NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Chemical shift assignments of calmodulin bound to a cytosolic domain of GluN2A (residues 1004-1024) from the NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the neuronal post-synaptic membrane are important for controlling neuroplasticity and synaptic transmission in the brain. Calmodulin (CaM) binds to the cytosolic C0 domains of both GluN1 (residues 841-865) and GluN2 (residues 1004-1024) that may play a role in the Ca2+-dependent desensitization of NMDAR channels. Mutations that disrupt Ca2+-dependent desensitization of NMDARs are linked to Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for Ca2+-saturated CaM bound to the GluN2A C0 domain of NMDAR (BMRB no. 51821).

Chemical shift assignments of calmodulin bound to the GluN1 C0 domain (residues 841-865) of the NMDA receptor.

Neuroplasticity and synaptic transmission in the brain are regulated by N-methyl-D-aspartate receptors (NMDARs) that consist of hetero-tetrameric combinations of the glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binds to the cytosolic C0 domain of GluN1 (residues 841-865) that may play a role in the Ca2+-dependent inactivation (CDI) of NMDAR channel activity. Dysregulation of NMDARs are linked to various neurological disorders, including Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the GluN1 C0 domain of the human NMDAR (BMRB no. 51715).

Retinal Cyclic Nucleotide-Gated Channel Regulation by Calmodulin.

Retinal cyclic nucleotide-gated (CNG) ion channels bind to intracellular cGMP and mediate visual phototransduction in photoreceptor rod and cone cells. Retinal rod CNG channels form hetero-tetramers comprised of three CNGA1 and one CNGB1 protein subunits. Cone CNG channels are similar tetramers consisting of three CNGA3 and one CNGB3 subunits. Calmodulin (CaM) binds to two distinct sites (CaM1: residues 565-587 and CaM2: residues 1120-1147) within the cytosolic domains of rod CNGB1. The binding of Ca2+-bound CaM to CNGB1 promotes the Ca2+-induced desensitization of CNG channels in retinal rods that may be important for photoreceptor light adaptation. Mutations that affect Ca2+-dependent CNG channel function are responsible for inherited forms of blindness. In this review, we propose structural models of the rod CNG channel bound to CaM that suggest how CaM might cause channel desensitization and how dysregulation of the channel may lead to retinal disease.

NMR Structures of Calmodulin Bound to Two Separate Regulatory Sites in the Retinal Cyclic Nucleotide-Gated Channel.

Retinal cyclic nucleotide-gated (CNG) channels (composed of three CNGA1 and one CNGB1 subunits) exhibit a Ca2+-induced reduction in channel open probability mediated by calmodulin (CaM). Defects in the Ca2+-dependent regulation of CNG channels may be linked to autosomal recessive retinitis pigmentosa and other inherited forms of blindness. Here, we report the NMR structure and binding analysis of CaM bound to two separate sites within CNGB1 (CaM1: residues 565-589 and CaM2: residues 1120-1147). Our binding studies reveal that CaM1 binds to the Ca2+-bound CaM N-lobe with at least fivefold higher affinity than it binds to the CaM C-lobe. By contrast, the CaM2 site binds to the Ca2+-bound CaM C-lobe with higher affinity than it binds to the N-lobe. CaM1 and CaM2 both exhibited very weak binding to Ca2+-free CaM. We present separate NMR structures of Ca2+-saturated CaM bound to CaM1 and CaM2 that define key intermolecular contacts: CaM1 residue F575 interacts with the CaM N-lobe while CaM2 residues L1129, L1132, and L1136 each make close contact with the CaM C-lobe. The CNGB1 mutation F575E abolishes CaM1 binding to the CaM N-lobe while L1132E and L1136E each abolish CaM2 binding to the CaM C-lobe. Thus, a single CaM can bind to both sites in CNGB1 in which the CaM N-lobe binds to CaM1 and the CaM C-lobe binds to CaM2. We propose a Ca2+-dependent conformational switch in the CNG channel caused by CaM binding, which may serve to attenuate cGMP binding to CNG channels at high cytosolic Ca2+ levels in dark-adapted photoreceptors.

Chemical shift assignments of calmodulin bound to a C-terminal site (residues 1120-1147) in the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565-587, called CaM1) decreases the open probability of CNG channels at elevated Ca2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120-1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

An integrated understanding of the evolutionary and structural features of the SARS-CoV-2 spike receptor binding domain (RBD).

