Transient Interactions Research Articles

Insights into the regulation of CHIP E3 ligase-mediated ubiquitination of neuronal protein BNIP-H

Abstract BCL2/adenovirus E1B 19-kDa protein-interacting protein 2 homolog (BNIP-H or Caytaxin), pivotal adaptor protein that facilitates cerebellar cortex growth and synaptic transmission, is post-translationally modified to regulate neuronal function. This study reports the ubiquitination of BNIP-H by CHIP (Carboxyl terminus of Hsc70-Interacting Protein), a U-box containing E3 ligase that is also regulated via autoubiquitination. Specifically, it was observed that CHIP autoubiquitinated itself primarily at Lys23 and Lys31 in-vitro. Mutation of these residues show the autoubiquitination of successive lysines of CHIP. In total, nine lysines on CHIP were identified as the autoubiquitination sites, the collective mutation of which completely terminated its autoubiquitination. Additionally, CHIP-mediated ubiquitination of BNIP-H is completely inhibited when BNIP-H bears arginine mutations at four key lysine residues. Next, using hydrogen deuterium exchange mass spectrometry a model of a plausible mechanism was proposed. The model suggests transient N-terminal interactions between the CHIP and BNIP-H which allows for the swinging of U-Box domain of CHIP to ubiquitinate BNIP-H. Following complex dissociation, BNIP-H population is regulated via the ubiquitin-proteasome pathway. Collectively, these results aid in our understanding of CHIP-mediated BNIP-H ubiquitination and provide further insight into the roles of these proteins in neuritogenesis and neurotransmission.

Research on unsteady flow characteristics of turbocharger turbine stage with coupling inlet and exhaust casings

The inlet and exhaust casings of marine turbochargers are closely coupled to the axial flow turbine. These are crucial parts that work with the axial flow turbine and have the ability to raise the power plant’s output power even higher. Their final stage of the axial flow turbine and the exhaust casing’s complex flow due to their close relationship will have a significant effect on each other’s aerodynamic performance. However, most existing studies have not paid sufficient attention to this coupled transient interaction. In this paper, the flow interaction between the axial flow turbine and the inlet and exhaust casings is studied. Particular attention is paid to the study of the coupled flow characteristics under design and variable operating conditions. This study is carried out using coupled computational fluid dynamics (CFD) simulation. The computational domain consists of an inlet casing, 28 stator blades, 43 rotor blades, and an exhaust casing. The commercial CFD software ANSYS CFX solver and the SST turbulence model are used to simulate the unsteady flow field of the turbocharger axial turbine coupled to the inlet and exhaust casings. In this paper, the turbine flow field and the inlet and exhaust casings flow fields are analyzed, and the influence of the exhaust casing on rotor surface pressure fluctuation is discussed. In addition, the flow field distribution and performance parameters of the axial flow turbine coupled with the inlet and exhaust casings under three different operating conditions have been calculated and analyzed. The results show that the inlet casing affects the circumferential distribution of the stator leading edge pressure, while the asymmetric back pressure caused by the exhaust casing flow field causes the low-frequency pressure fluctuation at the rotor blade surface. At the trailing edge of the suction surface, the complex flow separation and vortex structure in the exhaust casing are the main factors causing rotor-stator interaction. The low-frequency fluctuation amplitude caused by the exhaust casing is greater than that caused by the high-frequency fluctuation of the rotor-stator interaction. Moreover, as the flow coefficient increases, the low-frequency fluctuation amplitude is much greater than the high-frequency fluctuation amplitude.

Interaction with the cysteine-free protein HAX1 expands the substrate specificity and function of MIA40 beyond protein oxidation.

The mitochondrial disulphide relay machinery is essential for the import and oxidative folding of many proteins in the mitochondrial intermembrane space. Its core component, the import receptor MIA40 (also CHCHD4), serves as an oxidoreductase but also as a chaperone holdase, which initially interacts with its substrates non-covalently before introducing disulphide bonds for folding and retaining proteins in the intermembrane space. Interactome studies have identified diverse substrates of MIA40, among them the intrinsically disordered HCLS1-associated protein X-1 (HAX1). Interestingly, this protein does not contain cysteines, raising the question of how and to what end HAX1 can interact with MIA40. Here, we demonstrate that MIA40 non-covalently interacts with HAX1 independent of its redox-active cysteines. While HAX1 import is driven by its weak mitochondrial targeting sequence, its subsequent transient interaction with MIA40 stabilizes the protein in the intermembrane space. HAX1 solely depends on the holdase activity of MIA40, and the absence of MIA40 results in the aggregation, degradation and loss of HAX1. Collectively, our study introduces HAX1 as the first endogenous MIA40 substrate without cysteines and demonstrates the diverse functions of this highly conserved oxidoreductase and import receptor.

