Non-cognate Ligand Research Articles

On the function of TRAP substrate-binding proteins: Conformational variation of the sialic acid binding protein SiaP

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding protein to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 μM) compared to N-glycolylneuraminate (KD = 4.4 μM), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, we suggest that the open unliganded conformation may sample multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies.

Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified. In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes. In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro. Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

Natural and synthetic inhibitors of a phage-encoded quorum-sensing receptor affect phage–host dynamics in mixed bacterial communities

Viruses that infect bacteria, called phages, shape the composition of bacterial communities and are important drivers of bacterial evolution. We recently showed that temperate phages, when residing in bacteria (i.e., prophages), are capable of manipulating the bacterial cell-to-cell communication process called quorum sensing (QS). QS relies on the production, release, and population-wide detection of signaling molecules called autoinducers (AI). Gram-negative bacteria commonly employ N-acyl homoserine lactones (HSL) as AIs that are detected by LuxR-type QS receptors. Phage ARM81ld is a prophage of the aquatic bacterium Aeromonas sp. ARM81, and it encodes a homolog of a bacterial LuxR, called LuxRARM81ld. LuxRARM81ld detects host Aeromonas-produced C4-HSL, and in response, activates the phage lytic program, triggering death of its host and release of viral particles. Here, we show that phage LuxRARM81ld activity is modulated by noncognate HSL ligands and by a synthetic small molecule inhibitor. We determine that HSLs with acyl chain lengths equal to or longer than C8 antagonize LuxRARM81ld. For example, the C8-HSL AI produced by Vibrio fischeri that coexists with Aeromonads in aquatic environments, binds to and inhibits LuxRARM81ld, and consequently, protects the host from lysis. Coculture of V. fischeri with the Aeromonas sp. ARM81 lysogen suppresses phage ARM81ld virion production. We propose that the cell density and species composition of the bacterial community could determine outcomes in bacterial-phage partnerships.

X-Ray Crystallography to Study Conformational Changes in a TPP Riboswitch.

Conformational rearrangements are key to the function of riboswitches. These regulatory mRNA regions specifically bind to cellular metabolites using evolutionarily conserved sensing domains and modulate gene expression via adjacent downstream expression platforms, which carry gene expression signals. The regulation is achieved through the ligand-dependent formation of two alternative and mutually exclusive conformations involving the same RNA region. While X-ray crystallography cannot visualize dynamics of such dramatic conformational rearrangements, this method is pivotal to understand RNA-ligand interaction that stabilize the sensing domain and drive folding of the expression platform. X-ray crystallography can reveal local changes in RNA necessary for discriminating cognate and noncognate ligands. This chapter describes preparation of thiamine pyrophosphate riboswitch RNAs and its crystallization with different ligands, resulting in structures with local conformational changes in RNA. These structures can help to derive information on the dynamics of the RNA essential for specific binding to small molecules, with potential for using this information for developing designer riboswitch-ligand systems.

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2.

Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed fluorescence resonance energy transfer (FRET) sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM (egative allosteric modulator) increases the occupancy of one of the intermediate states while a positive allosteric modulator increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.

Bacterial 2'-Deoxyguanosine Riboswitch Classes as Potential Targets for Antibiotics: A Structure and Dynamics Study.

The spread of antibiotic-resistant bacteria represents a substantial health threat. Current antibiotics act on a few metabolic pathways, facilitating resistance. Consequently, novel regulatory inhibition mechanisms are necessary. Riboswitches represent promising targets for antibacterial drugs. Purine riboswitches are interesting, since they play essential roles in the genetic regulation of bacterial metabolism. Among these, class I (2′-dG-I) and class II (2′-dG-II) are two different 2′-deoxyguanosine (2′-dG) riboswitches involved in the control of deoxyguanosine metabolism. However, high affinity for nucleosides involves local or distal modifications around the ligand-binding pocket, depending on the class. Therefore, it is crucial to understand these riboswitches’ recognition mechanisms as antibiotic targets. In this work, we used a combination of computational biophysics approaches to investigate the structure, dynamics, and energy landscape of both 2′-dG classes bound to the nucleoside ligands, 2′-deoxyguanosine, and riboguanosine. Our results suggest that the stability and increased interactions in the three-way junction of 2′-dG riboswitches were associated with a higher nucleoside ligand affinity. Also, structural changes in the 2′-dG-II aptamers enable enhanced intramolecular communication. Overall, the 2′-dG-II riboswitch might be a promising drug design target due to its ability to recognize both cognate and noncognate ligands.