Conventional drug development strategies typically use pocket in protein structures as drug-target sites. They overlook the plausible effects of protein evolvability and resistant mutations on protein structure which in turn may impair protein-drug interaction. In this study, we used an integrated evolution and structure guided strategy to develop potential evolutionary-escape resistant therapeutics using receptor binding domain (RBD) of SARS-CoV-2 spike-protein/S-protein as a model. Deploying an ensemble of sequence space exploratory tools including co-evolutionary analysis and deep mutational scans we provide a quantitative insight into the evolutionarily constrained subspace of the RBD sequence-space. Guided by molecular simulation and structure network analysis we highlight regions inside the RBD, which are critical for providing structural integrity and conformational flexibility. Using fuzzy C-means clustering we combined evolutionary and structural features of RBD and identified a critical region. Subsequently, we used computational drug screening using a library of 1615 small molecules and identified one lead molecule, which is expected to target the identified region, critical for evolvability and structural stability of RBD. This integrated evolution-structure guided strategy to develop evolutionary-escape resistant lead molecules have potential general applications beyond SARS-CoV-2.

Chemical shift assignments of calmodulin under standard conditions at neutral pH.

The Ca2+ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca2+ transporters. The only published NMR chemical shift assignments for Ca2+-bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca2+-saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289).

Ligand-Dependent Modulation of the Dynamics of Intracellular Loops Dictates Functional Selectivity of 5-HT2AR.

The serotonin 2A receptor (5-HT2AR) subtype of the G protein-coupled receptor (GPCR) family is involved in a plethora of neuromodulatory functions (e.g., neurogenesis, sleep, and cognitive processes). 5-HT2AR is the target of pharmacologically distinct classes of ligands, binding of which either activate or inactivate the receptor. Although high-resolution structures of 5-HT2AR as well as several other 5-HT GPCRs provided snapshots of both active and inactive conformational states, these structures, representing a truncated form of the receptor, cannot fully explain the mechanism of conformational transitions during their function. Importantly, biochemical studies have suggested the importance of intracellular loops in receptor functions. In our previous study, a model of the ligand-free form of 5-HT2AR with the third intracellular loop (ICL3) has been meticulously built. Here, we have investigated the functional regulation of 5-HT2AR with intact intracellular loops in ligand-free and five distinct ligand-bound configurations using unbiased atomistic molecular dynamics (MD) simulations. The selected ligands belong to either of the full, partial, or inverse agonist classes, which exert distinct pharmacological responses. We have observed significant structural, dynamic, and thermodynamic differences within ligand-bound complexes. Our results revealed, for the first time, that either activation or inactivation of the receptor upon specific ligand binding is primarily achieved through conformational transitions of its second and third intracellular loops (ICL2 and ICL3). A remarkable allosteric cross-talk between the ligand-binding site and the distal intracellular parts of the receptor, where binding of a specific ligand thermodynamically controls (either stabilizes or destabilizes) the intracellular region, consisting of crucial dynamic elements ICL2 and ICL3, and differential conformational transitions of these loops determine ligand-dependent functional selectivity.

Chemical shift assignments of calmodulin bound to the ß-subunit of a retinal cyclic nucleotide-gated channel (CNGB1).

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679-702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

Structural Elucidation of Inter-CARD Interfaces involved in NOD2 Tandem CARD Association and RIP2 Recognition.

Nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) recognizes the muramyl dipeptide and activates the NF-κB signaling cascade following its interaction with receptor-interacting protein 2 (RIP2) via caspase recruitment domains (CARDs). The NOD2-RIP2 interaction is not understood well due to inadequate structural information. Using comparative modeling and multimicrosecond timescale molecular dynamics simulations, we have demonstrated the association of NOD2-CARDs (CARDa-CARDb) and their interaction with RIP2CARD. Our results suggest that a negatively charged interface of NOD2CARDa and positively charged type-Ia interface of NOD2CARDb are crucial for CARDa-CARDb association and the type-Ia interface of NOD2CARDa and type-Ib interface of RIP2CARD predicted to be involved in 1:1 CARD-CARD interaction. Moreover, the direct interaction of NOD2CARDb with RIP2CARD signifies the importance of both CARDs of NOD2 in RIP2-mediated CARD-CARD interaction. Altogether, the structural results could help in understanding the underlying molecular details of the NOD2-RIP2 association in higher and lower eukaryotes.

Structure, dynamics and lipid interactions of serotonin receptors: excitements and challenges.

