Mechanical forces impair antigen discrimination by reducing differences in T-cell receptor/peptide-MHC off-rates.

T cells use their T-cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains debated. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity TCR/pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds). This was confirmed by molecular dynamics simulations. Consistent with these findings, we show that the best-characterized catch bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination, and we suggest a role for the adhesion receptors CD2 and LFA-1 in force-shielding the TCR/pMHC interaction.

Crystal structure and allosteric activation of protein kinase C ßII.

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C ßII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCßII was derived from small-angle X-ray scattering. Together, these results show how PKCßII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.

Membrane budding.

Membrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms.

Molecular mechanisms and energetics of lipid droplet formation and directional budding.

The formation and budding of lipid droplets (LDs) are known to be governed by the LD size and by membrane tensions in the endoplasmic reticulum (ER) bilayer and LD-monolayers. Using coarse-grained simulations of an LD model, we first show that ER-embedded LDs of different sizes can form through a continuous transition from wide LD lenses to spherical LDs at a fixed LD size. The ER tendency to relax its bilayer modulates the transition via a subtle interplay between the ER and LD lipid densities. By calculating the energetic landscape of the LD transition, we demonstrate that this size-independent transition is regulated by the mechanical force balance of ER and LD-tensions, independent from membrane bending and line tension whose energetic contributions are negligible according to our calculations. Our findings explain experimental observation of stable LDs of various shapes. We then propose a novel mechanism for directional LD budding where the required membrane asymmetry is provided by the exchange of lipids between the LD-monolayers. Remarkably, we demonstrate that this budding process is energetically neutral. Consequently, LD budding can proceed by a modest energy input from proteins or other driving agents. We obtain equal lipid densities and membrane tensions in LD-monolayers throughout budding. Our findings indicate that unlike LD formation, LD budding by inter-monolayer lipid exchange is a tension-independent process.

A Practical Guide to All-Atom and Coarse-Grained Molecular Dynamics Simulations Using Amber and Gromacs: A Case Study of Disulfide-Bond Impact on the Intrinsically Disordered Amyloid Beta.

Intrinsically disordered proteins (IDPs) pose challenges to conventional experimental techniques due to their large-scale conformational fluctuations and transient structural elements. This work presents computational methods for studying IDPs at various resolutions using the Amber and Gromacs packages with both all-atom (Amber ff19SB with the OPC water model) and coarse-grained (Martini 3 and SIRAH) approaches. The effectiveness of these methodologies is demonstrated by examining the monomeric form of amyloid-ß (Aß42), an IDP, with and without disulfide bonds at different resolutions. Our results clearly show that the addition of a disulfide bond decreases the ß-content of Aß42; however, it increases the tendency of the monomeric Aß42 to form fibril-like conformations, explaining the various aggregation rates observed in experiments. Moreover, analysis of the monomeric Aß42 compactness, secondary structure content, and comparison between calculated and experimental chemical shifts demonstrates that all three methods provide a reasonable choice to study IDPs; however, coarse-grained approaches may lack some atomistic details, such as secondary structure recognition, due to the simplifications used. In general, this study not only explains the role of disulfide bonds in Aß42 but also provides a step-by-step protocol for setting up, conducting, and analyzing molecular dynamics (MD) simulations, which is adaptable for studying other biomacromolecules, including folded and disordered proteins and peptides.

Scrutinising the Conformational Ensemble of the Intrinsically Mixed-Folded Protein Galectin-3.

