Weak Protein-protein Interactions Research Articles

Heat shock-induced chaperoning by Hsp70 is enabled in-cell.

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a "preemptive holdase" mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function.

Genetically Encoded Residue-Selective Photo-Crosslinker to Capture Protein-Protein Interactions in Living Cells

Summary The genetically encoded non-selective photo-crosslinkers suffer from unpredictable crosslinking sites and false positive interacting proteins, thus, the identification of protein interactions and mapping accurate interaction interfaces remain largely elusive in living systems. Here, we report genetically encoded, residue-selective protein photo-crosslinker enabling covalent crosslinking of the interacting proteins with proximal lysine, upon UV light activation in vitro and in living cells. Using this photo-crosslinker, we demonstrate a crosslinking strategy to capture protein-protein interactions with residue selectivity in a temporal-resolved manner. In addition, this crosslinking strategy produces the lysine-selective crosslinking site and predictable crosslinked peptide, which serve as the direct evidence for protein-protein interactions and facilitate mass spectrometry analysis. More importantly, this photo-crosslinker enables the capture of elusive enzyme-substrate interaction with directly interacting lysine and validation of acetylation site of the substrate. This strategy represents higher spatiotemporal resolution, accuracy, and reliability for investigating dynamic, transient, and weak protein-protein interactions in living systems.

In-Cell Protein–Protein Contacts: Transient Interactions in the Crowd

Proteins in vivo are immersed in a crowded environment of water, ions, metabolites, and macromolecules. In-cell experiments highlight how transient weak protein-protein interactions promote (via functional "quinary structure") or hinder (via competitive binding or "sticking") complex formation. Computational models of the cytoplasm are expensive. We tackle this challenge with an all-atom model of a small volume of the E. coli cytoplasm to simulate protein-protein contacts up to the 5 μs time scale on the special-purpose supercomputer Anton 2. We use three CHARMM-derived force fields: C22*, C36m, and C36mCU (with CUFIX corrections). We find that both C36m and C36mCU form smaller contact surfaces than C22*. Although CUFIX was developed to reduce protein-protein sticking, larger contacts are observed with C36mCU than C36m. We show that the lifespan Δt of protein-protein contacts obeys a power law distribution between 0.03 and 3 μs, with ∼90% of all contacts lasting <1 μs (similar to the time scale for downhill folding).

X-ray Scattering and Coarse-Grained Simulations for Clustering and Interactions of Monoclonal Antibodies at High Concentrations.

Attractive protein?protein interactions (PPI) in concentrated monoclonal antibody (mAb) solutions may lead to reversible oligomers (clusters) that impact colloidal stability and viscosity. Herein, the PPI are tuned for two mAbs via the addition of arginine (Arg), NaCl, or ZnSO4 as characterized by the structure factor ( Seff( q)) with small-angle X-ray scattering (SAXS). The SAXS data are fit with molecular dynamics simulations by placing a physically relevant short-range attractive interaction on selected beads in coarse-grained 12-bead models of the mAb shape. The optimized 12-bead models are then used to differentiate key microstructural properties, including center of mass radial distribution functions ( gCOM( r)), coordination numbers, and cluster size distributions (CSD). The addition of cosolutes results in more attractive Seff( q) relative to the no cosolute control for all systems tested, with the most attractive systems showing an upturn at low q. Only the All1 model with an attractive site in each Fab and Fc region (possessing Fab?Fab, Fab?Fc, and Fc?Fc interactions) can reproduce this upturn, and the corresponding CSDs show the presence of larger clusters compared to the control. In general, for models with similar net attractions, i.e., second osmotic virial coefficients, the size of the clusters increases as the attraction is concentrated on a smaller number of evenly distributed beads. The cluster size distributions from simulations are used to improve the understanding and prediction of experimental viscosities. The ability to discriminate between models with bead interactions at particular Fab and Fc bead sites from SAXS simulations, and to provide real-space properties (CSD and gCOM( r)), will be of interest in engineering protein sequence and formulating protein solutions for weak PPI to minimize aggregation and viscosities.

Biogenesis, quality control, and structural dynamics of proteins as explored in living cells via site-directed photocrosslinking.

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the site-directed photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

Proteomic navigation using proximity-labeling.

The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells.

Light Scattering to Quantify Protein-Protein Interactions at High Protein Concentrations.

Static and dynamic (laser) light scattering (SLS and DLS, respectively) can be used to measure the so-called weak or colloidal protein-protein interactions in solution from low to high protein concentrations (c2). This chapter describes a methodology to measure protein-protein self-interactions using SLS and DLS, with illustrative examples for monoclonal antibody solutions from low to high protein concentrations (c2~1-102g/L).

Molecular snapshots of dynamic membrane-bound metabolons.

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

Expanding the Genetic Code to Study Protein-Protein Interactions.

Protein-protein interactions are central to many biological processes. A considerable challenge consists however in understanding and deciphering when and how proteins interact, and this can be particularly difficult when interactions are weak and transient. The site-specific incorporation of unnatural amino acids (UAAs) that crosslink with nearby molecules in response to light provides a powerful tool for mapping transient protein-protein interactions and for defining the structure and topology of protein complexes both in vitro and in vivo. Complementary strategies consist in site-specific incorporation of UAAs bearing electrophilic moieties that react with natural nucleophilic amino acids in a proximity-dependent manner, thereby chemically stabilizing low-affinity interactions and providing additional constraints on distances and geometries in protein complexes. Herein, we review how UAAs bearing fine-tuned chemical moieties that react with proteins in their vicinity can be utilized to map, study, and characterize weak and transient protein-protein interactions in living systems.

Weak IgG self- and hetero-association characterized by fluorescence analytical ultracentrifugation.

Weak protein-protein interactions may be important to binding cooperativity. A panel of seven fluorescently labeled tracer monoclonal IgG antibodies, differing in variable (V) and constant (C) region sequences, were sedimented in increasing concentrations of unlabeled IgGs of identical, similar, and different backgrounds. Weak IgG::IgG attractive interactions were detected and characterized by global analysis of the hydrodynamic nonideality coefficient, ks . The effects of salt concentration and temperature on ks suggest the interactions are predominantly enthalpic in origin. The interactions were found to be variable in strength, affected by both the variable and constant regions, but indiscriminate with respect to IgG subclass. Furthermore, weak attractive interactions were observed for all the mAbs with freshly purified human poly-IgG. The universality of the weak interactions suggest that they may contribute to effector function cooperativity in the normal immune response, and we postulate that the generality of the interactions allows for a broader range of epitope spacing for complement activation. These studies demonstrate the utility of analytical ultracentrifuge fluorescence detection in measuring weak protein-protein interactions. It also shows the strength of global analysis of sedimentation velocity data by SEDANAL to extract hydrodynamic nonideality ks to characterize weak macromolecular interactions.

Detection of membrane protein-protein interaction in planta based on dual-intein-coupled tripartite split-GFP association.

Despite the great interest in identifying protein-protein interactions (PPIs) in biological systems, only a few attempts have been made at large-scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs invivo at subcellular resolution. However, when the non-fluorescent fragments are highly expressed, spontaneous and irreversible self-assembly of the split halves can easily generate false positives. The recently developed tripartite split-GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β-estradiol-inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split-GFP association in plant cells and affirm that the tripartite split-GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split-GFP association and dual-intein-mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof-of-concept implementation of the tripartite split-GFP system as a potential tool for membrane PPI screens in planta.

Measuring Membrane Surface Reactions by Diffusion: Dimerization of Btk PH Domain and K-Ras

Transient protein-protein interactions on two-dimensional membrane surfaces, such as dimerization, play an important role in many signaling reactions. However, they are often difficult to quantitatively characterize in membrane environments. Here, the dimerization behavior of two proteins, the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (Btk) and K-Ras GTPase, investigated through the two-dimensional diffusion rates on planar supported lipid bilayers with fluorescence correlation spectroscopy and single-particle tracking, are presented. It is shown that the Btk PH domain dimerizes in a ligand-dependent manner, and K-Ras remains strictly monomeric over a wide range of surface densities, ranging from single-molecule level to dense packed. These works demonstrate how protein diffusion on homogeneous membranes can be used for quantitative measurement of weak protein-protein interactions.

Solvent saturation transfer to proteins (SSTP) for structural and functional characterization of proteins.

Protein structure determination using NMR is dependent on experimentally acquired distance restraints. Often, however, an insufficient number of these restraints are available for determining a protein's correct fold, much less its detailed three-dimensional structure. In consideration of this problem, we propose a simple means to acquire supplemental structural restraints from protein surface accessibilities using solvent saturation transfer to proteins (SSTP), based on the principles of paramagnetic chemical-exchange saturation transfer. Here, we demonstrate the utility of SSTP in structure calculations of two proteins, TSG101 and ubiquitin. The observed SSTP was found to be directly proportional to solvent accessibility. Since SSTP does not involve the direct excitation of water, which compromises the analysis of protein protons entangled in the breadth of the water resonance, it has an advantage over conventional water-based magnetization transfers. Inclusion of structural restraints derived from SSTP improved both the precision and accuracy of the final protein structures in comparison to those determined by traditional approaches, when using minimal amounts of additional structural data. Furthermore, we show that SSTP can detect weak protein-protein interactions which are unobservable by chemical shift perturbations.

Modulation of Weak Protein-Protein Interaction by Molecular Crowding

Modulation of Weak Protein-Protein Interaction by Molecular Crowding

Elucidating the weak protein-protein interaction mechanisms behind the liquid-liquid phase separation of a mAb solution by different types of additives

Liquid-liquid phase separation (LLPS) has long been observed during the physical stability investigation of therapeutic protein formulations. The buffer conditions and the presence of various excipients are thought to play important roles in the formulation development of monoclonal antibodies (mAbs). In this study, the effects of several small-molecule excipients (histidine, alanine, glycine, sodium phosphate, sodium chloride, sorbitol and sucrose) with diverse physical-chemical properties on LLPS of a model IgG1 (JM2) solutions were investigated by multiple techniques, including UV–vis spectroscopy, circular dichroism, differential scanning calorimetry/fluorimetry, size exclusion chromatography and dynamic light scattering. The LLPS of JM2 was confirmed to be a thermodynamic equilibrium process with no structural changes or irreversible aggregation of proteins. Phase diagrams of various JM2 formulations were constructed, suggesting that the phase behavior of JM2 was dependent on the solution pH, ionic strength and the presence of other excipients such as glycine, alanine, sorbitol and sucrose. Furthermore, we demonstrated that for this mAb, the interaction parameter (kD) determined at low protein concentration appeared to be a good predictor for the occurrence of LLPS at high concentration.

Correction to "Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy".

Correction to "Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy".

Chaperone–substrate interactions monitored via a robust TEM-1 β-lactamase fragment complementation assay

To investigate the application of the TEM-1 β-lactamase protein fragment complementation assay (PCA) in detecting weak and unstable protein-protein interactions as typically observed during chaperone-assisted protein folding in the periplasm of Escherichia coli. The TEM-1 β-lactamase PCA system effectively captured the interactions of three pairs of chaperones and substrates. Moreover, the strength of the interactions can be quantitatively analyzed by comparing different levels of penicillin resistance, and the assay can be performed under 0.5% butanol, a stress condition thought to be physiologically relevant. The β-lactamase PCA system faithfully reports chaperone-substrate interactions in the bacterial cell envelope, and therefore this system has the potential to map the complex protein homeostasis network under a fluctuating environment.

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System.

Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

What factors determine the stability of a weak protein-protein interaction in a charged aqueous droplet?

Maintaining the interface of a weak transient protein complex transferred from bulk solution to the gaseous state via evaporating droplets is a critical question in the detection of the complex association (dissociation) constant by using electrospray ionization mass spectrometry (ESI-MS). Here we explore the factors that may affect the stability of a protein-protein interaction (PPI) using atomistic molecular dynamics (MD) modelling of a complex of ubiquitin (Ub) and the ubiquitin-associated domain (UbA) (RCSB PDB code ) and a non-covalent complex of diubiquitin (RCSB PDB code ) in aqueous droplets. A general method is presented to determine the protonation states of the complexes we investigate in particular, and that of a protein in general, under various pH conditions that an evaporating droplet acquires due to its change in size. We find that the combination of high temperature and high charge states of the protein complexes may destabilize the interface by creating new interfaces instead of a direct rupture of the initial stable interface. We provide evidence that highly charged protein complexes are found in droplets that form conical extrusions of the solvent on the surface due to charge-induced instability. This distinct droplet morphology leads to a higher solvent evaporation rate that assists in transferring the complex in the gaseous state without dissociation. The conical solvent protrusions expose on the droplet surface certain amino acids that otherwise would be solvated in a droplet with the protein complex of low charge states. The new vapor-protein interface does not have a direct effect on the stability of the PPI. A common way in experiments to stabilize the protein complexes in droplets is to reduce the protonation state of the proteins. Here we find that weakly bound protein complexes even at high protonation states can be stabilized by the presence of a small number of counterions, without affecting the protonation state of the protein. Our findings may provide guiding principles in ESI-MS experiments to stabilize weak transient PPIs.