Specific Protein Interactions Research Articles

Genome-Wide Identification and Analysis of Phospholipase C Gene Family Reveals Orthologs, Co-Expression Networks, and Expression Profiling Under Abiotic Stress in Sorghum bicolor

Phospholipase C (PLC) is an essential enzyme involved in lipid signaling pathways crucial for regulating plant growth and responding to environmental stress. In sorghum, 11 PLC genes have been identified, comprising 6 PI-PLCs and 5 NPCs. Through phylogenetic and interspecies collinearity analyses, structural similarities between SbPLCs and ZmPLCs proteins have been observed, with a particularly strong collinearity between SbPLCs and OsPLCs. Promoter function analysis has shown that SbPLCs are significantly enriched under abiotic stress and hormonal stimuli, like ABA, jasmonic acid, drought, high temperature, and salt. Gene co-expression networks, constructed using a weighted gene co-expression network analysis (WGCNA), highlight distinct expression patterns of SbPLC1, SbPLC3a, and SbPLC4 in response to abiotic stress, providing further insights into the expression patterns and interactions of SbPLCs under various environmental stimuli. qRT-PCR results reveal variations in expression levels among most SbPLCs members under different stress conditions (drought, NaCl, NaHCO3), hormone treatments (ABA), and developmental stages, indicating both specific and overlapping expression patterns. This comprehensive analysis offers valuable insights into the roles of SbPLCs in sorghum, shedding light on their specific expression patterns, regulatory elements, and protein interactions across different environmental stimuli and developmental stages.

The genetic architecture of protein interaction affinity and specificity

The encoding and evolution of specificity and affinity in protein-protein interactions is poorly understood. Here, we address this question by quantifying how all mutations in one protein, JUN, alter binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. We fit a global thermodynamic model to the data to reveal that most affinity changing mutations equally affect JUN’s affinity to all its interaction partners. Mutations that alter binding specificity are relatively rare but distributed throughout the interaction interface. Specificity is determined both by features that promote on-target interactions and by those that prevent off-target interactions. Approximately half of the specificity-defining residues in JUN contribute both to promoting on-target binding and preventing off-target binding. Nearly all specificity-altering mutations in the interaction interface are pleiotropic, also altering affinity to all partners. In contrast, mutations outside the interface can tune global affinity without affecting specificity. Our results reveal the distributed encoding of specificity and affinity in an interaction interface and how coiled-coils provide an elegant solution to the challenge of optimizing both specificity and affinity in a large protein family.

Preparation of Polyclonal Antibodies to Barley Granule-Bound Amylopectin Synthase Ia and Their Application in the Characterization of Interacting Proteins

The production of amylose is facilitated by granule-bound starch synthase (GBSS). Despite its importance, the specific protein interactions involving barley grain-bound starch synthase Ia (HvGBSSIa) remain poorly understood. To elucidate this, we engineered a pET-32a-HvGBSSIa prokaryotic expression vector for specific expression in E. coli Rosetta cells. A rabbit anti-HvGBSSIa polyclonal antibody was generated and employed to enrich HvGBSSIa-binding proteins from barley grains through immunoprecipitation. The isolated complexes were then resolved through SDS-PAGE, and the constituent proteins were identified using mass spectrometry coupled with database searches. Our results confirmed the successful preparation of a highly specific polyclonal antibody against HvGBSSI. Furthermore, differential expression of HvGBSSIa was assessed across various barley tissues and developmental stages of the grain, revealing peak expression at 25 days post-flowering. Proteins interacting with HvGBSSIa, including sucrose synthase and starch branching enzyme, were identified through co-immunoprecipitation. This study lays the groundwork for further detailed analyses of the HvGBSSIa protein complex in barley.

Insights into structural vaccinology harnessed for universal coronavirus vaccine development.