Galectin-3 is a protein involved in many intra- and extra-cellular processes. It has been identified as a diagnostic or prognostic biomarker for certain types of heart disease, kidney disease and cancer. Galectin-3 comprises a carbohydrate recognition domain (CRD) and an N-terminal domain (NTD), which is unstructured and contains eight collagen-like Pro-Gly-rich tandem repeats. While the structure of the CRD has been solved using protein crystallography, current knowledge about conformations of full-length galectin-3 is limited. To fill in this knowledge gap, we performed molecular dynamics (MD) simulations of full-length galectin-3. We systematically re-scaled the solute-solvent interactions in the Martini 3 force field to obtain the best possible agreement between available data from SAXS experiments and the ensemble of conformations generated in the MD simulations. The simulation conformations were found to be very diverse, as reflected, e.g., by (i) large fluctuations in the radius of gyration, ranging from about 2 to 5 nm, and (ii) multiple transient contacts made by amino acid residues in the NTD. Consistent with evidence from NMR experiments, contacts between the CRD and NTD were observed to not involve the carbohydrate-binding site on the CRD surface. Contacts within the NTD were found to be made most frequently by aromatic residues. Formation of fuzzy complexes with unspecific stoichiometry was observed to be mediated mostly by the NTD. Taken together, we offer a detailed picture of the conformational ensemble of full-length galectin-3, which will be important for explaining the biological functions of this protein at the molecular level.

Membrane curvature sensing by model biomolecular condensates.

Biomolecular condensates (BCs) are fluid droplets that form in biological cells by liquid-liquid phase separation. Their major components are intrinsically disordered proteins. Vast attention has been given in recent years to BCs inside the cytosol and nucleus. BCs at the cell membrane have not been studied to the same extent so far. However, recent studies provide increasingly more examples of interfaces between BCs and membranes which function as platforms for diverse biomolecular processes. Galectin-3, for example, is known to mediate clathrin-independent endocytosis and has been recently shown to undergo liquid-liquid phase separation, but the function of BCs of galectin-3 in endocytic pit formation is unknown. Here, we use dissipative particle dynamics simulations to study a generic coarse-grained model for BCs interacting with lipid membranes. In analogy to galectin-3, we consider polymers comprising two segments - one of them mediates multivalent attractive interactions between the polymers, and the other one has affinity for association with specific lipid head groups. When these polymers are brought into contact with a multi-component membrane, they spontaneously assemble into droplets and, simultaneously, induce lateral separation of lipids within the membrane. Interestingly, we find that if the membrane is bent, the polymer droplets localize at membrane regions curved inward. Although the polymers have no particular shape or intrinsic curvature, they appear to sense membrane curvature when clustered at the membrane. Our results indicate toward a generic mechanism of membrane curvature sensing by BCs involved in such processes as endocytosis.

Convergent evolution in the mechanisms of ACBD3 recruitment to picornavirus replication sites.

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.

Interplay between cooperativity of intercellular receptor-ligand binding and coalescence of nanoscale lipid clusters in adhering membranes.

Adhesion of biological cells is mediated by the specific binding of receptors and ligands which are typically large proteins spanning through the plasma membranes of the contacting cells. The receptors and ligands can exhibit affinity for nanoscale lipid clusters that form within the plasma membrane. A central question is how these nanoscale lipid clusters physically affect and respond to the receptor-ligand binding during cell adhesion. Within the framework of classical statistical mechanics we find that the receptor-ligand binding reduces the threshold energy for lipid clusters to coalesce into mesoscale domains by up to ∼50%, and that the formation of these domains induces significant cooperativity of the receptor-ligand binding. The interplay between the receptor-ligand binding cooperativity and the lipid domain formation manifests acute sensitivity of the membrane system to changes in control parameters. This sensitivity can be crucial in cell signaling and immune responses.

Intercellular Receptor-Ligand Binding and Thermal Fluctuations Facilitate Receptor Aggregation in Adhering Membranes.

Nanoscale molecular clusters in cell membranes can serve as platforms to recruit membrane proteins for various biological functions. A central question is how these nanoclusters respond to physical contacts between cells. Using a statistical mechanics model and Monte Carlo simulations, we explore how the adhesion of cell membranes affects the stability and coalescence of clusters enriched in receptor proteins. Our results show that intercellular receptor-ligand binding and membrane shape fluctuations can lead to receptor aggregation within the adhering membranes even if large-scale clusters are thermodynamically unstable in nonadhering membranes.

