Pleomorphic Ensembles: Formation of Large Clusters Composed of Weakly Interacting Multivalent Molecules

Molecular interactions of importance to cell biology are subject to sol-gel transitions: large clusters of weakly interacting multivalent molecules (gel phase) are produced at a critical concentration of monomers. Examples include cell-cell and cell-matrix adhesions, nucleoprotein bodies, and cell signaling platforms. We use the term pleomorphic ensembles (PEs) to describe these clusters, because they have dynamic compositions and sizes and have rapid turnover of their molecular constituents; this plasticity can be highly responsive to cellular signals. The classical polymer physical chemistry theory developed by Flory and Stockmayer provides a brilliant framework for treating multivalent interactions for simple idealized systems. But the complexity and variability of PEs challenges existing modeling approaches. Here we describe and validate a computational algorithm that extends the Flory-Stockmayer formalism to overcome the limitations of analytic theories. We divide the problem by deterministically calculating the fraction of bound sites for each type of binding site, followed by the stochastic assignment of the bonds to a finite number of molecules. The method allows for high valency within many different kinds of interacting molecules and site types, permits simulation of steady-state distributions, as well as assembly kinetics, and can treat cooperative binding within one of the interacting molecules. We then apply our method to the analysis of interactions in the nephrin-Nck-N-Wasp signaling system, demonstrating how multivalent layered scaffolds produce PEs at low monomer concentrations despite weak binding interactions. We show how the experimental data for this system are most consistent with synergistic cooperative interactions between Nck and N-Wasp.     

The Interplay of Structural and Cellular Biophysics Controls Clustering of Multivalent Molecules

Dynamic molecular clusters are assembled through weak multivalent interactions and are platforms for cellular functions, especially receptor-mediated signaling. Clustering is also a prerequisite for liquid-liquid phase separation. It is not well understood, however, how molecular structure and cellular organization control clustering. Using coarse-grained kinetic Langevin dynamics, we performed computational experiments on a prototypical ternary system modeled after membrane-bound nephrin, the adaptor Nck1, and the actin nucleation promoting factor NWASP. Steady-state cluster size distributions favored stoichiometries that optimized binding (stoichiometry matching) but still were quite broad. At high concentrations, the system can be driven beyond the saturation boundary such that cluster size is limited only by the number of available molecules. This behavior would be predictive of phase separation. Domains close to binding sites sterically inhibited clustering much less than terminal domains because the latter effectively restrict access to the cluster interior. Increased flexibility of interacting molecules diminished clustering by shielding binding sites within compact conformations. Membrane association of nephrin increased the cluster size distribution in a density-dependent manner. These properties provide insights into how molecular ensembles function to localize and amplify cell signaling.     

Thermodynamic characterization of the multivalent binding of chartreusin to DNA

Characterization of the thermodynamics of DNA– drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 × 10⁵ M^–1 at 20°C, in a solution containing 18 mM Na⁺. The DNA–drug interaction was enthalpy driven, with a ΔH_b of –7.07 kcal/mol at 20°C. Binding enthalpies were determined by ITC in the 20–35°C range and used to calculate a binding-induced change in heat capacity (ΔCp) of –391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of ΔG_obs into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.     

Distinct Stages of Stimulated FcεRI Receptor Clustering and Immobilization Are Identified through Superresolution Imaging

Recent advances in fluorescence localization microscopy have made it possible to image chemically fixed and living cells at 20 nm lateral resolution. We apply this methodology to simultaneously record receptor organization and dynamics on the ventral surface of live RBL-2H3 mast cells undergoing antigen-mediated signaling. Cross-linking of IgE bound to FcεRI by multivalent antigen initiates mast cell activation, which leads to inflammatory responses physiologically. We quantify receptor organization and dynamics as cells are stimulated at room temperature (22°C). Within 2 min of antigen addition, receptor diffusion coefficients decrease by an order of magnitude, and single-particle trajectories are confined. Within 5 min of antigen addition, receptors organize into clusters containing ∼100 receptors with average radii of ∼70 nm. By comparing simultaneous measurements of clustering and mobility, we determine that there are two distinct stages of receptor clustering. In the first stage, which precedes stimulated Ca²⁺ mobilization, receptors slow dramatically but are not tightly clustered. In the second stage, receptors are tightly packed and confined. We find that stimulation-dependent changes in both receptor clustering and mobility can be reversed by displacing multivalent antigen with monovalent ligands, and that these changes can be modulated through enrichment or reduction in cellular cholesterol levels.     