Serotonin (5-hydroxytryptamine, 5-HT) is an intrinsically fluorescent neurotransmitter found in organisms spanning a wide evolutionary range. Serotonin exerts its diverse actions by binding to distinct cell membrane receptors which are classified into many groups. Serotonin receptors are involved in regulating a diverse array of physiological signaling pathways and belong to the family of either G protein-coupled receptors (GPCRs) or ligand-gated ion channels. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions such as sleep, mood, pain, anxiety, depression, aggression, and learning. Serotonin receptors act as drug targets for a number of diseases, particularly neuropsychiatric disorders. The signaling mechanism and efficiency of serotonin receptors depend on their amazing ability to rapidly access multiple conformational states. This conformational plasticity, necessary for the wide variety of functions displayed by serotonin receptors, is regulated by binding to various ligands. In this review, we provide a succinct overview of recent developments in generating and analyzing high-resolution structures of serotonin receptors obtained using crystallography and cryo-electron microscopy. Capturing structures of distinct conformational states is crucial for understanding the mechanism of action of these receptors, which could provide important insight for rational drug design targeting serotonin receptors. We further provide emerging information and insight from studies on interactions of membrane lipids (such as cholesterol) with serotonin receptors. We envision that a judicious combination of analysis of high-resolution structures and receptor-lipid interaction would allow a comprehensive understanding of GPCR structure, function and dynamics, thereby leading to efficient drug discovery.

Comprehensive structural modeling and preparation of human 5-HT2A G-protein coupled receptor in functionally active form.

The serotonin 2A receptor (5-HT2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT2A R has marked distinction with ligand-bound conformations of 5-HT2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier.

Conformational Entropy as a Determinant of the Thermodynamic Stability of the p53 Core Domain.

Mutations in the core domain of tumor suppressor protein p53 have been associated with ∼50% of the occurrences of human cancers. A majority of these mutations inactivate p53 function by destabilizing its native structure. Although studies have shown p53's function can be restored by stabilizing the mutants to their wild-type conformation with immense therapeutic potential, its applicability has been restricted because of our limited understanding of the precise nature of destabilization arising from changes in the mutant p53's structure and dynamics. Here, using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations, we have probed the conformational flexibility in three of the most widespread and clinically important "hot spot" mutants of the p53 core domain. Our results show that NMR order parameter-derived conformational entropy is linearly correlated with the change in free energy of urea-mediated denaturation, the latter being a well-established reporter of stability in p53 core domain mutants. Using a linear regression function, we show that the three parameters of equilibrium denaturation experiments, i.e., the free energy of denaturation (Δ GD-NH2O), the slope of the transition ( m), and the urea concentration at 50% denaturation ([urea]50%), can be used to predict the conformational entropy in p53 core domain mutants, thereby demonstrating a method for using these parameters as predictors of a protein's conformational entropy, which has been known to shape the functional properties of proteins.

Intrinsic Differences in Backbone Dynamics between Wild Type and DNA-Contact Mutants of the p53 DNA Binding Domain Revealed by Nuclear Magnetic Resonance Spectroscopy.

Mutations in p53's DNA binding domain (p53DBD) are associated with 50% of all cancers, making it an essential system to investigate and understand the genesis and progression of cancer. In this work, we studied the changes in the structure and dynamics of wild type p53DBD in comparison with two of its "hot-spot" DNA-contact mutants, R248Q and R273H, by analysis of backbone amide chemical shift perturbations and 15N spin relaxation measurements. The results of amide chemical shift changes indicated significantly more perturbations in the R273H mutant than in wild type and R248Q p53DBD. Analysis of 15N spin relaxation rates and the resulting nuclear magnetic resonance order parameters suggests that for most parts, the R248Q mutant exhibits limited conformational flexibility and is similar to the wild type protein. In contrast, R273H showed significant backbone dynamics extending up to its ß-sandwich scaffold in addition to motions along the DNA binding interface. Furthermore, comparison of rotational correlation times between the mutants suggests that the R273H mutant, with a higher correlation time, forms an enlarged structural fold in comparison to the R248Q mutant and wild type p53DBD. Finally, we identify three regions in these proteins that show conformational flexibility to varying degrees, which suggests that the R273H mutant, in addition to being a DNA-contact mutation, exhibits properties of a conformational mutant.

Structural and functional insights into CARDs of zebrafish (Danio rerio) NOD1 and NOD2, and their interaction with adaptor protein RIP2.

