term capturing the van der Waals volume of the protein and the particulate, finite-size nature of the water molecules, modeled together by the volume encased by the protein’s molecular surface, and (2) a physicochemical term for other solvation effects, accounted for by empirical proportionality relationships between experimental partial molar volumes and solvent-accessible surface areas of model compounds. We tested this construct by Langevin dynamics simulations of a 16-residue polyalanine. The simulated trajectories indicate an average volume decrease of 1.73 ± 0.1 A3/residue for coil–helix transition, ∼80% of which is caused by a decrease in geometric v...

We compared the folding pathways of selected mutational variants of the α-spectrin SH3 domain (Spc-SH3) by using a continuum model that combines a full atomistic protein representation with the Gō potential. Experimental data show that the N47G mutant shows very little tendency to aggregate while the N47A and triple mutant D48G(2Y) are both amyloidogenic, with the latter being clearly more aggregation prone. We identified a strikingly similar native-like folding intermediate across the three mutants, in which strand β1 is totally unstructured and more than half of the major hydrophobic core residues are highly solvent exposed. Results from extensive docking simulations show that the ability of the intermediates to dimerize is largely driven by strand β1 and is consistent with the in vitro aggregation behavior reported for the corresponding mutants. They further suggest that residues 44 and 53, which are key players in the nucleation–condensation mechanism of folding, are also important triggers of the aggregation process.

This work investigates the role of N- to C- termini coupling in the folding transition of small, single domain proteins via extensive Monte Carlo simulations of both lattice and off-lattice models. The reported results provide compelling evidence that the existence of native interactions between the terminal regions of the polypeptide chain (i.e. termini coupling) is a major determinant of the height of the free energy barrier that separates the folded from the denatured state in a two-state folding transition, therefore being a critical modulator of protein folding rates and thermodynamic cooperativity. We further report that termini interactions are able to substantially modify the kinetic behavior dictated by the full set of native interactions. Indeed, a native structure of high contact order with ‘‘switched-off’’ termini-interactions actually folds faster than its circular permutant of lowest CO.

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of β2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length β2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis., Author Summary Dialysis-related amyloidosis (DRA) is a conformational disease that affects individuals undergoing long-term haemodialysis. In DRA the progressive accumulation of protein human β2-microglobulin (Hβ2m) in the osteoarticular system, followed by its assembly into amyloid fibrils, eventually leads to tissue erosion and destruction. Disclosing the aggregation mechanism of Hβ2m under physiologically relevant conditions represents a major challenge due to the inability of the protein to efficiently nucleate fibrillogenesis in vitro at physiological pH. On the other hand, ΔN6, a truncated variant of Hβ2m, which is also a major component of ex vivo amyloid deposits extracted from DRA patients, is able to efficiently form amyloid fibrils de novo in physiological conditions. This amyloidogenic behavior is dramatically enhanced in a slightly more acidic pH (6.2) compatible with the mild acidification that occurs in the synovial fluid of DRA patients. In this work, an innovative three-stage methodological approach, relying on an array of molecular simulations, spanning different levels of resolution is used to investigate the initial stage of the de novo aggregation mechanism of ΔN6 in a physiologically relevant pH range. We identify an intermediate state for folding and aggregation, whose potential to dimerize is enhanced at pH 6.2. Our results provide rationalizations for previous experimental observations and new insights into the molecular basis of DRA.

We assessed the interplay of native topology and non-native interactions on surface-tethered protein folding via extensive Monte Carlo simulations of a simple lattice model. In particular, we investigated the thermodynamics and kinetics of protein-like sequences enclosing different amounts of non-native interactions to protein energetics, and which were designed to fold to distinct native topologies. Our results show that the high-contact order (CO) structure renders a folding transition that is robust to (external) steric constraints and non-native interactions. On the other hand, the folding process of the simple low-CO topology can be easily hampered by the presence of a nearby chemically inert plane. In this case, if non-native interactions are highly conspicuous during folding they can actually drive chain collapse into a very native-like trapped state, which impedes the formation of the native structure. The analysis of folding kinetics reveals that the empirical correlation between folding rate and CO may not apply to surface-tethered protein folding. Indeed, results reported here show that depending on the native environment of the tethered chain terminus the folding rate of a low-CO topology can become so drastically small that the high-CO topology actually folds faster under the same conditions. We predict that complex topologies are more likely to conserve their bulk folding mechanism upon surface tethering.