Membrane fluctuations and acidosis regulate cooperative binding of 'marker of self' protein CD47 with the macrophage checkpoint receptor SIRPα.

Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous 'marker of self' protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of 'self' cells - including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRPα immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces 'self' signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors - such as the acidity of tumors and other diseased or inflamed sites - can thus arise from the collective cooperative properties of flexible membranes.This article has an associated First Person interview with the first author of the paper.

Structural analysis of phosphatidylinositol 4-kinase IIIß (PI4KB) - 14-3-3 protein complex reveals internal flexibility and explains 14-3-3 mediated protection from degradation in vitro.

Phosphatidylinositol 4-kinase IIIß (PI4KB) is responsible for the synthesis of the Golgi and trans-Golgi network (TGN) pool of phosphatidylinositol 4-phospahte (PI4P). PI4P is the defining lipid hallmark of Golgi and TGN and also serves as a signaling lipid and as a precursor for higher phosphoinositides. In addition, PI4KB is hijacked by many single stranded plus RNA (+RNA) viruses to generate PI4P-rich membranes that serve as viral replication organelles. Given the importance of this enzyme in cells, it has to be regulated. 14-3-3 proteins bind PI4KB upon its phosphorylation by protein kinase D, however, the structural basis of PI4KB recognition by 14-3-3 proteins is unknown. Here, we characterized the PI4KB:14-3-3 protein complex biophysically and structurally. We discovered that the PI4KB:14-3-3 protein complex is tight and is formed with 2:2 stoichiometry. Surprisingly, the enzymatic activity of PI4KB is not directly modulated by 14-3-3 proteins. However, 14-3-3 proteins protect PI4KB from proteolytic degradation in vitro. Our structural analysis revealed that the PI4KB:14-3-3 protein complex is flexible but mostly within the disordered regions connecting the 14-3-3 binding site of the PI4KB with the rest of the PI4KB enzyme. It also predicted no direct modulation of PI4KB enzymatic activity by 14-3-3 proteins and that 14-3-3 binding will not interfere with PI4KB recruitment to the membrane by the ACBD3 protein. In addition, the structural analysis explains the observed protection from degradation; it revealed that several disordered regions of PI4KB become protected from proteolytical degradation upon 14-3-3 binding. All the structural predictions were subsequently biochemically validated.

The length but not the sequence of peptide linker modules exerts the primary influence on the conformations of protein domains in cellulosome multi-enzyme complexes.

Cellulosomes are large multi-protein catalysts produced by various anaerobic microorganisms to efficiently degrade plant cell-wall polysaccharides down into simple sugars. X-ray and physicochemical structural characterisations show that cellulosomes are composed of numerous protein domains that are connected by unstructured polypeptide segments, yet the properties and possible roles of these 'linker' peptides are largely unknown. We have performed coarse-grained and all-atom molecular dynamics computer simulations of a number of cellulosomal linkers of different lengths and compositions. Our data demonstrates that the effective stiffness of the linker peptides, as quantified by the equilibrium fluctuations in the end-to-end distances, depends primarily on the length of the linker and less so on the specific amino acid sequence. The presence of excluded volume - provided by the domains that are connected - dampens the motion of the linker residues and reduces the effective stiffness of the linkers. Simultaneously, the presence of the linkers alters the conformations of the protein domains that are connected. We demonstrate that short, stiff linkers induce significant rearrangements in the folded domains of the mini-cellulosome composed of endoglucanase Cel8A in complex with scaffoldin ScafT (Cel8A-ScafT) of Clostridium thermocellum as well as in a two-cohesin system derived from the scaffoldin ScaB of Acetivibrio cellulolyticus. We give experimentally testable predictions on structural changes in protein domains that depend on the length of linkers.

[Conformations of multi-domain and partially disordered proteins - simulations and experiments]. / Konformacje bialek wielodomenowych i czesciowo nieustrukturyzowanych - symulacje i eksperymenty.

