Showing papers on "Cooperativity published in 2022"

Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.

Abstract: Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Condensates formed by prion-like low-complexity domains have small-world network structures and interfaces defined by expanded conformations

Abstract: Abstract Biomolecular condensates form via coupled associative and segregative phase transitions of multivalent associative macromolecules. Phase separation coupled to percolation is one example of such transitions. Here, we characterize molecular and mesoscale structural descriptions of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). These systems conform to sticker-and-spacers architectures. Stickers are cohesive motifs that drive associative interactions through reversible crosslinking and spacers affect the cooperativity of crosslinking and overall macromolecular solubility. Our computations reproduce experimentally measured sequence-specific phase behaviors of PLCDs. Within simulated condensates, networks of reversible inter-sticker crosslinks organize PLCDs into small-world topologies. The overall dimensions of PLCDs vary with spatial location, being most expanded at and preferring to be oriented perpendicular to the interface. Our results demonstrate that even simple condensates with one type of macromolecule feature inhomogeneous spatial organizations of molecules and interfacial features that likely prime them for biochemical activity.

Cooperative approaches in catalytic hydrogenation and dehydrogenation.

Abstract: Metal-ligand cooperativity (MLC) is an established strategy for developing effective hydrogenation and dehydrogenation catalysts. Metal-metal cooperativity (MMC) in bimetallic complexes is not as well understood, and to date has had limited implementation in (de)hydrogenation. Herein we use (de)hydrogenation processes as a platform to examine modes of cooperativity, with a particular focus on catalytic mechanisms. We investigate how lessons learnt from the extensive development of metal-ligand cooperative catalysts can aid the ongoing development of metal-metal cooperative catalysts.

Cooperativity of steric bulk and H-bonding in coordination sphere engineering: heteroleptic PdII cages and bowls by design

Abstract: Recently developed self-assembly strategies allow to rationally reduce the symmetry of metallosupramolecular architectures. In addition, the combination of multiple ligand types without creating compound mixtures has become possible. Among several approaches to realize non-statistical heteroleptic assembly, Coordination Sphere Engineering (CSE) makes use of secondary repulsive or attractive interactions in direct vicinity of the metal nodes. Previously, we used steric congestion to turn dinuclear [Pd2L4] cages with fourfold symmetry into [Pd2L3X2] (X = solvent, halide) bowl structures. Here, we introduce a new subtype of this strategy based on balancing hydrogen bonding and repulsive interactions between ligands carrying quinoline (LQu) and 1,8-naphthyridine (LNa) donors to generate trans-[Pd2L2] and [Pd2L3L′] cages, assisted by templation of encapsulated fullerenes. Combined with steric congestion caused by acridine (LAc) donors, we further report the first example of a heteroleptic [Pd2L2L′X2] bowl. Formation, structure and fullerene binding ability of these metallo-supramolecular hosts were studied by NMR, mass spectrometry and single crystal X-ray diffraction.

SHAPE-enabled fragment-based ligand discovery for RNA

Abstract: Significance RNA molecules encode proteins and play numerous regulatory roles in cells. Targeting RNA with small molecules, as is routine with proteins, would create broad opportunities for modulating biology and creating new drugs. However, this opportunity has been difficult to realize because creating novel small molecules that bind RNA, especially using modest resources, is challenging. This study integrates two widely used technologies, SHAPE chemical probing of RNA and fragment-based ligand discovery, to craft an innovative strategy for creating small molecules that bind to and modulate the activity of a structured RNA. The anticipated impact is high because the methods are simple, can be implemented in diverse research and discovery contexts, and lead to realistic druglike molecules.

Subsite Ligand Recognition and Cooperativity in the TPP Riboswitch: Implications for Fragment-Linking in RNA Ligand Discovery.