Evolution of antiviral resistance captures a transient interdomain functional interaction between chikungunya virus envelope glycoproteins.

CHIKV is a reemergent pathogen that has caused large outbreaks in the twenty years. There are no available antiviral therapies and a vaccine has only recently been approved. Here, we describe the mode of action of a novel inhibitor designed against CHIKV envelope proteins heterodimer that blocks entry at the stage of fusion between virus and host membranes. Fusion is common to the entry of enveloped viruses. Virus envelope proteins drive fusion undergoing a series of transitions from an initial metastable conformational state to a more stable post-fusion state. Intermediate conformations are transient and have mostly remained inaccessible to structure determination. In this study, directed evolution of resistance to antiviral inhibition of fusion uncovered a functional interaction between two residues residing in domains that are apart in both the pre-fusion and post-fusion states. Thus, our approach allowed gaining insight into the molecular detail of the inner working of virus fusion machinery.

Pseudospins revealed through the giant dynamical Franz-Keldysh effect in massless Dirac materials

The dynamical Franz-Keldysh effect, indicative of the transient light-matter interaction regime between quantum and classical realms, is widely recognized as an essential signature in wide bandgap condensed matter systems such as dielectrics. In this theoretical study, we applied time-resolved transient absorption spectroscopy to investigate ultrafast optical responses in graphene, a zero-bandgap system. We observed in the gate-tuned graphene that the massless Dirac materials notably enhance intraband light-driven transitions, significantly leading to the giant dynamical Franz-Keldysh effect compared to the massive Dirac materials, a wide bandgap system. In addition, employing the angle-resolved spectroscopy, it is found that the unique polarimetry orientation, i.e., perpendicular polarizations for the pump and the probe, further pronounces the optical spectra to exhibit the complete fishbone structure, reflecting quantum pseudospin natures of Dirac cones. Our findings expand the establishment of emergent transient spectroscopy frameworks into not only zero-bandgap systems but also pseudospin-mediated quantum phenomena, moving beyond dielectrics.

The structures of protein kinase A in complex with CFTR: Mechanisms of phosphorylation and noncatalytic activation

Protein kinase A (PKA) is a key regulator of cellular functions by selectively phosphorylating numerous substrates, including ion channels, enzymes, and transcription factors. It has long served as a model system for understanding the eukaryotic kinases. Using cryoelectron microscopy, we present complex structures of the PKA catalytic subunit (PKA-C) bound to a full-length protein substrate, the cystic fibrosis transmembrane conductance regulator (CFTR)-an ion channel vital to human health. CFTR gating requires phosphorylation of its regulatory (R) domain. Unphosphorylated CFTR engages PKA-C at two locations, establishing two "catalytic stations" near to, but not directly involving, the R domain. This configuration, coupled with the conformational flexibility of the R domain, permits transient interactions of the eleven spatially separated phosphorylation sites. Furthermore, we determined two structures of the open-pore CFTR stabilized by PKA-C, providing a molecular basis to understand how PKA-C stimulates CFTR currents even in the absence of phosphorylation.

Revisiting Aspergillus terreus MTCC6324 recombinant alcohol oxidase (rAOx): enhanced in-silico insight into structure, function, and substrate sequestration mechanism

Alcohol oxidase (AOx) enzymes have gained significant attention for their potential in industrial applications due to their unique ability to catalyze irreversible oxidation of diverse alcohol substrates without external co-factors. This study revisits and enhances in-silico work on Aspergillus terreus MTCC6324 recombinant AOx (rAOx) enzyme, combining artificial intelligence (AlphaFold), molecular docking (AutoDock Vina) techniques and Molecular dynamics (MD) simulations (Desmond). Comprehensive sequence analysis revealed a high degree of conservation among 23 AOx amino acid sequences from various Aspergillus species highlights conserved regions, affirming its GXGXXG Rossmann fold motif. AlphaFold-predicted 3D structure of rAOx demonstrated improved stereo-chemical stability compared to I-TASSER predicted structure with 87.6% amino acid residues in most favourable region of Ramachandran plot compared to 79.5%, respectively. Molecular docking revealed the binding affinities of co-factors FAD and diverse alcohol substrates, with cinnamyl alcohol exhibiting robust interaction with rAOx holoenzyme. MD simulations further elucidate the stability and dynamics of rAOx-FAD-cinnamyl alcohol complex over 100 nanoseconds. The simulations showcase FAD’s stable binding within the protein core and highlights transient substrate interactions, dissociating within the active site after 75 ns suggesting a substrate sequestration mechanism. The study unveils substrate sequestration mechanism wherein cinnamyl alcohol exhibits temporary binding, leading to quick detachment from active site, mimicking reported exponential kinetics. This study not only validates previous findings but also offers a comprehensive understanding of intricate dynamics governing rAOx enzymatic activity. The improved sequence-to-structure prediction and detailed molecular insights into substrate sequestration provide a valuable foundation for future experimental investigations and rational design of bio-catalytic processes.