Structural vaccinology is pivotal in expediting vaccine design through high-throughput screening of immunogenic antigens. Leveraging the structural and functional characteristics of antigens and immune cell receptors, this approach employs protein structural comparison to identify conserved patterns in key pathogenic components. Molecular modeling techniques, including homology modeling and molecular docking, analyze specific three-dimensional (3D) structures and protein interactions and offer valuable insights into the 3D interactions and binding affinity between vaccine candidates and target proteins. In this review, we delve into the utilization of various immunoinformatics and molecular modeling tools to streamline the development of broad-protective vaccines against coronavirus disease 2019 variants. Structural vaccinology significantly enhances our understanding of molecular interactions between hosts and pathogens. By accelerating the pace of developing effective and targeted vaccines, particularly against the rapidly mutating severe acute respiratory syndrome coronavirus 2 and other prevalent infectious diseases, this approach stands at the forefront of advancing immunization strategies. The combination of computational techniques and structural insights not only facilitates the identification of potential vaccine candidates but also contributes to the rational design of vaccines, fostering a more efficient and targeted approach to combatting infectious diseases.

Self-Assembly of Single-Virus SERS Hotspots for Highly Sensitive In Situ Detection of SARS-CoV-2 on Solid Surfaces.

Microbial surface transmission has aroused great attention since the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Developing a simple in situ detection method for viruses on solid surfaces is of great significance for timely public health surveillance. Taking advantage of the natural structure of SARS-CoV-2, we reported the assembly of Au@AgNPs on the surface of a single virus by the specific aptamer-spike protein interaction. Multiple hotspots can be created between the neighboring Au@AgNPs for the highly sensitive surface-enhanced Raman scattering (SERS) detection of SARS-CoV-2. Using two different aptamers labeled with Cy3 and Au@AgNPs, in situ SERS detection of pseudotyped SARS-CoV-2 (PSV) on packaging surfaces was achieved within 20 min, with a detection limit of 5.26 TCID50/mL. For the blind testing of 20 PSV-contaminated packaging samples, this SERS aptasensor had a sensitivity of 100% and an accuracy of 100%. This assay has been successfully applied to in situ detection of PSV on the surfaces of different packaging materials, suggesting its potential applicability.

Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.

The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" (Jc-Craspase). We find that Jc-Craspase cleaves Jc-Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii, respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.

Simultaneous enhancement in phosphorescence and its lifetime of PtOEP-peptide assembly triggered by protein interaction

We fabricated hybrid nanoparticles consisting of organic semiconducting material with peptide sequence to reflect the target protein interaction. A phosphorescent OLED material, platinum octaethylporphyrin (PtOEP) was self-assembled by reprecipitation with the A17 peptide (YCAYYSPRHKTTF) selected as a probe ligand in order to recognize heat shock protein 70 (HSP70). The phosphorescence intensity of the PtOEP-A17 assembly was enhanced by 125 % after treatment with HSP70. The specificity of the protein interaction was confirmed in both solution and solid states of the PtOEP-A17 assembly against to BSA and nucleolin. We figured out that the phosphorescence lifetime of PtOEP-A17 assembly after exposed to HSP70 increased significantly to 153 ns from initial 115 ns. These simultaneous enhancements in phosphorescence and lifetime triggered by the specific protein interaction would open new applications of PtOEP, a representative material of light-emitting device fields.

SIRT4 as a novel interactor and candidate suppressor of C-RAF kinase in MAPK signaling.

Cellular responses leading to development, proliferation, and differentiation depend on RAF/MEK/ERK signaling, which integrates and amplifies signals from various stimuli for downstream cellular responses. C-RAF activation has been reported in many types of tumor cell proliferation and developmental disorders, necessitating the discovery of potential C-RAF protein regulators. Here, we identify a novel and specific protein interaction between C-RAF among the RAF kinase paralogs, and SIRT4 among the mitochondrial sirtuin family members SIRT3, SIRT4, and SIRT5. Structurally, C-RAF binds to SIRT4 through the N-terminal cysteine-rich domain, whereas SIRT4 predominantly requires the C-terminus for full interaction with C-RAF. Interestingly, SIRT4 specifically interacts with C-RAF in a pre-signaling inactive (serine 259-phosphorylated) state. Consistent with this finding, the expression of SIRT4 in HEK293 cells results in an up-regulation of pS259-C-RAF levels and a concomitant reduction in MAPK signaling as evidenced by strongly decreased phospho-ERK signals. Thus, we propose an additional extra-mitochondrial function of SIRT4 as a cytosolic tumor suppressor of C-RAF-MAPK signaling, besides its metabolic tumor suppressor role of glutamate dehydrogenase and glutamate levels in mitochondria.