Abstract: RNA molecules can show high levels of cooperativity in their global folding and interactions with divalent ions. However, cooperativity at individual ligand-RNA interaction sites remains poorly understood. Here, we investigated the binding of thiamine and methylene diphosphonic acid (MDP, a soluble structural analogue of pyrophosphate) to the thiamine pyrophosphate riboswitch. These ligands each bind weakly at proximal subsites, with 10 μM and 1 mM affinities, respectively. The affinity of MDP moderately improves when thiamine or thiamine-like fragments are pre-bound to the RNA. Covalent linking of thiamine and MDP substantially increases riboswitch binding to a notable high affinity of 20 nM. Crystal structures and single-molecule correlated chemical probing revealed favorable induced fit effects upon binding of individual ligands and, unexpectedly, a substantial thermodynamically unfavorable RNA structural rearrangement upon binding of the linked thiamine-MDP ligand. Thus, linking of two ligands of modest affinity, accompanied by an unfavorable structural rearrangement, still yields a potent linked RNA-binding compound. Since complex ligands often bind riboswitches and other RNAs at proximal subsites, principles derived from this work inform and support fragment-linking strategies for identifying small molecules that interact with RNA specifically and with high affinity.

Principles of Cation-π Interactions for Engineering Mussel-Inspired Functional Materials.

Abstract: ConspectusSupramolecular assembly is commonly driven by noncovalent interactions (e.g., hydrogen bonding, electrostatic, hydrophobic, and aromatic interactions) and plays a predominant role in multidisciplinary research areas ranging from materials design to molecular biology. Understanding these noncovalent interactions at the molecular level is important for studying and designing supramolecular assemblies in chemical and biological systems. Cation-π interactions, initially found through their influence on protein structure, are generally formed between electron-rich π systems and cations (mainly alkali, alkaline-earth metals, and ammonium). Cation-π interactions play an essential role in many biological systems and processes, such as potassium channels, nicotinic acetylcholine receptors, biomolecular recognition and assembly, and the stabilization and function of biomacromolecular structures. Early fundamental studies on cation-π interactions primarily focused on computational calculations, protein crystal structures, and gas- and solid-phase experiments. With the more recent development of spectroscopic and nanomechanical techniques, cation-π interactions can be characterized directly in aqueous media, offering opportunities for the rational manipulation and incorporation of cation-π interactions into the design of supramolecular assemblies. In 2012, we reported the essential role of cation-π interactions in the strong underwater adhesion of Asian green mussel foot proteins deficient in l-3,4-dihydroxyphenylalanine (DOPA) via direct molecular force measurements. In another study in 2013, we reported the experimental quantification and nanomechanics of cation-π interactions of various cations and π electron systems in aqueous solutions using a surface forces apparatus (SFA).Over the past decade, much progress has been achieved in probing cation-π interactions in aqueous solutions, their impact on the underwater adhesion and cohesion of different soft materials, and the fabrication of functional materials driven by cation-π interactions, including surface coatings, complex coacervates, and hydrogels. These studies have demonstrated cation-π interactions as an important driving force for engineering functional materials. Nevertheless, compared to other noncovalent interactions, cation-π interactions are relatively less investigated and underappreciated in governing the structure and function of supramolecular assemblies. Therefore, it is imperative to provide a detailed overview of recent advances in understanding of cation-π interactions for supramolecular assembly, and how these interactions can be used to direct supramolecular assembly for various applications (e.g., underwater adhesion). In this Account, we present very recent advances in probing and applying cation-π interactions for mussel-inspired supramolecular assemblies as well as their structural and functional characteristics. Particular attention is paid to experimental characterization techniques for quantifying cation-π interactions in aqueous solutions. Moreover, the parameters responsible for modulating the strengths of cation-π interactions are discussed. This Account provides useful insights into the design and engineering of smart materials based on cation-π interactions.

Janus Cross-links in Supramolecular Networks.