Evolving Small-Molecule Biosensors with Improved Performance and Reprogrammed Ligand Preference Using OrthoRep.

Genetically encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand preferences remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with a high dynamic range, reprogrammed activity toward desired noncognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that the propagation of host yeast cells resulted in BenM's rapid and continuous diversification. When these cells were subjected to cycles of culturing and sorting on BenM activity in the presence and absence of its cognate ligand, muconic acid, or the noncognate ligand, adipic acid, we obtained multiple BenM variants that respond to their corresponding ligands. These biosensors outperform previously engineered BenM-based biosensors by achieving a substantially greater dynamic range (up to ∼180-fold induction) and broadened operational range. The expression of select BenM variants in the presence of a muconic acid biosynthetic pathway demonstrated sensitive biosensor activation without saturating response, which should enable pathway and host engineering for higher production of muconic and adipic acids. Given the streamlined manner in which high-performance and versatile biosensors were evolved using OrthoRep, this study provides a template for generating custom biosensors for metabolic pathway engineering and other biotechnology goals.

A Thermodynamic Analysis of the Binding Specificity between Four Human PDZ Domains and Eight Host, Viral and Designed Ligands

PDZ domains are binding modules mostly involved in cell signaling and cell–cell junctions. These domains are able to recognize a wide variety of natural targets and, among the PDZ partners, viruses have been discovered to interact with their host via a PDZ domain. With such an array of relevant and diverse interactions, PDZ binding specificity has been thoroughly studied and a traditional classification has grouped PDZ domains in three major specificity classes. In this work, we have selected four human PDZ domains covering the three canonical specificity-class binding mode and a set of their corresponding binders, including host/natural, viral and designed PDZ motifs. Through calorimetric techniques, we have covered the entire cross interactions between the selected PDZ domains and partners. The results indicate a rather basic specificity in each PDZ domain, with two of the domains that bind their cognate and some non-cognate ligands and the two other domains that basically bind their cognate partners. On the other hand, the host partners mostly bind their corresponding PDZ domain and, interestingly, the viral ligands are able to bind most of the studied PDZ domains, even those not previously described. Some viruses may have evolved to use of the ability of the PDZ fold to bind multiple targets, with resulting affinities for the virus–host interactions that are, in some cases, higher than for host–host interactions.

A new cytokine target for chronic obstructive pulmonary disease?

A new cytokine target for chronic obstructive pulmonary disease?