Structural biology unravels three-dimensional structures of macromolecules such as proteins, DNA, RNA, and their complexes in the attempt to explain the basic mechanisms of their functions. Among the proteins that are most difficult to characterize structurally are those which have several large domains connected by long, unstructured polypeptide segments. Such proteins perform diverse functions in living organisms and, at the same time, they are very difficult to characterize using conventional methods of structural biology. This gap in the market has recently led to the development of hybrid methods that use state-of-the-art computational tools to combine complementary data from various experiments. This review article is focused on the implementation and usage of such hybrid methods. It includes a detailed description of how representative structures of multi-domain proteins are obtained using the so-called EROS (Ensemble Refinement of SAXS) hybrid method.

The crystal structure of the phosphatidylinositol 4-kinase IIα.

Phosphoinositides are a class of phospholipids generated by the action of phosphoinositide kinases with key regulatory functions in eukaryotic cells. Here, we present the atomic structure of phosphatidylinositol 4-kinase type IIα (PI4K IIα), in complex with ATP solved by X-ray crystallography at 2.8 Å resolution. The structure revealed a non-typical kinase fold that could be divided into N- and C-lobes with the ATP binding groove located in between. Surprisingly, a second ATP was found in a lateral hydrophobic pocket of the C-lobe. Molecular simulations and mutagenesis analysis revealed the membrane binding mode and the putative function of the hydrophobic pocket. Taken together, our results suggest a mechanism of PI4K IIα recruitment, regulation, and function at the membrane.

Membrane curvature generated by asymmetric depletion layers of ions, small molecules, and nanoparticles.

Biomimetic and biological membranes consist of molecular bilayers with two leaflets that are typically exposed to different aqueous solutions. We consider solutions of "particles" that experience effectively repulsive interactions with these membranes and form depletion layers in front of the membrane leaflets. The particles considered here are water-soluble, have a size between a few angstrom and a few nanometers as well as a rigid, more or less globular shape, and do neither adsorb onto the membranes nor permeate these membranes. Examples are provided by ions, small sugar molecules, globular proteins, or inorganic nanoparticles with a hydrophilic surface. We first study depletion layers in a hard-core system based on ideal particle solutions as well as hard-wall interactions between these particles and the membrane. For this system, we obtain exact expressions for the coverages and tensions of the two leaflets as well as for the spontaneous curvature of the bilayer membrane. All of these quantities depend linearly on the particle concentrations. The exact results for the hard-core system also show that the spontaneous curvature can be directly deduced from the planar membrane geometry. Our results for the hard-core system apply both to ions and solutes that are small compared to the membrane thickness and to nanoparticles with a size that is comparable to the membrane thickness, provided the particle solutions are sufficiently dilute. We then corroborate the different relationships found for the hard-core system by extensive simulations of a soft-core particle system using dissipative particle dynamics. The simulations confirm the linear relationships obtained for the hard-core system. Both our analytical and our simulation results show that the spontaneous curvature induced by a single particle species can be quite large. When one leaflet of the membrane is exposed, e.g., to a 100 mM solution of glucose, a lipid bilayer can acquire a spontaneous curvature of ±1/(270 nm). Our theoretical results can be scrutinized by systematic experimental studies using a large variety of different types of particles.

Large conformational fluctuations of the multi-domain xylanase Z of Clostridium thermocellum.

The cellulosome is a multi-enzyme machinery which efficiently degrades plant cell-wall polysaccharides. The multiple domains of the cellulosome complexes are often tethered to one another by intrinsically disordered regions. The properties and functions of these disordered linkers are unknown to a large extent. In this work, we study the conformational variability of one component of the cellulosome - the multi-domain xylanase Z (XynZ) of Clostridium thermocellum. We use a coarse-grained protein model to efficiently simulate conformations of the enzyme. Our simulation results are in excellent agreement with data from small angle X-ray scattering experiments, which validates the simulation outcome. Both in the presence and absence of the cohesin domain, the XynZ enzyme appears to be flexible in the sense that it takes various compact and extended conformations. The physical interactions between the individual domains are rather weak and transient, and the XynZ enzyme is held together mainly by the flexible linkers connecting the domains. The end-to-end distance distributions for the flexible linkers can be rationalized by the excluded volume effect. Taken together, our results provide a detailed picture of the conformational ensemble of the XynZ enzyme in solution.