Structural and Functional Relevance of Charge Based Transient Interactions inside Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) perform a wide range of biological functions without adopting stable, well-defined, three-dimensional structures. Instead, IDPs exist as dynamic ensembles of flexible conformations, traditionally thought to be governed by weak, nonspecific interactions, which are well described by homopolymer theory. However, recent research highlights the presence of transient, specific interactions in several IDPs, suggesting that factors beyond overall size influence their conformational behavior. In this study, we investigate how the spatial arrangement of charged amino acids within IDP sequences shapes the prevalence of transient, specific interactions. Through a series of model peptides, we establish a quantitative empirical relationship between the fraction of transient interactions and a novel sequence metric, termed effective charged patch length, which characterizes the ability of charged patches to drive these interactions. By examining IDP ensembles with varying levels of transient interactions, we further explore their heteropolymeric structural behavior in phase-separated condensates, where we observe the formation of a condensate-spanning network structure. Additionally, we perform a proteome-wide scan for charge-based transient interactions within disordered regions of the human proteome, revealing that approximately 10% of these regions exhibit such charge-driven transient interactions, leading to heteropolymeric behaviors in their conformational ensembles. Finally, we examine how these charge-based transient interactions correlate with molecular functions, identifying specific biological roles in which these interactions are enriched.

AlphaFold opens the doors to deorphanizing secreted proteins

Danneskiold-Samsøe and coworkers1 have developed an in silico screening pipeline based on AlphaFold2 for identifying single-pass transmembrane receptors for secreted peptides that play important roles in cell-cell signaling. Their approach can be used to deorphanize a diverse range of ligands. The overall strategy can be valuable in screening for weak and transient interactions.

A quantitative intracellular peptide binding assay reveals recognition determinants and context dependence of short linear motifs.

Transient protein-protein interactions play key roles in controlling dynamic cellular responses. Many examples involve globular protein domains that bind to peptide sequences known as Short Linear Motifs (SLiMs), which are enriched in intrinsically disordered regions of proteins. Here we describe a novel functional assay for measuring SLiM binding, called Systematic Intracellular Motif Binding Analysis (SIMBA). In this method, binding of a foreign globular domain to its cognate SLiM peptide allows yeast cells to proliferate by blocking a growth arrest signal. A high-throughput application of the SIMBA method involving competitive growth and deep sequencing provides rapid quantification of the relative binding strength for thousands of SLiM sequence variants, and a comprehensive interrogation of SLiM sequence features that control their recognition and potency. We show that multiple distinct classes of SLiM-binding domains can be analyzed by this method, and that the relative binding strength of peptides in vivo correlates with their biochemical affinities measured in vitro. Deep mutational scanning provides high-resolution definitions of motif recognition determinants and reveals how sequence variations at non-core positions can modulate binding strength. Furthermore, mutational scanning of multiple parent peptides that bind human tankyrase ARC or YAP WW domains identifies distinct binding modes and uncovers context effects in which the preferred residues at one position depend on residues elsewhere. The findings establish SIMBA as a fast and incisive approach for interrogating SLiM recognition via massively parallel quantification of protein-peptide binding strength in vivo.

A three-way interface of the Nipah virus phosphoprotein X-domain coordinates polymerase movement along the viral genome.

Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors.