Abstract: Thermosets composed of cross-linked polymers demonstrate enhanced thermal, solvent, chemical, and dimensional stability as compared to their non-cross-linked counterparts. However, these often-desirable material properties typically come at the expense of reprocessability, recyclability, and healability. One solution to this challenge comes from the construction of polymers that are reversibly cross-linked. We relied on lessons from Nature to present supramolecular polymer networks comprised of cooperative Janus-faced hydrogen bonded cross-links. A triazine-based guanine-cytosine base (GCB) with two complementary faces capable of self-assembly through three hydrogen bonding sites was incorporated into poly(butyl acrylate) to create a reprocessable and recyclable network. Rheological experiments and dynamic mechanical analysis (DMA) were employed to investigate the flow behavior of copolymers with randomly distributed GCB units of varying incorporation. Our studies revealed that the cooperativity of multiple hydrogen bonding faces yields excellent network integrity evidenced by a rubbery plateau that spanned the widest temperature range yet reported for any supramolecular network. To verify that each Janus-faced motif engages in multiple cross-links, we studied the effects of local concentration of the incorporated GCB units within the polymer chain. Mechanical strength improved by colocalizing the GCB within a block copolymer morphology. This enhanced performance revealed that the number of effective cross-links in the network increased with the local concentration of hydrogen bonding units. Overall, this study demonstrates that cooperative noncovalent interactions introduced through Janus-faced hydrogen bonding moieties confers excellent network stability and predictable viscoelastic flow behavior in supramolecular networks.

Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors

Abstract: Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.

Heterogeneity in Protein Folding and Unfolding Reactions.

Abstract: Proteins have dynamic structures that undergo chain motions on time scales spanning from picoseconds to seconds. Resolving the resultant conformational heterogeneity is essential for gaining accurate insight into fundamental mechanistic aspects of the protein folding reaction. The use of high-resolution structural probes, sensitive to population distributions, has begun to enable the resolution of site-specific conformational heterogeneity at different stages of the folding reaction. Different states populated during protein folding, including the unfolded state, collapsed intermediate states, and even the native state, are found to possess significant conformational heterogeneity. Heterogeneity in protein folding and unfolding reactions originates from the reduced cooperativity of various kinds of physicochemical interactions between various structural elements of a protein, and between a protein and solvent. Heterogeneity may arise because of functional or evolutionary constraints. Conformational substates within the unfolded state and the collapsed intermediates that exchange at rates slower than the subsequent folding steps give rise to heterogeneity on the protein folding pathways. Multiple folding pathways are likely to represent distinct sequences of structure formation. Insight into the nature of the energy barriers separating different conformational states populated during (un)folding can also be obtained by resolving heterogeneity.

Combining metal–metal cooperativity, metal–ligand cooperativity and chemical non-innocence in diiron carbonyl complexes

Abstract: Several metalloenzymes, including [FeFe]-hydrogenase, employ cofactors wherein multiple metal atoms work together with surrounding ligands that mediate heterolytic and concerted proton-electron transfer (CPET) bond activation steps. Herein, we report a new dinucleating PNNP expanded pincer ligand, which can bind two low-valent iron atoms in close proximity to enable metal-metal cooperativity (MMC). In addition, reversible partial dearomatization of the ligand's naphthyridine core enables both heterolytic metal-ligand cooperativity (MLC) and chemical non-innocence through CPET steps. Thermochemical and computational studies show how a change in ligand binding mode can lower the bond dissociation free energy of ligand C(sp3)-H bonds by ∼25 kcal mol-1. H-atom abstraction enabled trapping of an unstable intermediate, which undergoes facile loss of two carbonyl ligands to form an unusual paramagnetic (S = ) complex containing a mixed-valent iron(0)-iron(i) core bound within a partially dearomatized PNNP ligand. Finally, cyclic voltammetry experiments showed that these diiron complexes show catalytic activity for the electrochemical hydrogen evolution reaction. This work presents the first example of a ligand system that enables MMC, heterolytic MLC and chemical non-innocence, thereby providing important insights and opportunities for the development of bimetallic systems that exploit these features to enable new (catalytic) reactivity.

Mechanistic Aspects of Cobalt-Oxo Cubane Clusters in Oxidation Chemistry.