Manganese- and zinc-coordinated interaction of Schistosoma japonicum glutathione S-transferase with neurotransmitter transporters GlyT1 and GAT3 in vitro

Glutathione S-transferases (GSTs) are a family of multifunctional isoenzymes involved in the neutralization of toxic compounds, drug resistance and several other cellular functions. The glutathione S-transferase enzyme of Schistosoma japonicum (SjGST-26) plays a role in human schistosomiasis and is also a frequently used fusion partner in mammalian and bacterial expression and pull-down systems. GSTs seem not to be naturally associated with metal ions. Exceptionally, in vitro, metal binding sites have been previously described in some schistosome GSTs; however, their possible physiological role is unclear. Molecules of several neurotransmitter transporters also contain a regulatory zinc binding site, which affects their transport cycle. Here we show that among several metals, manganese and zinc are able to induce a specific protein interaction of SjGST-26 with the glycine transporter GlyT1 and the GABA transporter GAT3 in vitro. The results suggest that metal-binding sites on SjGST-26 and neurotransmitter transporters might function in metal-coordinated interactions with other metalloproteins. Our results additionally indicate that the presence of metal ions in SjGST-26-based GST protein pull-down assays may lead to a false-positive interaction if the potential interacting target is the metalloprotein.

Specific protein interactions between rice members of the GT43 and GT47 families form various central cores of putative xylan synthase complexes.

Members of the glycosyltransferase (GT)43 and GT47 families have been associated with heteroxylan synthesis in both dicots and monocots and are thought to assemble into central cores of putative xylan synthase complexes (XSCs). Currently, it is unknown whether protein-protein interactions within these central cores are specific, how many such complexes exist, and whether these complexes are functionally redundant. Here, we used gene association network and co-expression approaches in rice to identify four OsGT43s and four OsGT47s that assemble into different GT43/GT47 complexes. Using two independent methods, we showed that (i) these GTs assemble into at least six unique complexes through specific protein-protein interactions and (ii) the proteins interact directly in vitro. Confocal microscopy showed that, when alone, all OsGT43s were retained in the endoplasmic reticulum (ER), while all OsGT47s were localized in the Golgi. co-expression of OsGT43s and OsGT47s displayed complexes that form in the ER but accumulate in Golgi. ER-to-Golgi trafficking appears to require interactions between OsGT43s and OsGT47s. Comparison of the central cores of the three putative rice OsXSCs to wheat, asparagus, and Arabidopsis XSCs, showed great variation in GT43/GT47 combinations, which makes the identification of orthologous central cores between grasses and dicots challenging. However, the emerging picture is that all central cores from these species seem to have at least one member of the IRX10/IRX10-L clade in the GT47 family in common, suggesting greater functional importance for this family in xylan synthesis. Our findings provide a new framework for future investigation of heteroxylan biosynthesis and function in monocots.

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously invitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.