Controlled Assembly of Lipid Molecules via Regulating Transient Spatial Confinement

The constructs of lipid molecules follow self-assembly, driven by intermolecular interactions, forming stacking of lipid bilayer films. Achieving designed geometry at nano- to micro-levels with packing deviating from the near-equilibrium structure is difficult to achieve due to the strong tendency of lipid molecules to self-assemble. Using ultrasmall (<fL) droplets containing designed molecules, our prior work has demonstrated that molecular assembly, in principle, is governed mainly by transient inter-molecular interactions under their dynamic spatial confinement, i.e., tri-phase boundaries during drying. As a result, the assemblies can deviate, sometimes significantly, from the near-equilibrium structures of self-assembly. The present work applies the approach and concept to lipid molecules using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Taking advantage of the high spatial precision and the minute size of the delivery probe in our combined atomic force microscopy and microfluidic delivery, the transient shape of each liquid droplet is regulated. In doing so, the final geometry of the POPC assemblies has been regulated to the designed geometry with nanometer precision. The results extend the concept of controlled assembly of molecules to amphiphilic systems. The outcomes exhibit high potential in lipid-based biomaterial science and biodevice engineering.

Use of Proximity Ligation Assay to Study Lymphoid Malignancies.

The advent of high-throughput and unbiased proteogenomic screens promises to rapidly advance our understanding of the molecular mechanisms underpinning pathogenesis of lymphoid malignancies. The wealth of data generated from these studies requires methods to rapidly confirm and extend findings into cell line models and primary patient samples. The proximity ligation assay (PLA) is a method that can visualize protein-protein interactions in situ. PLA can capture transient interactions and characterize constituents of stable biomolecular condensates, both of which pose technical difficulties for traditional biochemical and fluorescence imaging techniques.

P57Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions.

p57Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21Cip1/WAF1 and p27Kip1. Different from its siblings, few data are available about the p57Kip2 protein, especially in humans. Structurally, p57Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21Cip1/WAF1 and p27Kip1. We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57Kip2 influences the binding with some p57Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified.

Binding dynamics of linear alkyl-sulfates of different chain lengths on a protein surface

Sulfate-based anionic surfactants, such as sodium dodecyl sulfate (SDS), are widely recognized for their ability to deactivate and denature proteins. Despite extensive research, the molecular mechanisms governing the preferential binding of surfactants to protein surfaces remain incompletely understood. This study employs molecular dynamics (MD) simulations to investigate interactions between subtilisin E (SubE) and two anionic surfactants: SDS and sodium hexyl sulfate (SHS), with SHS possessing alkyl chains half the length of SDS. Simulations conducted at a low surfactant concentration (0.5% w/v) reveal distinct binding dynamics and interaction preferences of these surfactants with the SubE protein surface. Results demonstrate that the presence of SDS and SHS does not induce significant alterations in the overall secondary structure of SubE compared to simulations in pure water. The residue-level analysis identifies SDS as having greater contact with surface residues than SHS, particularly in helical and turn regions of SubE. SDS exhibits persistent binding sites in proximity to active site residues, suggesting potential implications for the observed experimental reduction in enzymatic activity. Dynamic interaction analysis indicates rapid and sustained binding of nearly all surfactant molecules to the protein within the initial 10-25 ns, with SDS displaying more transient interactions compared to SHS. Specific interaction patterns are observed between SDS and catalytic triad residues Asp32, His64, and Ser221, as well as Asn155 in the oxyanion hole, contrasting with the minimal interaction observed with SHS. These findings offer insights into the molecular mechanisms underlying surfactant-protein interactions, underscoring opportunities for engineering protein stability and functionality in the presence of surfactants.

Transient promoter interactions modulate developmental gene activation

Transcriptional induction coincides with the formation of various chromatin topologies. Strong evidence supportsthat gene activation is accompanied by a general increase in promoter-enhancer interactions. However,it remains unclear how these topological changes are coordinated across time and space during transcriptional activation. Here, we combine chromatin conformation capture with transcription and chromatin profiling during an embryonic stem cell (ESC) differentiation time course to determine how 3D genome restructuring is related to transcriptional transitions. This approach allows us to identify distinct topological alterationsthat are associated with the magnitude of transcriptional induction. We detect transiently formed interactions and demonstrate by genetic deletions that associated distal regulatory elements (DREs), as well as appropriate formation and disruption of these interactions, can contribute to the transcriptional induction of linked genes. Together, our study links topological dynamics to the magnitude of transcriptional induction and detects an uncharacterized type of transcriptionally important DREs.