Nucleotide-binding and oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern-recognition receptors (PRRs) composed of an N-terminal caspase activation and recruitment domain (CARD), a central NACHT domain and C-terminal leucine-rich repeats (LRRs). They play a vital role in innate immune signaling by activating the NF-κB pathway via recognition of peptidoglycans by LRRs, and ATP-dependent self-oligomerization of NACHT followed by downstream signaling. After oligomerization, CARD/s play a crucial role in activating downstream signaling via the adaptor molecule, RIP2. Due to the inadequacy of experimental 3D structures of CARD/s of NOD2 and RIP2, and results from differential experimental setups, the RIP2-mediated CARD-CARD interaction has remained as a contradictory statement. We employed a combinatorial approach involving protein modeling, docking, molecular dynamics simulation, and binding free energy calculation to illuminate the molecular mechanism that shows the possible involvement of either the acidic or basic patch of zebrafish NOD1/2-CARD/a and RIP2-CARD in CARD-CARD interaction. Herein, we have hypothesized 'type-I' mode of CARD-CARD interaction in NOD1 and NOD2, where NOD1/2-CARD/a involve their acidic surfaces to interact with RIP2. Asp37 and Glu51 (of NOD1) and Arg477, Arg521 and Arg529 (of RIP2) were identified to be crucial for NOD1-RIP2 interaction. However, in NOD2-RIP2, Asp32 (of NOD2) and Arg477 and Arg521 (of RIP2) were anticipated to be significant for downstream signaling. Furthermore, we found that strong electrostatic contacts and salt bridges are crucial for protein-protein interactions. Altogether, our study has provided novel insights into the RIP2-mediated CARD-CARD interaction in zebrafish NOD1 and NOD2, which will be helpful to understand the molecular basis of the NOD1/2 signaling mechanism.

Structural models of zebrafish (Danio rerio) NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Conformational flexibility tunes the propensity of transthyretin to form fibrils through non-native intermediate states.

The formation of partially unfolded intermediates through conformational excursions out of the native state is the starting point of many diseases involving protein aggregation. Therapeutic strategies often aim to stabilize the native structure and prevent the formation of intermediates that are also cytotoxic in vivo. However, their transient nature and low population makes it difficult to characterize these intermediates. We have probed the backbone dynamics of transthyretin (TTR) over an extended timescale by using NMR spectroscopy and MD simulations. The location and extent of these motions indicates that the backbone flexibility of TTR is a cause of dissociation and destabilization, both of which are responsible for fibril formation. Importantly, approximately 10 % of wild-type TTR exists in an intermediate state, which increased to up to 28 % for pathogenic TTR mutants, for which the formation of the intermediate state is shown to be energetically more favorable compared to the wild type. This result suggests an important role for the intermediates in TTR amyloidosis.

LRRsearch: An asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors.

The leucine-rich repeat (LRR) motifs of the nucleotide-binding oligomerization domain like receptors (NLRs) play key roles in recognizing and binding various pathogen associated molecular patterns (PAMPs) resulting in the activation of downstream signaling and innate immunity. Therefore, identification of LRR motifs is very important to study ligand-receptor interaction. To date, available resources pose restrictions including both false negative and false positive prediction of LRR motifs from the primary protein sequence as their algorithms are relied either only on sequence based comparison or alignment techniques or are over biased for a particular LRR containing protein family. Therefore, to minimize the error (≤5%) and to identify a maximum number of LRR motifs in the wide range of proteins, we have developed "LRRsearch" web-server using position specific scoring matrix (PSSM) of 11 residue LRR-HCS (highly conserved segment) which are frequently observed motifs in the most divergent classes of LRR containing proteins. A data library of 421 proteins, distributed among five known NLR families has also been integrated with the "LRRsearch" for the rich user experience. The access to the "LRRsearch" program is freely available at http://www.lrrsearch.com/.

Structural and functional investigation of zebrafish (Danio rerio) NOD1 leucine rich repeat domain and its interaction with iE-DAP.

Nucleotide binding and oligomerization domain 1 (NOD1), a cytoplasmic pattern recognition receptor (PRR) and is a key component for modulating innate immunity and signaling. It is highly specific to γ-D-Glu-mDAP (iE-DAP), a cell wall component of Gram-negative and few Gram-positive bacteria. In the absence of the experimental structure of NOD1 leucine rich repeat (NOD1-LRR) domain, the NOD signaling cascade mediated through NOD1 and iE-DAP interaction is poorly understood. Herein, we modeled 3D structure of zebrafish NOD1-LRR (zNOD1-LRR) through a protein-threading approach and structural integrity of the model was assessed using molecular dynamics simulations. Molecular interaction analysis of iE-DAP and zNOD1-LRR, their complex stability and binding free energy studies were conducted to anticipate the ligand binding residues in zNOD1. Our study revealed that His775, Lys777, Asp803, Gly805, Trp807, Asn831, Ser833, Ile859 and Trp861 situated in the ß-sheet region of zNOD1-LRR could be involved in iE-DAP recognition, which correlates the earlier findings in human. Comparison of binding free energies of native and mutant zNOD1-iE-DAP complexes delineated His775, Lys777, Asp803, Ser833 and Ile859 as the pivotal residues for energetic stability of NOD1 and iE-DAP interaction. This study provides the first comprehensive description of biophysical and biochemical parameters responsible for NOD1 and iE-DAP interaction in zebrafish, which is expected to shed more light on NOD1 signaling and therapeutic applications in other organisms.