Gō models are exceedingly popular tools in computer simulations of protein folding. These models are native-centric, i.e., they are directly constructed from the protein's native structure. Therefore, it is important to understand up to which extent the atomistic details of the native structure dictate the folding behavior exhibited by Gō models. Here we address this challenge by performing exhaustive discrete molecular dynamics simulations of a Gō potential combined with a full atomistic protein representation. In particular, we investigate the robustness of this particular type of Gō models in predicting the existence of intermediate states in protein folding. We focus on the N47G mutational form of the Spc-SH3 folding domain (x-ray structure) and compare its folding pathway with that of alternative native structures produced in silico. Our methodological strategy comprises equilibrium folding simulations, structural clustering, and principal component analysis.

The adhesion of cells is mediated by receptors and ligands anchored in apposing membranes. A central question is how to characterize the binding affinity of these membrane-anchored molecules. For soluble molecules, the binding affinity is typically quantified by the binding equilibrium constant K3D in the linear relation [RL] = K3D [R][L] between the volume concentration [RL] of bound complexes and the volume concentrations [R] and [L] of unbound molecules. For membrane-anchored molecules, it is often assumed by analogy that the area concentration of bound complexes [RL] is proportional to the product [R][L] of the area concentrations for the unbound receptor and ligand molecules. We show here (i) that this analogy is only valid for two planar membranes immobilized on rigid surfaces, and (ii) that the thermal roughness of flexible membranes leads to cooperative binding of receptors and ligands. In the case of flexible membranes, the area concentration [RL] of receptor-ligand bonds is proportional to the square of [R][L] for typical lengths and concentrations of receptors and ligands in cell adhesion zones. The cooperative binding helps to understand why different experimental methods for measuring the binding affinity of membrane-anchored molecules have led to values differing by several orders of magnitude., 9 pages, 4 figures; to appear in Soft Matter

We performed extensive lattice Monte Carlo simulations of ribosome-bound stalled nascent chains (RNCs) to explore the relative roles of native topology and non-native interactions in co-translational folding of small proteins. We found that the formation of a substantial part of the native structure generally occurs towards the end of protein synthesis. However, multi-domain structures, which are rich in local interactions, are able to develop gradually during chain elongation, while those with proximate chain termini require full protein synthesis to fold. A detailed assessment of the conformational ensembles populated by RNCs with different lengths reveals that the directionality of protein synthesis has a fine-tuning effect on the probability to populate low-energy conformations. In particular, if the participation of non-native interactions in folding energetics is mild, the formation of native-like conformations is majorly determined by the properties of the contact map around the tethering terminus. Likewise, a pair of RNCs differing by only 1-2 residues can populate structurally well-resolved low energy conformations with significantly different probabilities. An interesting structural feature of these low-energy conformations is that, irrespective of native structure, their non-native interactions are always long-ranged and marginally stabilizing. A comparison between the conformational spectra of RNCs and chain fragments folding freely in the bulk reveals drastic changes amongst the two set-ups depending on the native structure. Furthermore, they also show that the ribosome may enhance (up to 20%) the population of low energy conformations for chains folding to native structures dominated by local interactions. In contrast, a RNC folding to a non-local topology is forced to remain largely unstructured but can attain low energy conformations in bulk.

Cell membranes interact via anchored receptor and ligand molecules. Central questions on cell adhesion concern the binding affinity of these membrane-anchored molecules, the mechanisms leading to the receptor-ligand domains observed during adhesion, and the role of cytoskeletal and other active processes. In this review, these questions are addressed from a theoretical perspective. We focus on models in which the membranes are described as elastic sheets, and the receptors and ligands as anchored molecules. In these models, the thermal membrane roughness on the nanometer scale leads to a cooperative binding of anchored receptor and ligand molecules, since the receptor-ligand binding smoothens out the membranes and facilitates the formation of additional bonds. Patterns of receptor domains observed in Monte Carlo simulations point towards a joint role of spontaneous and active processes in cell adhesion. The interactions mediated by the receptors and ligand molecules can be characterized by effective membrane adhesion potentials that depend on the concentrations and binding energies of the molecules., Review article, 13 pages, 9 figures, to appear in Soft Matter

The adhesion of cells is mediated by membrane receptors that bind to complementary ligands in apposing cell membranes. It is generally assumed that the lateral diffusion of mobile receptor-ligand bonds in membrane-membrane adhesion zones is slower than the diffusion of unbound receptors and ligands. We find that this slowing-down is not only caused by the larger size of the bound receptor-ligand complexes, but also by thermal fluctuations of the membrane shape. We model two adhering membranes as elastic sheets pinned together by receptor-ligand bonds and study the diffusion of the bonds using Monte Carlo simulations. In our model, the fluctuations reduce the bond diffusion constant in planar membranes by a factor close to 2 in the biologically relevant regime of small bond concentrations.