Selectivity by Small-Molecule Inhibitors of Protein Interactions Can Be Driven by Protein Surface Fluctuations

Small-molecules that inhibit interactions between specific pairs of proteins have long represented a promising avenue for therapeutic intervention in a variety of settings. Structural studies have shown that in many cases, the inhibitor-bound protein adopts a conformation that is distinct from its unbound and its protein-bound conformations. This plasticity of the protein surface presents a major challenge in predicting which members of a protein family will be inhibited by a given ligand. Here, we use biased simulations of Bcl-2-family proteins to generate ensembles of low-energy conformations that contain surface pockets suitable for small molecule binding. We find that the resulting conformational ensembles include surface pockets that mimic those observed in inhibitor-bound crystal structures. Next, we find that the ensembles generated using different members of this protein family are overlapping but distinct, and that the activity of a given compound against a particular family member (ligand selectivity) can be predicted from whether the corresponding ensemble samples a complementary surface pocket. Finally, we find that each ensemble includes certain surface pockets that are not shared by any other family member: while no inhibitors have yet been identified to take advantage of these pockets, we expect that chemical scaffolds complementing these “distinct” pockets will prove highly selective for their targets. The opportunity to achieve target selectivity within a protein family by exploiting differences in surface fluctuations represents a new paradigm that may facilitate design of family-selective small-molecule inhibitors of protein-protein interactions.     

Protein Interactions and Fluctuations in a Proteomic Network using an Elastic Network Model

Abstract

A set of protein conformations are analyzed by normal mode analysis. An elastic network model is used to obtain fluctuation and cooperativity of residues with low amplitude fluctuations across different species. Slow modes that are associated with the function of proteins have common features among different protein structures. We show that the degree of flexibility of the protein is important for proteins to interact with other proteins and as the species gets more complex its proteins become more flexible. In the complex organism, higher cooperativity arises due to protein structure and connectivity.     

Community-Based Network Study of Protein-Carbohydrate Interactions in Plant Lectins Using Glycan Array Data

Lectins play major roles in biological processes such as immune recognition and regulation, inflammatory responses, cytokine signaling, and cell adhesion. Recently, glycan microarrays have shown to play key roles in understanding glycobiology, allowing us to study the relationship between the specificities of glycan binding proteins and their natural ligands at the omics scale. However, one of the drawbacks in utilizing glycan microarray data is the lack of systematic analysis tools to extract information. In this work, we attempt to group various lectins and their interacting carbohydrates by using community-based analysis of a lectin-carbohydrate network. The network consists of 1119 nodes and 16769 edges and we have identified 3 lectins having large degrees of connectivity playing the roles of hubs. The community based network analysis provides an easy way to obtain a general picture of the lectin-glycan interaction and many statistically significant functional groups.     

A Dynamic Study of Protein Secretion and Aggregation in the Secretory Pathway

Precise coordination of protein biogenesis, traffic and homeostasis within the early secretory compartment (ESC) is key for cell physiology. As a consequence, disturbances in these processes underlie many genetic and chronic diseases. Dynamic imaging methods are needed to follow the fate of cargo proteins and their interactions with resident enzymes and folding assistants. Here we applied the Halotag labelling system to study the behavior of proteins with different fates and roles in ESC: a chaperone, an ERAD substrate and an aggregation-prone molecule. Exploiting the Halo property of binding covalently ligands labelled with different fluorochromes, we developed and performed non-radioactive pulse and chase assays to follow sequential waves of proteins in ESC, discriminating between young and old molecules at the single cell level. In this way, we could monitor secretion and degradation of ER proteins in living cells. We can also follow the biogenesis, growth, accumulation and movements of protein aggregates in the ESC. Our data show that protein deposits within ESC grow by sequential apposition of molecules up to a given size, after which novel seeds are detected. The possibility of using ligands with distinct optical and physical properties offers a novel possibility to dynamically follow the fate of proteins in the ESC.     

