Specific Protein Interactions Research Articles

Novel Roles of SH2 and SH3 Domains in Lipid Binding.

Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.

A diffusiophoretic mechanism for ATP-driven transport without motor proteins

The healthy growth and maintenance of a biological system depends on the precise spatial organization of molecules within the cell through the dissipation of energy. Reaction–diffusion mechanisms can facilitate this organization, as can directional cargo transport orchestrated by motor proteins, by relying on specific protein interactions. However, transport of material through the cell can also be achieved by active processes based on non-specific, purely physical mechanisms, a phenomenon that remains poorly explored. Here, using a combined experimental and theoretical approach, we discover and describe a hidden function of the Escherichia coli MinDE protein system: in addition to forming dynamic patterns, this system accomplishes the directional active transport of functionally unrelated cargo on membranes. Remarkably, this mechanism enables the sorting of diffusive objects according to their effective size, as evidenced using modular DNA origami–streptavidin nanostructures. We show that the diffusive fluxes of MinDE and non-specific cargo couple via density-dependent friction. This non-specific process constitutes a diffusiophoretic mechanism, as yet unknown in a cell biology setting. This nonlinear coupling between diffusive fluxes could represent a generic physical mechanism for establishing intracellular organization.

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions

SummarySARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.

Intrinsic disorder in integral membrane proteins.

The well-defined roles and specific protein-protein interactions of many integral membrane proteins (IMPs), such as those functioning as receptors for extracellular matrix proteins and soluble growth factors, easily align with considering IMP structure as a classical "lock-and-key" concept. Nevertheless, continued advances in understanding protein conformation, such as those which established the widespread existence of intrinsically disordered proteins (IDPs) and especially intrinsically disordered regions (IDRs) in otherwise three-dimensionally organized proteins, call for ongoing reevaluation of transmembrane proteins. Here, we present basic traits of IDPs and IDRs, and, for some select single-span IMPs, consider the potential functional advantages intrinsic disorder might provide and the possible conformational impact of disease-associated mutations. For transmembrane proteins in general, we highlight several investigational approaches, such as biophysical and computational methods, stressing the importance of integrating them to produce a more-complete mechanistic model of disorder-containing IMPs. These procedures, when synergized with in-cell assessments, will likely be key in translating in silico and in vitro results to improved understanding of IMP conformational flexibility in normal cell physiology as well as disease, and will help to extend their potential as therapeutic targets.

Protein residues determining interaction specificity inparalogous families.

Predicting the residues controlling a protein's interaction specificity is important not only to better understand its interactions but also to design mutations aimed at fine-tuning or swapping them as well. In this work, we present a methodology that combines sequence information (in the form of multiple sequence alignments) with interactome information to detect that kind of residues in paralogous families of proteins. The interactome is used to define pairwise similarities of interaction contexts for the proteins in the alignment. The method looks for alignment positions with patterns of amino-acid changes reflecting the similarities/differences in the interaction neighborhoods of the corresponding proteins. We tested this new methodology in a large set of human paralogous families with structurally characterized interactions, and discuss in detail the results for the RasH family. We show that this approach is a better predictor of interfacial residues than both, sequence conservation and an equivalent 'unsupervised' method that does not use interactome information. http://csbg.cnb.csic.es/pazos/Xdet/. Supplementary data are available at Bioinformatics online.

Structural dynamics govern substrate recruitment and catalytic turnover in H/ACA RNP pseudouridylation

ABSTRACT H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.

HIV‐1 infection of CD4 T cells impairs antigen‐specific B cell function

Failures to produce neutralizing antibodies upon HIV‐1 infection result in part from B‐cell dysfunction due to unspecific B‐cell activation. How HIV‐1 affects antigen‐specific B‐cell functions remains elusive. Using an adoptive transfer mouse model and ex vivo HIV infection of human tonsil tissue, we found that expression of the HIV‐1 pathogenesis factor NEF in CD4 T cells undermines their helper function and impairs cognate B‐cell functions including mounting of efficient specific IgG responses. NEF interfered with T cell help via a specific protein interaction motif that prevents polarized cytokine secretion at the T‐cell–B‐cell immune synapse. This interference reduced B‐cell activation and proliferation and thus disrupted germinal center formation and affinity maturation. These results identify NEF as a key component for HIV‐mediated dysfunction of antigen‐specific B cells. Therapeutic targeting of the identified molecular surface in NEF will facilitate host control of HIV infection.