Karyopherin-Centric Control of Nuclear Pores Based on Molecular Occupancy and Kinetic Analysis of Multivalent Binding with FG Nucleoporins

Intrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes exert multivalent interactions with transport receptors (Karyopherins (Kaps)) that orchestrate nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps with the FG Nups due to avidity contradicts rapid Kap translocation in vivo. Here, using surface plasmon resonance, we innovate a means to correlate in situ mechanistic (molecular occupancy and conformational changes) with equilibrium (binding affinity) and kinetic (multivalent binding kinetics) aspects of Karyopherinβ1 (Kapβ1) binding to four different FG Nups. A general feature of the FxFG domains of Nup214, Nup62, and Nup153 is their capacity to extend and accommodate large numbers of Kapβ1 molecules at physiological Kapβ1 concentrations. A notable exception is the GLFG domain of Nup98, which forms a partially penetrable cohesive layer. Interestingly, we find that a slowly exchanging Kapβ1 phase forms an integral constituent within the FG Nups that coexists with a fast phase, which dominates transport kinetics due to limited binding with the pre-occupied FG Nups at physiological Kapβ1 concentrations. Altogether, our data reveal an emergent Kap-centric barrier mechanism that may underlie mechanistic and kinetic control in the nuclear pore complex.     

Trapping by Clusters of Channels,Receptors, and Transporters: Quantitative Description

Various membrane functional units such as receptors, transporters, and channels, whose action necessarily involves capturing diffusing molecules, are often organized into multimeric complexes forming clusters on the cell and organelle membranes. These functional units themselves are usually oligomers of several integral proteins, which have their own symmetry. Depending on the symmetry, they form clusters on different packing lattices. Moreover, local membrane inhomogeneities, e.g., the so-called membrane domains, rafts, stalks, etc., lead to different patterns even within the structures on the same packing lattice. Units in the cluster compete for diffusing molecules and screen each other. Here we propose a general approach that allows one to quantify the screening effects. The approach is used to derive simple approximate formulas giving the trapping rates of diffusing molecules by clusters of absorbers on lattices of different packing symmetries. The obtained results describe smooth variation of the trapping rate from the sum of the rates of individual absorbers forming the cluster to the effective collective rate. The latter shows how the trapping efficiency of an individual absorber decreases as the number of absorbers in the cluster increases and/or the inter-absorber distance decreases. Numerical tests demonstrate good agreement between the rates predicted by the theory and obtained from Brownian dynamics simulations for clusters of different shapes and sizes.     

Structure of the RZZ complex and molecular basis of Spindly‐driven corona assembly at human kinetochores

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein–dynactin adaptor Spindly and the ROD–Zwilch–ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high‐resolution cryo‐EM structure that captures the essential features of the RZZ complex, including a farnesyl‐binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ–Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation‐dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long‐sought‐for molecular basis of corona assembly on metazoan kinetochores.     

Brownian-dynamics simulations of metal-ion binding to four-way junctions

Four-way junctions (4Hs) are important intermediates in DNA rearrangements such as genetic recombination. Under the influence of multivalent cations these molecules undergo a conformational change, from an extended planar form to a quasi-continuous stacked X-structure. Recently, a number of X-ray structures and a nuclear magnetic resonance (NMR) structure of 4Hs have been reported and in three of these the position of multivalent cations is revealed. These structures belong to two main families, characterized by the angle between the two co-axial stacked helices, which is either around +40 to +55° or around –70 to –80°. To investigate the role of metal-ion binding on the conformation of folded 4Hs we performed Brownian-dynamics simulations on the set of available structures. The simulations confirm the proposed metal-ion binding sites in the NMR structure and in one of the X-ray structures. Furthermore, the calculations suggest positions for metal-ion binding in the other X-ray structures. The results show a striking dependence of the ion density on the helical environment (B-helix or A-helix) and the structural family.     