Co-Expression and Functional Interaction between CXCR4 and CD74 in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by progressive accumulation of small, mature B-lymphocytes aberrantly expressing CD5. One of the crucial factors in B-CLL development is the decreased apoptosis of malignant B-Cells.The chemokine receptor CXCR4 and the MHC class II invariant chain (Ii) CD74, are expressed on CLL B-cells and play a role in cells' migration and survival. CXCL12, the cognate ligand of CXCR4, promotes homing of CLL cells into the bone marrow (BM) and survival within the niche. Furthermore, CXCR4 is activated by macrophage migration inhibitory factor (MIF), a chemokine-like inflammatory cytokine and non-cognate ligand of CXCR4. MIF and its homolog MIF2 are bona fide ligands of CD74.CD74 is highly expressed on CLL cells and expression is correlated with the adversely prognostic factor ZAP 70. Interestingly, MIF plays a role in the survival of CLL cells by binding to CD74. Furthermore, increased MIF levels are found in the plasma of CLL patients and genetic Mif deficiency leads to delayed development of CLL in the Eµ-TCL1 mouse model. Finally, the aim of our study was to investigate the expression and interaction between CXCR4 and CD74 as well as their potential contribution to B-Cell survival in B-CLL.An initial cohort of 350 patients with different backgrounds of hematological malignancies was analyzed for CXCR4 and CD74 expression on mononuclear cells. Interestingly, the highest coexpression of both receptors was observed in B-CLL (n=10) by flow cytometry (FCM), where cells were stained for CD3+, CD14+ and CD19+/CD5+ and compared to cells from healthy donors (n=20). For in vivo and in vitro studies cell lines and a mice model were chosen, which express both receptors CXCR4 and CD74 on the surface of B-Cells.The human CLL line JVM3 and primary CLL cells from aged leukemic Eμ-TCL1 mice were used to study the cross-talk of CXCR4 and CD74 in regulation of CLL survival after incubation with rhMIF, rhCXCL12, or rhMIF-2 in conjunction with Bcl2-inhibitor treatment (Venetoclax, 30µM) or pharmacological blockade of CXCR4 and/or CD74 (AMD3100 30µM, neutralizing CD74 antibody LN2 5µg/ml). Potential direct CXCR4/CD74 interactions were studied by confocal laser scanning microscopy (CLSM) and coimmunoprecipitation (CoIP). Chemotaxis towards CXCL12, MIF and MIF-2 were analyzed to understand homing behavior, which contributes to B-Cell survival.CXCR4 and CD74 were found significantly coexpressed on B-cells from CLL patients compared to controls (p< 0.0001), suggesting a functional interaction between these receptors. Colocalization was confirmed by CLSM and CoIP detected receptor complexes between CD74 and CXCR4 on B- Cells from the JVM3 and OSU CLL cell lineFunctionally, inhibition of either receptor substantially impaired survival rates that did not differ from a simultaneous co-blockade of CD74 and CXCR4, indicating a crosstalk and/or merging of downstream signaling pathways. Apoptosis assays indicated that inhibition of CXCR4 and/or CD74 triggered substantial cell death (78,17 +/- 1,84%). Of note, single vs. double blockade experiments confirmed that CD74 and CXCR4 functionally interact to control CLL survival. Inversely, pre-incubation of JVM3 cells with CXCL12, MIF and MIF-2 decreased apoptosis by 43, 36 % (MIF) and 51, 50 % (CXCL12) compared to the control.The ligands MIF, MIF2 and CXCL12 promoted chemotactic migration of JVM3 B-cells that contributes to the B-Cell survival mechanism.Together, our results suggest an important role of MIF proteins in the survival and homing of malignant CLL cells that is mediated through a functional interaction between the CXCR4 and CD74 survival receptors. In conclusion, high levels of MIF attract B-Cells with the coexpression of CXCR4 and CD74, which build a complex after binding to their ligands and activate survival pathways to inhibit cell death. The MIF/CXCR4/CD74 axis could represent a novel target for therapeutic intervention. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Koschmieder:Roche: Other: Clinical Trial participation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding.

Myomedin scaffold variants targeted to 10E8 HIV-1 broadly neutralizing antibody mimic gp41 epitope and elicit HIV-1 virus-neutralizing sera in mice

ABSTRACT One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this “non-cognate ligand” concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

Correlation Patterns Among B7 Family Ligands and Tryptophan Degrading Enzymes in Hepatocellular Carcinoma.

Mechanisms of dysfunctional T cell immunity in Hepatocellular Carcinoma (HCC) need to be well defined. B7 family molecules provide both co-stimulatory and co-inhibitory signals to T cells while tryptophan degrading enzymes like Indoleamine 2,3 dioxygenase (IDO) and Tryptophan 2,3 Dioxygenase (TDO) mediate tumor immune tolerance. It is necessary to identify their in situ correlative expression, which informs targets for combined immunotherapy approaches. We investigated B7 family molecules, IDO, TDO and immune responsive effectors in the tumor tissues of patients with HCC (n = 28) using a pathway-focused quantitative nanoscale chip real-time PCR. Four best correlative expressions, namely (1) B7-1 & PD-L2, (2) B7-H2 & B7-H3, (3) B7-2 & PD-L1, (4) PD-L1 & PD-L2, were identified among B7 family ligands, albeit they express at different levels. Although TDO expression is higher than IDO, PD-L1 correlates only with IDO but not TDO. Immune effector (Granzyme B) and suppressive (PD-1 and TGF-β) genes correlate with IDO and B7-1, B7-H5, PD-L2. Identification of the in situ correlation of PD-L1, PD-L2 and IDO suggest their cumulative immuno suppressive role in HCC. The distinct correlations among B7-1, B7-2, B7-H2, and B7-H3, correlation of PD-1 with non-cognate ligands such as B7-1 and B7-H5, and correlation of tumor lytic enzyme Granzyme B with IDO and PD-L2 suggest that HCC microenvironment is complexly orchestrated with both stimulatory and inhibitory molecules which together neutralize and blunt anti-HCC immunity. Functional assays demonstrate that both PDL-1 and IDO synergistically inhibit T cell responses. Altogether, the present data suggest the usage of combined immune checkpoint blocking strategies targeting co-inhibitory B7 molecules and IDO for HCC management.