Multi-omics analysis profile oral tumor module clusters to reveal the potential pathogenic mechanism

Oral tumors are malignant cancers caused by abnormal proliferation or pathological changes of soft or hard tissues in the oral cavity. Serious cases may pose a threat to life. However, its precancerous lesions remain unclear. This study is based on a comprehensive strategy to explore a multi-factor-driven oral cancer barrier module, which is an attempt to describe the pathogenesis of the disease and potential regulatory drugs from a global perspective. Functional disease modules were identified by constructing a protein-specific interaction network in patients' oral tissues. Then, comprehensive pathogenesis was explored through combination with analysis of functional and signaling pathway enrichment, prediction of key regulatory factors. It was found that these specifically expressed proteins and their interactions often play a pivotal part in oral tumors. This is reflected in the results of functional and pathway enrichment of modulating genes, which show that they are mainly involved in various immune responses, inflammatory reactions, oral plaque, and oral ulcer-related regulatory processes. This may represent the potential pathogenesis of oral tumors. On the predictive analysis of regulators, a series of ncRNAs (including miR-590, CRNDE and miR-340) and transcription factors (including E2F1, MYC and TP53) were identified that have potential important regulatory effects on oral tumors. These key regulators may manipulate a crucial part of the module sub-network and then work together to mediate the occurrence of oral tumors. On the comprehensive Multi-omics module analysis, the specific proteins and their interactions in patients' oral tissues were identified, while the prominent pivotal regulators were involved in the different pathogenic functions of oral tumors.

The cruciform DNA-binding protein Crp1 stimulates the endonuclease activity of Mus81-Mms4 in Saccharomycescerevisiae.

The Saccharomycescerevisiae Mus81-Mms4 complex is a highly conserved DNA structure-specific endonuclease that plays essential roles in the processing of recombination intermediates that arise during the repair of stalled replication forks and double-stranded breaks. To identify novel factors functioning conjointly with Mus81-Mms4, we performed a biochemical screen and found that Crp1, a cruciform DNA-recognizing protein that specifically binds to DNA four-way junction structures, could stimulate the Mus81-Mms4 endonuclease. The specific protein interaction between Mus81-Mms4 and Crp1 was responsible for the stimulation observed. Multicopy expression of Crp1 could partially rescue the sensitivity to DNA-damaging agents of the sgs1∆mus81∆21-24N mutant. Our results provide insight into the functional role and interaction of Crp1 with other proteins involved in DNA repair.

Controlling protein function by fine-tuning conformational flexibility.

In a living cell, protein function is regulated in several ways, including post-translational modifications (PTMs), protein-protein interaction, or by the global environment (e.g. crowding or phase separation). While site-specific PTMs act very locally on the protein, specific protein interactions typically affect larger (sub-)domains, and global changes affect the whole protein non-specifically. Herein, we directly observe protein regulation under three different degrees of localization, and present the effects on the Hsp90 chaperone system at the levels of conformational steady states, kinetics and protein function. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We disentangle specific and non-specific effects that control Hsp90's ATPase function, which has remained a puzzle up to now. Lastly, we introduce a new mechanistic concept: functional stimulation through conformational confinement. Our results demonstrate how cellular protein regulation works by fine-tuning the conformational state space of proteins.

Identification of a Novel NtLRR-RLK and Biological Pathways That Contribute to Tolerance of TMV in Nicotiana tabacum.

Tobacco mosaic virus (TMV) infection can causes serious damage to tobacco crops. To explore the approach of preventing TMV infection of plants, two tobacco cultivars with different resistances to TMV were used to analyze transcription profiling before and after TMV infection. The involvement of biological pathways differed between the tolerant variety (Yuyan8) and the susceptible variety (NC89). In particular, the plant-virus interaction pathway was rapidly activated in Yuyan8, and specific resistance genes were enriched. Liquid chromatography tandem mass spectrometry analysis detected large quantities of antiviral substances in the tolerant Yuyan8. A novel Nicotiana tabacum leucine-rich repeat receptor kinase (NtLRR-RLK) gene was identified as being methylated and this was verified using bisulfite sequencing. Transient expression of TMV-green fluorescent protein in pRNAi-NtLRR-RLK transgenic plants confirmed that NtLRR-RLK was important for susceptibility to TMV. The specific protein interaction map generated from our study revealed that levels of BIP1, E3 ubiquitin ligase, and LRR-RLK were significantly elevated, and all were represented at node positions in the protein interaction map. The same expression tendency of these proteins was also found in pRNAi-NtLRR-RLK transgenic plants at 24 h after TMV inoculation. These data suggested that specific genes in the infection process can activate the immune signal cascade through different resistance genes, and the integration of signal pathways could produce resistance to the virus. These results contribute to the overall understanding of the molecular basis of plant resistance to TMV and in the long term could identify new strategies for prevention and control virus infection.