Crystal Structure of Reaction Intermediates in Pyruvate Class II Aldolase: SUBSTRATE CLEAVAGE,ENOLATE STABILIZATION,AND SUBSTRATE SPECIFICITY*

Crystal structures of divalent metal-dependent pyruvate aldolase, HpaI, in complex with substrate and cleavage products were determined to 1.8–2.0 Å resolution. The enzyme·substrate complex with 4-hydroxy-2-ketoheptane-1,7-dioate indicates that water molecule W2 bound to the divalent metal ion initiates C3–C4 bond cleavage. The binding mode of the aldehyde donor delineated a solvent-filled capacious binding locus lined with predominantly hydrophobic residues. The absence of direct interactions with the aldehyde aliphatic carbons accounts for the broad specificity and lack of stereospecific control by the enzyme. Enzymatic complex structures formed with keto acceptors, pyruvate, and 2-ketobutyrate revealed bidentate interaction with the divalent metal ion by C1-carboxyl and C2-carbonyl oxygens and water molecule W4 that is within close contact of the C3 carbon. Arg⁷⁰ assumes a multivalent role through its guanidinium moiety interacting with all active site enzymatic species: C2 oxygen in substrate, pyruvate, and ketobutyrate; substrate C4 hydroxyl; aldehyde C1 oxygen; and W4. The multiple interactions made by Arg⁷⁰ stabilize the negatively charged C4 oxygen following proton abstraction, the aldehyde alignment in aldol condensation, and the pyruvate enolate upon aldol cleavage as well as support proton exchange at C3. This role is corroborated by loss of aldol cleavage ability and pyruvate C3 proton exchange activity and by a 730-fold increase in the dissociation constant toward the pyruvate enolate analog oxalate in the R70A mutant. Based on the crystal structures, a mechanism is proposed involving the two enzyme-bound water molecules, W2 and W4, in acid/base catalysis that facilitates reversible aldol cleavage. The same reaction mechanism promotes decarboxylation of oxaloacetate.     

Who's In and Who's Out—Compositional Control of Biomolecular Condensates

Biomolecular condensates are two- and three-dimensional compartments in eukaryotic cells that concentrate specific collections of molecules without an encapsulating membrane. Many condensates behave as dynamic liquids and appear to form through liquid–liquid phase separation driven by weak, multivalent interactions between macromolecules. In this review, we discuss current models and data regarding the control of condensate composition, and we describe our current understanding of the composition of representative condensates including PML nuclear bodies, P-bodies, stress granules, the nucleolus, and two-dimensional membrane localized LAT and nephrin clusters. Specific interactions, such as interactions between modular binding domains, weaker interactions between intrinsically disorder regions and nucleic acid base pairing, and nonspecific interactions, such as electrostatic interactions and hydrophobic interactions, influence condensate composition. Understanding how specific condensate composition is determined is essential to understanding condensates as biochemical entities and ultimately discerning their cellular and organismic functions.     

Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.     

Two Classes of Cholesterol Binding Sites for the β2AR Revealed by?Thermostability and NMR

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the β₂ adrenergic receptor (β₂AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β₂AR. By analyzing the β₂AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β₂AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.     

Cooperation effects in binding of large ligands to DNA. II. Contact interactions between adsorbed ligands

Cooperative effects arising upon binding of biologically active ligands to DNA are considered. Equations are derived which enable one to describe the binding of two different ligands to DNA. We also consider the case when ligand can form two type of DNA complexes. The cooperative binding of the ligand in the vicinity of saturation level of binding can be described with a good accuracy by equation derived for the non-cooperative adsorption of the same ligand with some effective binding constant Keff. It is shown that cooperative effects arising upon binding of proteins and other ligands to DNA can be divided into two groups depending on the symmetry of interactions between the bound ligand molecules. In particular, if such interactions favor the formation of dimeric ligand species on the DNA, Keff approximately a1/2, where a is the ligand-ligand interaction constant. If cooperative interactions favor the formation of aggregates of unrestricted size, then Keff approximately aL+Y, where L is the size of the binding site for the ligand on DNA.     