Single-Molecule Super-Resolution Microscopy Reveals Heteromeric Complexes of MET and EGFR upon Ligand Activation

Receptor tyrosine kinases (RTKs) orchestrate cell motility and differentiation. Deregulated RTKs may promote cancer and are prime targets for specific inhibitors. Increasing evidence indicates that resistance to inhibitor treatment involves receptor cross-interactions circumventing inhibition of one RTK by activating alternative signaling pathways. Here, we used single-molecule super-resolution microscopy to simultaneously visualize single MET and epidermal growth factor receptor (EGFR) clusters in two cancer cell lines, HeLa and BT-20, in fixed and living cells. We found heteromeric receptor clusters of EGFR and MET in both cell types, promoted by ligand activation. Single-protein tracking experiments in living cells revealed that both MET and EGFR respond to their cognate as well as non-cognate ligands by slower diffusion. In summary, for the first time, we present static as well as dynamic evidence of the presence of heteromeric clusters of MET and EGFR on the cell membrane that correlates with the relative surface expression levels of the two receptors.

Novel Immunomodulatory Proteins Generated via Directed Evolution of Variant IgSF Domains.

Immunoglobulin superfamily member (IgSF) proteins play a significant role in regulating immune responses with surface expression on all immune cell subsets, making the IgSF an attractive family of proteins for therapeutic targeting in human diseases. We have developed a directed evolution platform capable of engineering IgSF domains to increase affinities for cognate ligands and/or introduce binding to non-cognate ligands. Using this scientific platform, ICOSL domains have been derived with enhanced binding to ICOS and with additional high-affinity binding to the non-cognate receptor, CD28. Fc-fusion proteins containing these engineered ICOSL domains significantly attenuate T cell activation in vitro and in vivo and can inhibit development of inflammatory diseases in mouse models. We also present evidence that engineered ICOSL domains can be formatted to selectively provide costimulatory signals to augment T cell responses. Our scientific platform thus provides a system for developing therapeutic protein candidates with selective biological impact for treatments of a wide array of human disorders including cancer and autoimmune/inflammatory diseases.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Non-cognate ligands of Procrustean paratopes as potential vaccine components.

Animals immunized with specific antibodies - idiotypes - produce anti-antibodies, some of them directed to the idiotypic antigen-binding sites, i.e., the paratopes [1,2]. When the paratopes of one idiotype ligate B-cell receptors, they elicit anti-idiotypes and in a subsequent round anti-anti-idiotypes that might resemble the first idiotypes [3]. With this rationale, anti-idiotypic paratopes were propounded as substitutes for pathogen epitopes in immunization. The aim is to elicit anti-anti-idiotypic antibodies that mimic the idiotype in anti-microbial effects [4], e.g., neutralization of virus infectivity [5].