RNA binding tunes the conformational plasticity and intradomain stability of TDP-43 tandem RNA recognition motifs

TAR DNA binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein with pivotal roles in RNA-related processes such as splicing, transcription, transport, and stability. The high binding affinity and specificity of TDP-43 toward its cognate RNA sequences (GU-rich) is mediated by highly conserved residues in its tandem RNA recognition motif (RRM) domains (aa: 104–263). Importantly, the loss of RNA binding to the tandem RRMs caused by physiological stressors and chemical modifications promotes cytoplasmic mislocalization and pathological aggregation of TDP-43. Despite the substantial implications of RNA binding in TDP-43 function and pathology, its precise effects on the intradomain stability, and conformational dynamics of the tandem RRMs is not properly understood. Here, we employed all-atom molecular dynamics (MD) simulations to assess the effect of RNA binding on the conformational landscape and intradomain stability of TDP-43 tandem RRMs. RNA limits the overall conformational space of the tandem RRMs and promotes intradomain stability through a combination of specific base stacking interactions and transient electrostatic interactions. In contrast, tandem RRMs exhibit a high intrinsic conformational plasticity in the absence of RNA, which, surprisingly, is accompanied by a tendency of RRM1 to adopt partially unfolded conformations. Overall, our simulations reveal how RNA binding dynamically tunes the structural and conformational landscape of TDP-43 tandem RRMs, contributing to physiological function and mitigating pathological aggregation.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase-Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein that binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full-length human protein tau and other negatively charged binding substrates, as revealed by differential interference contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase-separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes (i.e. tau-lipoplexes) exhibited distinct types of morphologies. This included large ∼20-30 μm tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles and tau-mediated finite-size assemblies of small liposomes. As the salt concentration was increased to near and above 150 mM for 1:1 electrolytes, AL-tau complexes remained stable, while tau self-coacervate droplets were found to dissolve, indicative of the breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen-facing monolayer of the axon's plasma membrane, suggesting the possibility of transient yet robust interactions near relevant ionic strengths found in neurons.

Regulatory mechanisms of miR171d-SCL6 module in the rooting process of Acer rubrum L.

MiR171d and SCL6 are induced by the plant hormone auxin. MiR171d negatively regulates the expression of SCL6, thereby regulating the growth and development of plant adventitious roots. Under natural conditions, it is difficult to induce rooting in the process of propagating Acer rubrum L. via branches, which seriously limits its wide application in landscaping construction. In this study, the expression of Ar-miR171d was downregulated and the expression of ArSCL6 was upregulated after 300mg/L indole-3-butyric acid (IBA) treatment. The transient interaction of Ar-miR171d and ArSCL6 in tobacco cells further confirmed their cleavage activity. Transgenic function verification confirmed that OE-Ar-miR171d inhibited adventitious root (AR) development, while OE-ArSCL6 promoted AR development. Tissue-specific expression verification of the ArSCL6 promoter demonstrated that it was specifically expressed in the plant root and leaf organs. Subcellular localization and transcriptional activation assays revealed that both ArSCL6 and ArbHLH089 were located in the nucleus and exhibited transcriptional activation activity. The interaction between the two was verified by bimolecular fluorescence complementarity (BIFC) experiments. These results help elucidate the regulatory mechanisms of the Ar-miR171d-ArSCL6 module during the propagation of A. rubrum and provide a molecular basis for the rooting of branches.

Coupling of the catalytic reactions from formate dehydrogenase and hydrogenase in solution: Insights from computations and in situ IR spectroscopy

Sophisticated enzymatic systems have evolved in nature to efficiently couple distinct biochemical reactions in form of cascades. As such they serve as reference models to understand the indirect interactions of catalytic centers. Herein, we studied, in solution, the coupling of the reactions from membrane‐bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (reversible H2 splitting into H+ and e‐) and the molybdenum‐dependent formate dehydrogenase from Rhodobacter capsulatus (reversible formate to CO2 interconversion). To follow their interplay via the characteristic absorptions from the MBH's active site or the respective substrate and product bands of FDH, we utilized in situ IR spectrocopy and GC(‐MS), in the absence or presence of soluble redox mediators. Coarse grained molecular dynamics (cgMD) computations revealed the lack of productive enzyme complexes for direct electron transfer (ET). Thus, the observed minor amounts of H2 or formate were produced from transient interactions between the two enzymes. On the contrary, the significantly increased product formation in the presence of methylene viologen can be related to the putative multiple interaction sites of the redox mediator with FDH identified by cgMD. Our study represents a proof‐of‐concept approach that can be used in future to develop novel coupled biocatalytic systems by identifying potential ET pathways.