Evolution of a plant growth-regulatory protein interaction specificity

Specific protein–protein interactions (PPIs) enable biological regulation. However, the evolution of PPI specificity is little understood. Here we trace the evolution of the land-plant growth-regulatory DELLA–SLY1/GID2 PPI, revealing progressive increase in specificity of affinity of SLY1/GID2 for a particular DELLA form. While early-diverging SLY1s display relatively broad-range DELLA affinity, later-diverging SLY1s tend towards increasingly stringent affinity for a specific DELLA A’ form generated by the growth-promoting phytohormone gibberellin (GA). Our novel mutational strategy reveals amino acid substitutions contributing to the evolution of Arabidopsis thaliana SLY1 A’ specificity, also showing that routes permitting reversion to broader affinity became increasingly constrained over evolutionary time. We suggest that progressive affinity narrowing may be an important evolutionary driver of PPI specificity and that increase in SLY1/GID2-DELLA specificity enabled the enhanced flexibility of plant physiological environmental adaptation conferred by the GA-DELLA growth-regulatory mechanism.

THU558 Combination Therapy Of Acetyl-CoA Synthetase Inhibitor And Fulvestrant Reverse Epigenetic Alterations Of Metastatic Breast Cancer Cells And Reduce Cancer Cell Proliferation

Abstract Disclosure: A.N. Mogol: None. Z. Madak Erdogan: None. Endocrine therapies exploit the hormone dependencies of the cancer cells in various ways, including by targeting estrogen receptor alpha (ERα) that is expressed by approximately 70% of human breast cancers. After treatment, recurrence and metastasis are still observed in 30-40% of ER+ patients and the 5-year relative overall survival rate for these patients is just 24%. Acetyl-CoA production significantly regulates histone acetylation and gene regulation, which support tumor progression. In our previous study, we showed the synergistic effect of the ERα agonist Fulvestrant (Fulv) and Acetyl-CoA Synthase Inhibitor (ACSI) in reducing cell viability in ER+ metastatic breast cancer (MBC) cells in vitro. To study the synergistic contribution of ACSS2 to endocrine resistance, we used ChIP analysis, which showed that ACSS2 was recruited to ERα binding sites with Fulv treatment in cell line xenograft tumors and PDX samples. We used the CUT&RUN method to investigate specific protein (H3k27ac, ERα and ACSS2) and DNA interactions in ER+ MBC cells globally. Enriched chromatin samples were then used for qPCR and sequencing. Combination therapy of the ACSI and Fulv significantly altered the epigenetic landscape of the breast cancer cells in a manner to decrease cell viability. High histone acetylation at lysine 27 residue was observed in Veh treatment group and combination therapy of ACSI+Fulv further decreased the enrichment of this histone modification in classical ER binding sites. Interestingly, ACSS2 binding was increased with Fulv treatment. Targeting epigenetic alterations caused by cancer cell adaptations is a novel approach that could potentially lead to new therapies to reduce metastasis mortality. Presentation: Thursday, June 15, 2023

Evolved distal tail protein of skunaviruses facilitates adsorption to exopolysaccharide-encoding lactococci.

Aim: Lactococcal skunaviruses are diverse and problematic in the industrial dairy environment. Host recognition involves the specific interaction of phage-encoded proteins with saccharidic host cell surface structures. Lactococcal plasmid pEPS6073 encodes genes required for the biosynthesis of a cell surface-associated exopolysaccharide (EPS), designated 6073-like. Here, the impact of this EPS on Skunavirus sensitivity was assessed. Methods: Conjugal transfer of pEPS6073 into two model strains followed by phage plaque assays and adsorption assays were performed to assess its effect on phage sensitivity. Phage distal tail proteins were analyzed bioinformatically using HHpred and modeling with AlphaFold. Construction of recombinant phages carrying evolved Dits was performed by supplying a plasmid-encoded template for homologous recombination. Results: pEPS6073 confers resistance against a subset of skunaviruses via adsorption inhibition. IFF collection skunaviruses that infect strains encoding the 6073-like eps gene cluster carry insertions in their distal tail protein-encoding (dit) genes that result in longer Dit proteins (so-called evolved Dits), which encode carbohydrate-binding domains. Three skunaviruses with classical Dits (no insertion) were unable to fully infect their hosts following the conjugal introduction of pEPS6073, showing reductions in both adsorption and efficiency of plaquing. Cloning the evolved Dit into these phages enabled full infectivity on their host strains, both wild type and transconjugant carrying pEPS6073, with recombinant phages adsorbing slightly better to the EPS+ host than wild type. Conclusion: The 6073-like EPS potentially occludes the phage receptor for skunaviruses that encode a classical Dit protein. Skunaviruses that infect strains encoding the 6073-like EPS harbor evolved Dits, which likely help promote phage adsorption rather than just allow the phage to circumvent the putative EPS barrier. This work furthers our knowledge of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of a subset of skunaviruses.