Abstract: Cobalt oxides are recognized as one of the most efficient earth-abundant catalysts for challenging oxidation chemistry, with substrates ranging from water to organic compounds. In these oxidations, cobalt-oxo species with formal oxidation states greater than 3 are commonly invoked as reactive intermediates. However, there is a dearth of mechanistic information regarding how these high-valent cobalt catalysts operate. This Perspective describes how the study of molecular cobalt oxo clusters, with an emphasis on [Co4O4] oxo cubane complexes, has helped to shed light on the operative mechanisms of cobalt-catalyzed oxidation reactions. Implications for high-valent CoIV-oxo and CoV-oxo intermediates and remaining mechanistic questions concerning how these intermediates mediate O-O bond formation are also discussed. Furthermore, structural modifications of these oxo cubane clusters (i.e., incorporation of heteroatoms and modulation of ligands) have provided insight into multimetallic cooperativity, but the influence of such metal-metal interactions on oxidation activity remains to be explored. A more detailed understanding of these structure-activity relationships may enable fine-tuning of reactivity and stability of synthetic multimetallic catalysts for energy storage and challenging organic transformations.

Catalytic Synergies in Bimetallic RuPt Single–Atom Catalysts via Speciation Control

Abstract: Bimetallic single–atom catalysts (b–SACs) have recently gained prominence by virtue of the unique catalytic cooperative interactions they can exhibit, intertwining electronic and geometric effects. To date, research efforts have exclusively focused on direct mechanisms such as electron density transfer or sequential reactivity. Herein, the first study on indirect, coordination–induced catalytic synergies in carbon‐supported RuPt SACs is conducted. To this end, a holistic approach is developed, combining i) precision synthesis, ii) advanced characterization, iii) exploration of single–site adsorption properties via the hydrogen evolution reaction, and iv) modeling through density functional theory. Despite the lack of both intermetallic coordination in the first or second shell and charge redistribution effects, the RuPt SACs exhibit a H2 formation rate enhanced up to 15–fold compared with their monometallic counterparts. To unfold the origin of the intermetallic cooperativity, modifications of the structural and catalytic properties induced by the integration of a second metal species are investigated. Thus, Pt atoms are found to selectively occupy the most energeticallyfavorable cavities in the support, prompting Ru atoms to assume a distinct, more active, configuration. This contribution unveils a novel principle of bimetallic cooperativity, demonstrating the key role of integrative experimental and computational analyses in studying b–SACs.

Developmental disorders caused by haploinsufficiency of transcriptional regulators: a perspective based on cell fate determination

Abstract: ABSTRACT Many human birth defects and neurodevelopmental disorders are caused by loss-of-function mutations in a single copy of transcription factor (TF) and chromatin regulator genes. Although this dosage sensitivity has long been known, how and why haploinsufficiency (HI) of transcriptional regulators leads to developmental disorders (DDs) is unclear. Here I propose the hypothesis that such DDs result from defects in cell fate determination that are based on disrupted bistability in the underlying gene regulatory network (GRN). Bistability, a crucial systems biology concept to model binary choices such as cell fate decisions, requires both positive feedback and ultrasensitivity, the latter often achieved through TF cooperativity. The hypothesis explains why dosage sensitivity of transcriptional regulators is an inherent property of fate decisions, and why disruption of either positive feedback or cooperativity in the underlying GRN is sufficient to cause disease. I present empirical and theoretical evidence in support of this hypothesis and discuss several issues for which it increases our understanding of disease, such as incomplete penetrance. The proposed framework provides a mechanistic, systems-level explanation of HI of transcriptional regulators, thus unifying existing theories, and offers new insights into outstanding issues of human disease. This article has an associated Future Leader to Watch interview with the author of the paper.