A solid-phase method for synthesis of dimeric and trimeric ligands: Identification of potent bivalent ligands of 14-3-3σ

Multivalent protein-protein interactions including bivalent and trivalent interactions play a critical role in mediating a wide range of biological processes. Hence, there is a significant interest in developing molecules that can modulate those signaling pathways mediated by multivalent interactions. For example, multimeric molecules capable of binding to a receptor protein through a multivalent interaction could serve as modulators of such interactions. However, it is challenging to efficiently generate such multimeric ligands. Here, we have developed a facile solid-phase method that allows for the rapid generation of (homo- and hetero-) dimeric and trimeric protein ligands. The feasibility of this strategy was demonstrated by efficiently synthesizing fluorescently-labeled dimeric peptide ligands, which led to dramatically increased binding affinities (~400-fold improvement) relative to a monomeric 14-3-3σ protein ligand.     

Molecular Mechanisms of Cell-cell Recognition

Cell-cell recognition is the key for muhicellular organisms to survive. This recognition critically depends on protein-protein interactions from opposing cell surfaces. Recent structural investigations reveal unique features of these cell surface receptors and how they interact. These interactions are specific, but usually relatively weak, with more hydrophilic forces involved in binding. The receptors appear to have specialized ways to present their key interacting elements for ligand-binding from the cell surface. Cell-cell contacts are multivalent. A large group of cell surface molecules are engaged in interactions. Characteristic weak interactions make possible for each individual molecule pair within the group to constantly associate-dissociate-reassociate, such that the cell-cell recognition becomes a dynamic process. The immunological synapse is a good example for immune receptors to be orchestrated in performing immunological function in a collective fashion.     

Mechanisms orchestrating the enzymatic activity and cellular functions of deubiquitinases

Deubiquitinases (DUBs) are required for the reverse reaction of ubiquitination and act as major regulators of ubiquitin signaling processes. Emerging evidence suggests that these enzymes are regulated at multiple levels in order to ensure proper and timely substrate targeting and to prevent the adverse consequences of promiscuous deubiquitination. The importance of DUB regulation is highlighted by disease-associated mutations that inhibit or activate DUBs, deregulating their ability to coordinate cellular processes. Here, we describe the diverse mechanisms governing protein stability, enzymatic activity, and function of DUBs. In particular, we outline how DUBs are regulated by their protein domains and interacting partners. Intramolecular interactions can promote protein stability of DUBs, influence their subcellular localization, and/or modulate their enzymatic activity. Remarkably, these intramolecular interactions can induce self-deubiquitination to counteract DUB ubiquitination by cognate E3 ubiquitin ligases. In addition to intramolecular interactions, DUBs can also oligomerize and interact with a wide variety of cellular proteins, thereby forming obligate or facultative complexes that regulate their enzymatic activity and function. The importance of signaling and post-translational modifications in the integrated control of DUB function will also be discussed. While several DUBs are described with respect to the multiple layers of their regulation, the tumor suppressor BAP1 will be outlined as a model enzyme whose localization, stability, enzymatic activity, and substrate recognition are highly orchestrated by interacting partners and post-translational modifications.     

An entropic mechanism to generate highly cooperative and specific binding from protein phosphorylations

Cooperative interactions are essential to the operation of many biochemical networks. Such networks then respond ultrasensitively in a nonlinear manner to linear changes in network input, and network output, for example, levels of a phosphorylated protein or of gene expression, becomes a sigmoidal function of concentrations of input molecules. We present a novel, entropic ultrasensitivity mechanism that generates highly cooperative and specific binding between two proteins. We consider a disordered protein with multiple phosphorylation sites that binds to a single binding site on an interacting protein. We assume that each phosphorylation locally orders the protein. Such local order affects protein conformational entropy nonlinearly and generates binding that is a highly cooperative function of the number of protein phosphorylations (with Hill coefficients well above 10). Substantial binding may only occur once the disordered protein is phosphorylated a critical number of times or more. Cooperativity is determined by the size of the disordered region of the protein, the binding affinity, and unusually the concentration of the interacting protein. Given the widespread occurrence of disordered, multiply phosphorylated proteins, its highly ultrasensitive character, and the ease of its control, entropic, phosphorylation-driven cooperativity may be extensively exploited intracellularly.