The Vital Role Played by Deferiprone in the Transition of Thalassaemia from a Fatal to a Chronic Disease and Challenges in Its Repurposing for Use in Non-Iron-Loaded Diseases.

The iron chelating orphan drug deferiprone (L1), discovered over 40 years ago, has been used daily by patients across the world at high doses (75-100 mg/kg) for more than 30 years with no serious toxicity. The level of safety and the simple, inexpensive synthesis are some of the many unique properties of L1, which played a major role in the contribution of the drug in the transition of thalassaemia from a fatal to a chronic disease. Other unique and valuable clinical properties of L1 in relation to pharmacology and metabolism include: oral effectiveness, which improved compliance compared to the prototype therapy with subcutaneous deferoxamine; highly effective iron removal from all iron-loaded organs, particularly the heart, which is the major target organ of iron toxicity and the cause of mortality in thalassaemic patients; an ability to achieve negative iron balance, completely remove all excess iron, and maintain normal iron stores in thalassaemic patients; rapid absorption from the stomach and rapid clearance from the body, allowing a greater frequency of repeated administration and overall increased efficacy of iron excretion, which is dependent on the dose used and also the concentration achieved at the site of drug action; and its ability to cross the blood-brain barrier and treat malignant, neurological, and microbial diseases affecting the brain. Some differential pharmacological activity by L1 among patients has been generally shown in relation to the absorption, distribution, metabolism, elimination, and toxicity (ADMET) of the drug. Unique properties exhibited by L1 in comparison to other drugs include specific protein interactions and antioxidant effects, such as iron removal from transferrin and lactoferrin; inhibition of iron and copper catalytic production of free radicals, ferroptosis, and cuproptosis; and inhibition of iron-containing proteins associated with different pathological conditions. The unique properties of L1 have attracted the interest of many investigators for drug repurposing and use in many pathological conditions, including cancer, neurodegenerative conditions, microbial conditions, renal conditions, free radical pathology, metal intoxication in relation to Fe, Cu, Al, Zn, Ga, In, U, and Pu, and other diseases. Similarly, the properties of L1 increase the prospects of its wider use in optimizing therapeutic efforts in many other fields of medicine, including synergies with other drugs.

Dynamic remodeling of Escherichia coli interactome in response to environmental perturbations.

Proteins play an essential role in the vital biological processes governing cellular functions. Most proteins function as members of macromolecular machines, with the network of interacting proteins revealing the molecular mechanisms driving the formation of these complexes. Profiling the physiology-driven remodeling of these interactions within different contexts constitutes a crucial component to achieving a comprehensive systems-level understanding of interactome dynamics. Here, we apply co-fractionation mass spectrometry and computational modeling to quantify and profile the interactions of ∼2000 proteins in the bacterium Escherichia coli cultured under 10 distinct culture conditions. The resulting quantitative co-elution patterns revealed large-scale condition-dependent interaction remodeling among protein complexes involved in diverse biochemical pathways in response to the unique environmental challenges. The network-level analysis highlighted interactome-wide biophysical properties and structural patterns governing interaction remodeling. Our results provide evidence of the local and global plasticity of the E. coli interactome along with a rigorous generalizable framework to define protein interaction specificity. We provide an accompanying interactive web application to facilitate the exploration of these rewired networks.