Influence of the spatial distribution of copper sites on the selectivity of the oxygen reduction reaction

Abstract: Moving towards a hydrogen economy raises the demand for affordable and efficient catalysts for the oxygen reduction reaction. Cu-bmpa (bmpa = bis(2-picolyl)amine) is shown to have moderate activity, but poor selectivity for the 4-electron reduction of oxygen to water. To enhance the selectivity towards water formation, the cooperative effect of three Cu-bmpa binding sites in a single trinuclear complex is investigated. The catalytic currents in the presence of the trinuclear sites are lower, possibly due to the more rigid structure and therefore higher reorganization energies and/or slower diffusion rates of the catalytic species. Although the oxygen reduction activity of the trinuclear complexes is lower than that of mononuclear Cu-bmpa, the selectivity of the copper mediated oxygen reduction was significantly enhanced towards the 4-electron process due to a cooperative effect between three copper centers that have been positioned in close proximity. These results indicate that the cooperativity between metal ions within biomimetic sites can greatly enhance the ORR selectivity.

Characterization of Type I and II Interactions between Halogen Atoms

Abstract: The noncovalent interactions between pairs of halogen (X) atoms on separate molecules are commonly categorized as Type I or II (T1 or T2). T2 contains a standard halogen bond where the two molecules are roughly perpendicular to one another, while the two units in T1 are nearly antiparallel. Quantum calculations reveal that the T2 geometry is more stable than T1 for FX pairs (X = Cl, Br, I) but only by a small amount. In mixed heterodimers FX1···X2F, there is a preference for the heavier X atom to serve as an electron acceptor. The T1 conformation is bound by a pair of bent halogen bonds, along with some positive cooperativity between them. The internal FX bond is stretched within the acidic subunit and contracted in the base, accompanied respectively by red and blue shifts of the FX stretching frequency. The NMR chemical shielding of the X atoms is increased by the formation of each complex, with a smaller deshielding observed on the F atoms. These changes rise rapidly in the order Cl < Br < I.

Assessment of Hydrogen Bond Strengths and Cooperativity in Self- and Cross-Associating Cyclic (HF)m(H2O)n, (m + n) = 2 to 8 Clusters

Abstract: In this work, we investigate the strength of various self- and cross-associating hydrogen bonds (HBs) in mixed hydrogen fluoride-water cyclic (HF)m(H2O)n, (m + n = 2 to 8) clusters, employing...

Cooperativity as quantification and optimization paradigm for nuclear receptor modulators

Abstract: Nuclear Receptors (NRs) are highly relevant drug targets, for which small molecule modulation goes beyond a simple ligand/receptor interaction. NR-ligands modulate Protein-Protein Interactions (PPIs) with coregulator proteins. Here we bring forward a cooperativity mechanism for small molecule modulation of NR PPIs, using the Peroxisome Proliferator Activated Receptor γ (PPARγ), which describes NR-ligands as allosteric molecular glues. The cooperativity framework uses a thermodynamic model based on three-body binding events, to dissect and quantify reciprocal effects of NR-coregulator binding (KID) and NR-ligand binding (KIID), jointly recapitulated in the cooperativity factor (α) for each specific ternary ligand·NR·coregulator complex formation. These fundamental thermodynamic parameters allow for a conceptually new way of thinking about structure-activity-relationships for NR-ligands and can steer NR modulator discovery and optimization via a completely novel approach.

Supramolecular polymerization of electronically complementary linear motifs: anti-cooperativity by attenuated growth

Abstract: Anti-cooperative supramolecular polymerization by attenuated growth exhibited by self-assembling units of two electron-donor benzo[1,2-b:4,5-b']dithiophene (BDT) derivatives (compounds 1a and 1b) and the electron-acceptor 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) (compound 2) is reported. Despite the apparent cooperative mechanism of 1 and 2, AFM imaging and SAXS measurements reveal the formation of small aggregates that suggest the operation of an anti-cooperative mechanism strongly conditioned by an attenuated growth. In this mechanism, the formation of the nuclei is favoured over the subsequent addition of monomeric units to the aggregate, which finally results in short aggregates. Theoretical calculations show that both the BDT and BODIPY motifs, after forming the initial dimeric nuclei, experience a strong distortion of the central aromatic backbone upon growth, which makes the addition of successive monomeric units unfavourable and impedes the formation of long fibrillar structures. Despite the anti-cooperativity observed in the supramolecular polymerization of 1 and 2, the combination of both self-assembling units results in the formation of small co-assembled aggregates with a similar supramolecular polymerization behaviour to that observed for the separate components.