Spontaneous curvature of bilayer membranes from molecular simulations: asymmetric lipid densities and asymmetric adsorption.

Biomimetic and biological membranes consist of molecular bilayers with two leaflets which are typically exposed to different aqueous environments and may differ in their molecular density or composition. Because of these asymmetries, the membranes prefer to curve in a certain manner as quantitatively described by their spontaneous curvature. Here, we study such asymmetric membranes via coarse-grained molecular dynamics simulations. We consider two mechanisms for the generation of spontaneous curvature: (i) different lipid densities within the two leaflets and (ii) leaflets exposed to different concentrations of adsorbing particles. We focus on membranes that experience no mechanical tension and describe two methods to compute the spontaneous curvature. The first method is based on the detailed structure of the bilayer's stress profile which can hardly be measured experimentally. The other method starts from the intuitive view that the bilayer represents a thin fluid film bounded by two interfaces and reduces the complexity of the stress profile to a few membrane parameters that can be measured experimentally. For the case of asymmetric adsorption, we introduce a simulation protocol based on two bilayers separated by two aqueous compartments with different adsorbate concentrations. The adsorption of small particles with a size below 1 nm is shown to generate large spontaneous curvatures up to about 1/(24 nm). Our computational approach is quite general: it can be applied to any molecular model of bilayer membranes and can be extended to other mechanisms for the generation of spontaneous curvatures as provided, e.g., by asymmetric lipid composition or depletion layers of solute molecules.

Unbinding and unfolding of adhesion protein complexes through stretching: interplay between shear and tensile mechanical clamps.

Using coarse-grained molecular dynamics simulations, we analyze mechanically induced dissociation and unfolding of the protein complex CD48-2B4. This heterodimer is an indispensable component of the immunological system: 2B4 is a receptor on natural killer cells whereas CD48 is expressed on surfaces of various immune cells. So far, its mechanostability has not been assessed either experimentally or theoretically. We find that the dissociation processes strongly depend on the direction of pulling and may take place in several pathways. Interestingly, the CD48-2B4 interface can be divided into three distinct patches that act as units when resisting the pulling forces. At experimentally accessible pulling speeds, the characteristic mechanostability forces are in the range between 100 and 200 pN, depending on the pulling direction. These characteristic forces need not be associated with tensile forces involved in the act of separation of the complex because prior shear-involving unraveling within individual proteins may give rise to a higher force peak.

Citrate synthase proteins in extremophilic organisms: studies within a structure-based model.

We study four citrate synthase homodimeric proteins within a structure-based coarse-grained model. Two of these proteins come from thermophilic bacteria, one from a cryophilic bacterium and one from a mesophilic organism; three are in the closed and two in the open conformations. Even though the proteins belong to the same fold, the model distinguishes the properties of these proteins in a way which is consistent with experiments. For instance, the thermophilic proteins are more stable thermodynamically than their mesophilic and cryophilic homologues, which we observe both in the magnitude of thermal fluctuations near the native state and in the kinetics of thermal unfolding. The level of stability correlates with the average coordination number for amino acid contacts and with the degree of structural compactness. The pattern of positional fluctuations along the sequence in the closed conformation is different than in the open conformation, including within the active site. The modes of correlated and anticorrelated movements of pairs of amino acids forming the active site are very different in the open and closed conformations. Taken together, our results show that the precise location of amino acid contacts in the native structure appears to be a critical element in explaining the similarities and differences in the thermodynamic properties, local flexibility, and collective motions of the different forms of the enzyme.