Evaluation and extension of the two-site, two-step model for binding and activation of the chemokine receptor CCR1

Interactions between secreted immune proteins called chemokines and their cognate G protein-coupled receptors regulate the trafficking of leukocytes in inflammatory responses. The two-site, two-step model describes these interactions. It involves initial binding of the chemokine N-loop/β3 region to the receptor's N-terminal region and subsequent insertion of the chemokine N-terminal region into the transmembrane helical bundle of the receptor concurrent with receptor activation. Here, we test aspects of this model with C-C motif chemokine receptor 1 (CCR1) and several chemokine ligands. First, we compared the chemokine-binding affinities of CCR1 with those of peptides corresponding to the CCR1 N-terminal region. Relatively low affinities of the peptides and poor correlations between CCR1 and peptide affinities indicated that other regions of the receptor may contribute to binding affinity. Second, we evaluated the contributions of the two CCR1-interacting regions of the cognate chemokine ligand CCL7 (formerly monocyte chemoattractant protein-3 (MCP-3)) using chimeras between CCL7 and the non-cognate ligand CCL2 (formerly MCP-1). The results revealed that the chemokine N-terminal region contributes significantly to binding affinity but that differences in binding affinity do not completely account for differences in receptor activation. On the basis of these observations, we propose an elaboration of the two-site, two-step model-the "three-step" model-in which initial interactions of the first site result in low-affinity, nonspecific binding; rate-limiting engagement of the second site enables high-affinity, specific binding; and subsequent conformational rearrangement gives rise to receptor activation.

Receptor crosstalk improves concentration sensing of multiple ligands.

Cells need to reliably sense external ligand concentrations to achieve various biological functions such as chemotaxis or signaling. The molecular recognition of ligands by surface receptors is degenerate in many systems, leading to crosstalk between ligand-receptor pairs. Crosstalk is often thought of as a deviation from optimal specific recognition, as the binding of noncognate ligands can interfere with the detection of the receptor's cognate ligand, possibly leading to a false triggering of a downstream signaling pathway. Here we quantify the optimal precision of sensing the concentrations of multiple ligands by a collection of promiscuous receptors. We demonstrate that crosstalk can improve precision in concentration sensing and discrimination tasks. To achieve superior precision, the additional information about ligand concentrations contained in short binding events of the noncognate ligand should be exploited. We present a proofreading scheme to realize an approximate estimation of multiple ligand concentrations that reaches a precision close to the derived optimal bounds. Our results help rationalize the observed ubiquity of receptor crosstalk in molecular sensing.

Protein-protein binding supersites.

The lack of a deep understanding of how proteins interact remains an important roadblock in advancing efforts to identify binding partners and uncover the corresponding regulatory mechanisms of the functions they mediate. Understanding protein-protein interactions is also essential for designing specific chemical modifications to develop new reagents and therapeutics. We explored the hypothesis of whether protein interaction sites serve as generic biding sites for non-cognate protein ligands, just as it has been observed for small-molecule-binding sites in the past. Using extensive computational docking experiments on a test set of 241 protein complexes, we found that indeed there is a strong preference for non-cognate ligands to bind to the cognate binding site of a receptor. This observation appears to be robust to variations in docking programs, types of non-cognate protein probes, sizes of binding patches, relative sizes of binding patches and full-length proteins, and the exploration of obligate and non-obligate complexes. The accuracy of the docking scoring function appears to play a role in defining the correct site. The frequency of interaction of unrelated probes recognizing the binding interface was utilized in a simple prediction algorithm that showed accuracy competitive with other state of the art methods.

The Limited Information Capacity of Cross-Reactive Sensors Drives the Evolutionary Expansion of Signaling.

Signaling systems expand by duplications of various components, be it receptors or downstream effectors. However, whether and how duplicated components contribute to higher signaling capacity is unclear, especially because in most cases, their specificities overlap. Using information theory, we found that augmentation of capacity by an increase in the copy number is strongly limited by logarithmic diminishing returns. Moreover, counter to conventional biochemical wisdom, refinements of the response mechanism, e.g., by cooperativity or allostery, do not increase the overall signaling capacity. However, signaling capacity nearly doubles when a promiscuous, non-cognate ligand becomes explicitly recognized via duplication and partial divergence of signaling components. Our findings suggest that expansion of signaling components via duplication and enlistment of promiscuously acting cues is virtually the only accessible evolutionary strategy to achieve overall high-signaling capacity despite overlapping specificities and molecular noise. This mode of expansion also explains the highly cross-wired architecture of signaling pathways.