Supramolecular polymerization of electronically complementary linear motifs: anti-cooperativity by attenuated growth

Abstract: Anti-cooperative supramolecular polymerization by attenuated growth exhibited by self-assembling units of two electron-donor benzo[1,2-b:4,5-b′]dithiophene (BDT) derivatives (compounds 1a and 1b) and the electron-acceptor 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) (compound 2) is reported. Despite the apparent cooperative mechanism of 1 and 2, AFM imaging and SAXS measurements reveal the formation of small aggregates that suggest the operation of an anti-cooperative mechanism strongly conditioned by an attenuated growth. In this mechanism, the formation of the nuclei is favoured over the subsequent addition of monomeric units to the aggregate, which finally results in short aggregates. Theoretical calculations show that both the BDT and BODIPY motifs, after forming the initial dimeric nuclei, experience a strong distortion of the central aromatic backbone upon growth, which makes the addition of successive monomeric units unfavourable and impedes the formation of long fibrillar structures. Despite the anti-cooperativity observed in the supramolecular polymerization of 1 and 2, the combination of both self-assembling units results in the formation of small co-assembled aggregates with a similar supramolecular polymerization behaviour to that observed for the separate components.

TF-COMB – Discovering grammar of transcription factor binding sites

Abstract: Cooperativity between transcription factors is important to regulate target gene expression. In particular, the binding grammar of TFs in relation to each other, as well as in the context of other genomic elements, is crucial for TF functionality. However, tools to easily uncover co-occurrence between DNA-binding proteins, and investigate the regulatory modules of TFs, are limited. Here we present TF-COMB (Transcription Factor Co-Occurrence using Market Basket analysis) - a tool to investigate co-occurring TFs and binding grammar within regulatory regions. We found that TF-COMB can accurately identify known co-occurring TFs from ChIP-seq data, as well as uncover preferential localization to other genomic elements. With the use of ATAC-seq footprinting and TF motif locations, we found that TFs exhibit both preferred orientation and distance in relation to each other, and that these are biologically significant. Finally, we extended the analysis to not only investigate individual TF pairs, but also TF pairs in the context of networks, which enabled the investigation of TF complexes and TF hubs. In conclusion, TF-COMB is a flexible tool to investigate various aspects of TF binding grammar.

Cooperative binding of T cell receptor and CD4 to peptide-MHC enhances antigen sensitivity

Abstract: Antigen recognition by the T cell receptor (TCR) of CD4+ T cells can be greatly enhanced by the coreceptor CD4. Yet, understanding of the molecular mechanism is hindered by the ultra-low affinity of CD4 binding to class-II peptide-major histocompatibility complexes (pMHC). Here we show, using two-dimensional (2D) mechanical-based assays, that the affinity of CD4-pMHC interaction is 3-4 logs lower than that of cognate TCR-pMHC interactions, and it is more susceptible to increased dissociation by forces (slip bond). In contrast, CD4 binds TCR-pre-bound pMHC at 3-6 logs higher affinity, forming TCR-pMHC-CD4 tri-molecular bonds that are prolonged by force (catch bond), and modulated by protein mobility on the cell membrane, indicating profound TCR-CD4 cooperativity. Consistent with a tri-crystal structure, using DNA origami as a molecular ruler to titrate spacing between TCR and CD4 we show that 7-nm proximity optimizes TCR-pMHC-CD4 tri-molecular bond formation with pMHC. Our results thus provide deep mechanistic insight into CD4 enhancement of TCR antigen recognition.