错误折叠蛋白质的聚集效应及其对策

错误折叠蛋白质大量聚集将引发蛋白质构象紊乱症(protein conformational disorder,PCD)。目前的研究发现,蛋白质聚集过程中的中间体(纤维前体,pre—fibfillar)导致细胞膜结构受损,从而诱发细胞凋亡。根据这一原理设计出的抗纤维前体抗体和干扰肽可以作为PCD治疗的一般性方法。此外,该就消除错误折叠蛋白质聚集的研究方向作了展望。     

ATP-Dependent Dynamic Protein Aggregation Regulates Bacterial Dormancy Depth Critical for Antibiotic Tolerance

1. Download high-res image (143KB)
2. Download full-size image

     

Mapping Dynamic Protein Interactions to Insulin Secretory Granule Behavior with TIRF-FRET

Biological processes are governed by extensive networks of dynamic molecular interactions. Yet, establishing a spatial and temporal map of these interactions and their direct relationship to specific cell functions has remained a challenge. Here, we implement sensitized emission Förster resonance energy transfer (FRET) stoichiometry under total internal reflection fluorescence (TIRF) microscopy. We demonstrate through quantitative analysis and modeling that evanescent fields must be precisely matched between FRET excitation wavelengths to isolate dynamic interactions between bimolecular FRET pairs that are not entirely membrane-delimited. We then use TIRF-FRET to monitor the behavior of individual insulin-containing secretory granules at the plasma membrane of living cells, while simultaneously tracking the dynamic interaction between the GTPase Rab27A and its effector Slp4A, on those same granules. Notably, insulin granules that underwent exocytosis demonstrated a specific increase in Rab27A-GTP/Slp4A FRET in the 5 s before membrane fusion, which coincided temporally with an increase in granule displacement and mobility. These results demonstrate an initial spatiotemporal mapping of a dynamic protein-protein interaction on individual secretory granules that is linked to a specific granule behavior in living cells.     

Dynamic Light Scattering Application to Study Protein Interactions in Electrolyte Solutions

The concentration dependence of the diffusion coefficient of particles suspended in solution depends primarily on the occupied volume fraction and on repulsive and attractive forces. This dependency is expressed by the interaction parameter, which can be assessed experimentally by light scattering measurements and have been determined for the diffusion coefficient of BSA under different salt concentration conditions in the present work. The result shows that the diffusion coefficient of protein grows up with increasing protein concentration, and when the ionic strength turns up gradually the diffusion coefficient decreases with protein concentrations increasing. The concentration dependence of BSA diffusion coefficients is interpreted in the context of a two-body potential of mean force, which includes repulsive hard-sphere and Coulombic interactions and attractive dispersion. With the increase of ionic strength, Debye screening decreases, protein interaction changes from repulsion to attraction, and protein begins to aggregate. By means of the concentration dependence of BSA diffusion coefficients, one can obtain the parameters of protein interactions and can find that protein bears a net effective charge of –9.0 e and has a Hamaker constant of 2.8k_BT. This work demonstrates that DLS is an effective technique of studying protein interactions.     

Quantification of Dynamic Protein Interactions and Phosphorylation in LPS Signaling Pathway by SWATH-MS

1. Download : Download high-res image (142KB)
2. Download : Download full-size image

Highlights

• •Intensive investigation of dynamic interactions in MyD88, TRAF6, and NEMO complexes.
• •Mechanistic insights into IRAKs proteins' assembly.
• •A signal amplification mechanism for MyD99-denpendent TLR signaling disclosed by stoichiometry of complexes.
• •Quantitative measurement of multiple phosphorylation sites on the key components in TLR signaling pathway.

     

Dynamic Fluctuations Provide the Basis of a Conformational Switch Mechanism in Apo Cyclic AMP Receptor Protein

Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP''s allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.     