Narrow funnel-like interaction energy distribution is an indicator of specific protein interaction partner

Protein interaction networks underlie countless biological mechanisms. However, most protein interaction predictions are based on biological evidence that are biased to well-known protein interaction or physical evidence that exhibits low accuracy for weak interactions and requires high computational power. In this study, a novel method has been suggested to predict protein interaction partners by investigating narrow funnel-like interaction energy distribution. In this study, it was demonstrated that various protein interactions including kinases and E3 ubiquitin ligases have narrow funnel-like interaction energy distribution. To analyze protein interaction distribution, modified scores of iRMS and TM-score are introduced. Then, using these scores, algorithm and deep learning model for prediction of protein interaction partner and substrate of kinase and E3 ubiquitin ligase were developed. The prediction accuracy was similar to or even better than that of yeast two-hybrid screening. Ultimately, this knowledge-free protein interaction prediction method will broaden our understanding of protein interaction networks.

Pleiotropy of C1QL proteins across physiological systems and their emerging role in synapse homeostasis.

The C1q/TNF superfamily of proteins engages in a pleiotropy of physiological functions associated with various diseases. C1QL proteins demonstrate important protective and regulatory roles in the endocrine, immune, cardiovascular, and nervous systems in both human and rodent studies. Studies in the central nervous system (CNS), adipose, and muscle tissue reveal several C1QL protein and receptor pathways altering multiple cellular responses, including cell fusion, morphology, and adhesion. This review examines C1QL proteins across these systems, summarizing functional and disease associations and highlighting cellular responses based on in vitro and in vivo data, receptor interaction partners, and C1QL-associated protein signaling pathways. We highlight the functions of C1QL proteins in organizing CNS synapses, regulating synapse homeostasis, maintaining excitatory synapses, and mediating signaling and trans-synaptic connections. Yet, while these associations are known, present studies provide insufficient insight into the underlying molecular mechanism of their pleiotropy, including specific protein interactions and functional pathways. Thus, we suggest several areas for more in-depth and interdisciplinary hypothesis testing.

Thymosin α1 interacts with Galectin-1 modulating the β-galactosides affinity and inducing alteration in the biological activity.

The study of mechanism of action of Thymosin alpha 1 (Tα1) and the basis of the pleiotropic effect in health and disease, is one of the main focus of our ongoing research. Tα1 is a thymic peptide that demonstrates a peculiar ability to restore homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination, and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. However, few are the information about mechanisms of action mediated by specific Tα1-target protein interaction that could explain its pleiotropic effect. We investigated the interaction of Tα1 with Galectin-1 (Gal-1), a protein belonging to an oligosaccharide binding protein family involved in a variety of biological and pathological processes, including immunoregulation, infections, cancer progression and aggressiveness. Using molecular and cellular methodological approaches, we demonstrated the interaction between these two proteins. Tα1 specifically inhibited the hemagglutination activity of Gal-1, the Gal-1 dependent in vitro formation of endothelial cell tubular structures, and the migration of cancer cells in wound healing assay. Physico-chemical methods revealed the details of the molecular interaction of Tα1 with Gal-1. Hence, the study allowed the identification of the not known until now specific interaction between Tα1 and Gal-1, and unraveled a novel mechanism of action of Tα1 that could support understanding of its pleiotropic activity.

Marginal specificity in protein interactions constrains evolution of a paralogous family

The evolution of novel functions in biology relies heavily on gene duplication and divergence, creating large paralogous protein families. Selective pressure to avoid detrimental cross-talk often results in paralogs that exhibit exquisite specificity for their interaction partners. But how robust or sensitive is this specificity to mutation? Here, using deep mutational scanning, we demonstrate that a paralogous family of bacterial signaling proteins exhibits marginal specificity, such that many individual substitutions give rise to substantial cross-talk between normally insulated pathways. Our results indicate that sequence space is locally crowded despite overall sparseness, and we provide evidence that this crowding has constrained the evolution of bacterial signaling proteins. These findings underscore how evolution selects for "good enough"rather than optimized phenotypes, leading to restrictions on the subsequent evolution of paralogs.