Constraints on the expansion of paralogous protein families.

Duplication and divergence is a major mechanism by which new proteins and functions emerge in biology. Consequently, most organisms, in all domains of life, have genomes that encode large paralogous families of proteins. For recently duplicated pathways to acquire different, independent functions, the two paralogs must acquire mutations that effectively insulate them from one another. For instance, paralogous signaling proteins must acquire mutations that endow them with different interaction specificities such that they can participate in different signaling pathways without disruptive cross-talk. Although duplicated genes undoubtedly shape each other's evolution as they diverge and attain new functions, it is less clear how other paralogs impact or constrain gene duplication. Does the establishment of a new pathway by duplication and divergence require the system-wide optimization of all paralogs? The answer has profound implications for molecular evolution and our ability to engineer biological systems. Here, we discuss models, experiments, and approaches for tackling this question, and for understanding how new proteins and pathways are born.

Biomimetic Models to Investigate Membrane Biophysics Affecting Lipid-Protein Interaction.

Biological membranes are highly dynamic in their ability to orchestrate vital mechanisms including cellular protection, organelle compartmentalization, cellular biomechanics, nutrient transport, molecular/enzymatic recognition, and membrane fusion. Controlling lipid composition of different membranes allows cells to regulate their membrane characteristics, thus modifying their physical properties to permit specific protein interactions and drive structural function (membrane deformation facilitates vesicle budding and fusion) and signal transduction. Yet, how lipids control protein structure and function is still poorly understood and needs systematic investigation. In this review, we explore different in vitro membrane models and summarize our current understanding of the interplay between membrane biophysical properties and lipid–protein interaction, taken as example few proteins involved in muscular activity (dystrophin), digestion and Legionella pneumophila effector protein DrrA. The monolayer model with its movable barriers aims to mimic any membrane deformation while surface pressure modulation imitates lipid packing and membrane curvature changes. It is frequently used to investigate peripheral protein binding to the lipid headgroups. Examples of how lipid lateral pressure modifies protein interaction and organization within the membrane are presented using various biophysical techniques. Interestingly, the shear elasticity and surface viscosity of the monolayer will increase upon specific protein(s) binding, supporting the importance of such mechanical link for membrane stability. The lipid bilayer models such as vesicles are not only used to investigate direct protein binding based on the lipid nature, but more importantly to assess how local membrane curvature (vesicles with different size) influence the binding properties of a protein. Also, supported lipid bilayer model has been used widely to characterize diffusion law of lipids within the bilayer and/or protein/biomolecule binding and diffusion on the membrane. These membrane models continue to elucidate important advances regarding the dynamic properties harmonizing lipid–protein interaction.

Biomembrane-based organic electronic devices for ligand-receptor binding studies.

We present a simple, rapid method for forming supported lipid bilayers on organic electronic devices composed of conducting polymer electrodes using a solvent-assisted lipid bilayer formation method. These supported bilayers present protein recognition elements that are mobile, critical for multivalent binding interactions. Because these polymers are transparent and conducting, we demonstrate, by optical and electrical detection, the specific interactions of proteins with these biomembrane-based bioelectronic devices. This work paves the way for easy formation of biomembrane mimetics for sensing and detection of binding events in a label-free manner on organic electronic devices of more sophisticated architectures. Graphical abstract.

Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes.

Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species.IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

Study of Acetolactate Synthase and its Mechanism of Inhibition by Sulfonylurea Active Ingredients: Amidosulfuron, Nicosulfuron, Cyclosulfuron – In-silico Approach

Acetolactate synthase (ALS) or Acetohydroxyacid synthase (AHAS) catalyzes the first step in the synthesis of the branched-chain amino acids i.e., valine (2-amino-3-methylbutanoic acid), leucine (2-amino-4-methylpentanoic acid), and isoleucine (2-amino-3-methylpentanoic acid), in plants, bacteria, algae and fungi but not in humans. AHAS is the main target enzyme for sulfonylurea active ingredients; Amidosulfuron, Nicosulfuron and Cyclosulfuron those assist in lowering branched-chain amino acid synthesis through inhibition to form the complex of Lactyl-ThDP(TDL) to ALS with great practical importance. Amino acid composition, evolutionary and sequence analysis of the ALS protein from Arabidopsis thaliana and its homologous were systematically studied. Composition analysis reveals that ALS is a soluble protein. Moreover, the phylogenetic tree showed different clusters based on the source organism and multiple sequence alignment depicts conservative nature in amino acid residues. Furthermore, molecular docking has been conducted to study the interactions between ALS of Arabidopsis thaliana and TDL in presence/absence of the active ingredients of sulfonylurea herbicide groups. Molecular docking studies confirm active ingredients are effective to inhibit the binding of TDL to ALS. Our obtained results can be very useful to study specific protein interactions along with developing new herbicides using computational methods.

Study of the noncovalent interactions of ginsenosides and amyloid-β-peptide by CSI-MS and molecular docking.

Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI-MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI-MS). A cold spray ionization providing a stable solvation-ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug-protein complexes with exact stoichiometries. In this paper, we apply CSI-MS to explore the interactions of ginsenosides toward amyloid-β-peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI-MS and ESI-MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI-MS, while dehydration products are more readily available by ESI-MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI-MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.

An Immune-Stimulatory Helix-Loop-Helix Peptide: Selective Inhibition of CTLA-4-B7 Interaction.

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC50 of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

A Compact Biosensor for Binding Kinetics Analysis of Protein–Protein Interaction

Kinetics analysis of specific interactions of proteins is important for elucidating the molecular mechanism and the cellular process. In this work, we demonstrated a portable sensor based on fiber-optic (FO) surface plasmon resonance (SPR) sensing elements which can be used to detect the kinetics of protein-protein interaction. The sensor was built by FO-SPR sensing elements, an optical fiber, a red light-emitting diode (LED) and a detection platform based on smartphone. We set reference channel and control channel to compensate the fluctuations of the LED power, ambient temperature and refractive index (RI) of the buffer. Our sensor can monitor the interaction process in real-time and give valuable dynamic information. To verify its performance, the interactions between Ranse B/protein A and Con A/IgG were used as model systems. The sensor was examined by the recognitions of Con A and IgG on functionalized gold film, and the detection data was used to calculate the kinetic rate constant and binding constant of the reaction. The results demonstrate the superiority of our sensor in biomolecular recognition and kinetics analysis.

A New Strategy for Reporting Specific Protein Binding Events at Aqueous-Liquid Crystal Interfaces in the Presence of Non-Specific Proteins.

Aqueous-liquid crystal (LC) interfaces offer promise as responsive interfaces at which biomolecular recognition events can be amplified into macroscopic signals. However, the design of LC interfaces that distinguish between specific and non-specific protein interactions remains an unresolved challenge. Herein, we report the synthesis of amphiphilic monomers, dimers, and trimers conjugated to sulfonamide ligands via triazole rings, their assembly at aqueous-LC interfaces, and the orientational response of LCs to the interactions of carbonic anhydrase II (CAII) and serum albumin with the oligomer-decorated LC interfaces. Of six oligomers synthesized, only dimers without amide methylation were found to assemble at aqueous interfaces of nematic 4-cyano-4'-pentylbiphenyl (5CB) to induce perpendicular LC orientations. At dimer-decorated LC interfaces, we found that concentrations of CAII less than 4 μM did not measurably perturb the LC but prevented non-specific adsorption and penetration of serum albumin into the dimer-decorated interface that otherwise triggered bright, globular LC optical domains. These experiments and others (including competitive adsorption of CAII, BSA, and lysozyme) support our hypothesis that specific binding of CAII to the dimer prevents LC anchoring transitions triggered by non-specific adsorption of serum albumin. We illustrate the utility of the approach by reporting (i) the relative activity of two small-molecule inhibitors (6-ethoxy-2-benzothiazolesulfonamide and benzenesulfonamide) of CAII to sulfonamide and (ii) proteolytic digestion of a protein (CAII) by thermolysin. Overall, the results in this paper provide new insight into the interactions of proteins at aqueous-LC interfaces and fresh ideas for either blocking non-specific interactions of proteins at surfaces or reporting specific binding events at LC interfaces in the presence of non-specific proteins.