蛋白质聚集的形成与调控机制

大肠杆菌是外源蛋白质的首选表达系统,但蛋白质易被宿主细胞蛋白酶降解或聚集形成包含体。包含体与淀粉样蛋白纤维的形成过程相似,都依赖于特异性氨基酸序列的分子间相互作用。因此,淀粉样蛋白质抗聚集的方法也可用于防止细菌表达蛋白质的聚集。另外,基于序列的新型方法也能调节蛋白质聚集。     

规模化蛋白质相互作用的研究技术

蛋白质相互作用既是蛋白质执行功能的主要方式,也是细胞功能调控网络的结构基础。蛋白质间异常的相互作用及其连锁网络的紊乱是引起许多病理改变的原因。作为功能基因组和蛋白质组研究的重要内容,规模化蛋白质相互作用研究已成为近年国际上研究的热点之一。文章综述了当前规模化蛋白质相互作用研究中的常用技术和常用蛋白质相互作用数据库,研究者可根据研究需要和技术特点利用这些资源。     

Zinc Oxide Nanoparticles Promote the Aggregation of Concanavalin A

The ability of nanoparticles to influence protein folding and aggregation is interesting, not only because of the potential beneficial applications, but also the potential risks to human health and the environment. The interactions of concanavalin A (Con A) with zinc oxide nanoparticles (ZnO-NPs) were investigated by using fluorescence, fourier transform infrared spectroscopy, circular dichroism (CD) and dynamic light scattering techniques. ANS fluorescence and CD spectroscopy authenticated the formation of molten globule state of Con A after its incubation with ZnO-NPs for 36 h. Further incubation of 48 h resulted in the aggregation of unadsorbed Con A, proved by decrease in ANS fluorescence while an increase in thioflavin T fluorescence, characteristic of an aggregates. Moreover, Fourier transform-infrared spectroscopy confirmed the aggregation of unadsorbed Con A. The aggregated products were negligible genotoxic as analyzed by pUC19 plasmid degradation and comet assay. It is clear that ZnO-NPs morphology affect unadsorbed proteins structure. A better understanding of these differences will be essential to engineer fully functional nanobioconjugates and NPs which do not damage the proteins present in the biological system.     

Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

We develop a theoretical model of a clathrin protein lattice on a flexible cell membrane. The clathrin subunit is modeled as a three-legged pinwheel with elastic deformation modes and intersubunit binding interactions. The pinwheels are constrained to lie on the surface of an elastic sheet that opposes bending deformation and is subjected to tension. Through Monte Carlo simulations, we predict the equilibrium phase behavior of clathrin lattices at various levels of tension. High membrane tensions, which correspond to suppressed membrane fluctuations, tend to stabilize large, flat crystalline structures similar to plaques that have been observed in vivo on cell membranes that are adhered to rigid surfaces. Low tensions, on the other hand, give rise to disordered, defect-ridden lattices that behave in a fluidlike manner. The principles of two-dimensional melting theory are applied to our model system to further clarify how high tensions can stabilize crystalline order on flexible membranes. These results demonstrate the importance of environmental physical cues in dictating the collective behavior of self-assembled protein structures.     

Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

We develop a theoretical model of a clathrin protein lattice on a flexible cell membrane. The clathrin subunit is modeled as a three-legged pinwheel with elastic deformation modes and intersubunit binding interactions. The pinwheels are constrained to lie on the surface of an elastic sheet that opposes bending deformation and is subjected to tension. Through Monte Carlo simulations, we predict the equilibrium phase behavior of clathrin lattices at various levels of tension. High membrane tensions, which correspond to suppressed membrane fluctuations, tend to stabilize large, flat crystalline structures similar to plaques that have been observed in vivo on cell membranes that are adhered to rigid surfaces. Low tensions, on the other hand, give rise to disordered, defect-ridden lattices that behave in a fluidlike manner. The principles of two-dimensional melting theory are applied to our model system to further clarify how high tensions can stabilize crystalline order on flexible membranes. These results demonstrate the importance of environmental physical cues in dictating the collective behavior of self-assembled protein structures.     

Electrostatic Effects in Filamentous Protein Aggregation

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules.