In silico design of potential Mcl-1 peptide-based inhibitors.

CONTEXT: BIM (Bcl-2 interacting mediator of apoptosis)-derived peptides that specifically target over-expressed Mcl-1 (myeloid cell leukemia-1) protein and induce apoptosis are potentially anti-cancer agents. Since the helicity of BIM-derived peptides has a crucial role in their functionality, a range of strategies have been used to increase the helicity including the introduction of unnatural residues and stapling methods that have some drawbacks such as the accumulation in the liver. To avoid these drawbacks, this study aimed to design a more helical peptide by utilizing bioinformatics algorithms and molecular dynamics simulations without exploiting unnatural residues and stapling methods. MM-PBSA results showed that the mutations of A4fE and A2eE in analogue 5 demonstrate a preference towards binding with Mcl-1. As evidenced by Circular dichroism results, the helicity increases from 18 to 34%, these findings could enhance the potential of analogue 5 as an anti-cancer agent targeting Mcl-1. The applied strategies in this research could shed light on the in silico peptide design. Moreover, analogue 5 as a drug candidate can be evaluated in vitro and in vivo studies. METHODS: The sequence of the lead peptide was determined using the ApInAPDB database and PRALINE program. Contact finder and PDBsum web server softwares were used to determine the contact involved amino acids in complex with Mcl-1. All identified salt bridge contributing residues were unaltered to preserve the binding affinity. After proposing novel analogues, their secondary structures were predicted by Cham finder web server software and GOR, Neural Network, and Chou-Fasman algorithms. Finally, molecular dynamics simulations run for 100 ns were done using the GROMACS, version 5.0.7, with the CHARMM36 force field. MM-PBSA was used to assess binding affinity specificity in targeting Mcl-1 and Bcl-xL (B-cell lymphoma extra-large).

The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Reconciling ASPP-p53 binding mode discrepancies through an ensemble binding framework that bridges crystallography and NMR data.

ASPP2 and iASPP bind to p53 through their conserved ANK-SH3 domains to respectively promote and inhibit p53-dependent cell apoptosis. While crystallography has indicated that these two proteins employ distinct surfaces of their ANK-SH3 domains to bind to p53, solution NMR data has suggested similar surfaces. In this study, we employed multi-scale molecular dynamics (MD) simulations combined with free energy calculations to reconcile the discrepancy in the binding modes. We demonstrated that the binding mode based solely on a single crystal structure does not enable iASPP's RT loop to engage with p53's C-terminal linker-a verified interaction. Instead, an ensemble of simulated iASPP-p53 complexes facilitates this interaction. We showed that the ensemble-average inter-protein contacting residues and NMR-detected interfacial residues qualitatively overlap on ASPP proteins, and the ensemble-average binding free energies better match experimental KD values compared to single crystallgarphy-determined binding mode. For iASPP, the sampled ensemble complexes can be grouped into two classes, resembling the binding modes determined by crystallography and solution NMR. We thus propose that crystal packing shifts the equilibrium of binding modes towards the crystallography-determined one. Lastly, we showed that the ensemble binding complexes are sensitive to p53's intrinsically disordered regions (IDRs), attesting to experimental observations that these IDRs contribute to biological functions. Our results provide a dynamic and ensemble perspective for scrutinizing these important cancer-related protein-protein interactions (PPIs).

Identification of anti-cancer organometallic compounds by inhibition of BCL-2/Bax interactions.

Apoptosis is regulated by the BCL-2 family, which includes the anti-apoptotic and pro-apoptotic proteins (Bax, Bok, Bak, etc.). These proteins often interact in dimers and act as apoptotic switches. Anti-apoptotic proteins, such as BCL-2, block the functions of these pro-apoptotic proteins. The pro-apoptotic and anti-apoptotic protein-protein interactions must be inhibited to prevent tumor cells from escaping apoptosis. This method has been used to develop anticancer drugs by inhibiting BCL-2 with both natural and synthetic compounds. Metal-containing compounds were used as pharmaceuticals for human cancer patients for a long time, and cisplatin was the first candidate of this class. Drug design, however, needs to pay more attention to metal complexes. We have studied the X-ray crystal structure of the BCL-2 protein in detail and identified the hydrophobic nature of the site with two less solvent-accessible sites. Based on the hydrophobic nature of the compounds, 74 organometallic compounds with X-ray crystallographically characterized bioactivity (including anticancer activity) were selected from the Cambridge crystallographic database. For testing, molecular docking was used to determine which compound was most effective against the BCL-2 protein. Organometallic compounds (benzene)-chloro-(1-{[(9H-fluoren-2-yl)imino]methyl}naphthalen-2-olato)-ruthenium (2), (1-((1,1'-biphenyl)-4-yl)-2,3,4,5-tetramethylcyclopentadienyl)-chloro-(4,4'-dimethyl-2,2'-bipyridine)-rhodium hexafluorophosphate (37), (µ-1,1'-(butane-1,4-diyl)bis(3-oxy-2-methylpyridin-4(1H)-one))-dichloro-bis(pentamethyl-cyclopentadienyl)-di-rhodium tetrahydrate (46), (µ-1,1'-(butane-1,4-diyl)bis(3-oxy-2-methylpyridin-4(1H)-one))-dichloro-bis(pentamethyl-cyclopentadienyl)-di-iridium (47) etc are found to be important compounds in this study. The capability of different types of complex interactions was identified using Hirshfeld surface analysis of the complexes. A NCI plot was conducted to understand the nature of the interaction between complex amino acids and active-site amino acids. A DFT study was conducted to examine the stability and chemical reactivity of the selected complexes. Using this study, one suitable hydrophobic lead anti-cancer organometallic pharmaceutical was found that binds at the less solvent-accessible hydrophobic site of BCL-2.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

A Step Forward toward Selective Activation/Inhibition of Bak, a Pro-Apoptotic Member of the Bcl-2 Protein Family: Discovery of New Prospective Allosteric Sites Using Molecular Dynamics.

Bak is a pro-apoptotic protein and a member of the Bcl-2 family that plays a key role in apoptosis, a programmed cell death mechanism of multicellular organisms. Its activation under death stimuli triggers the permeabilization of the mitochondrial outer membrane that represents a point of no return in the apoptotic pathway. This process is deregulated in many tumors where Bak is inactivated, whereas in other cases like in neurodegeneration, it exhibits an excessive response leading to disorders such as the Alzheimer disease. Members of the Bcl-2 family share a common 3D structure, exhibiting an extremely similar orthosteric binding site, a place where both pro and antiapoptotic proteins bind. This similarity raises a selectivity issue that hampers the identification of new drugs, capable of altering Bak activation in a selective manner. An alternative activation site triggered by antibodies has been recently identified, opening the opportunity to undertake new drug discovery studies. Despite this recent identification, an exhaustive study to identify cryptic pockets as prospective allosteric sites has not been yet performed. Thus, the present study aims to characterize novel hotspots in the Bak structure. For this purpose, we have carried out extensive molecular dynamics simulations using three different Bak systems including Bak in its apo form, Bak in complex with its endogen activator Bim and an intermediate form, set up by removing Bim from the previous complex. The results reported in the present work shed some light on future docking studies on Bak through the identification of new prospective allosteric sites, not previously described in this protein.

[CD5L is elevated in the serum of patients with candidemia and promotes disease progression in mouse models].

OBJECTIVE: To investigate the changes of CD5L levels in patients with candidemia and explore the role of CD5L in progression of candidemia. METHODS: Twenty healthy control individuals, 27 patients with bacteremia and 35 patients with candidemia were examined for serum CD5L levels using ELISA, and the correlations of CD5L level with other serological indicators were analyzed. A C57BL/6 mouse model of candidemia induced by intravenous injection of Candida albicans were treated with intraperitoneal injection of recombinant CD5L protein, and renal histopathological and serological changes were analyzed to assess renal injures. The effects of CD5L treatment on general condition, fungal burden, of survival of the mice were observed, and the changes in serum IL-6 and IL-8 levels of the mice were detected using ELISA. RESULTS: CD5L levels were significantly elevated in patients with candidemia and positively correlated with WBC, BDG, Scr and PCT levels. The mouse model of candidemia also showed significantly increased serum and renal CD5L levels, and CD5L treatment significantly increased fungal burden in the renal tissue, elevated IL-6 and IL-8 levels in the serum and kidney, aggravated renal tissue damage, and reduced survival rate of candidemia mice. CONCLUSION: Serum CD5L levels are increased in patients with candidemia, and treatment with CD5L aggravates candidemia in mouse models.

Immunoglobulin M perception by FcµR.

Immunoglobulin M (IgM) is the first antibody to emerge during embryonic development and the humoral immune response1. IgM can exist in several distinct forms, including monomeric, membrane-bound IgM within the B cell receptor (BCR) complex, pentameric and hexameric IgM in serum and secretory IgM on the mucosal surface. FcµR, the only IgM-specific receptor in mammals, recognizes different forms of IgM to regulate diverse immune responses2-5. However, the underlying molecular mechanisms remain unknown. Here we delineate the structural basis of the FcµR-IgM interaction by crystallography and cryo-electron microscopy. We show that two FcµR molecules interact with a Fcµ-Cµ4 dimer, suggesting that FcµR can bind to membrane-bound IgM with a 2:1 stoichiometry. Further analyses reveal that FcµR-binding sites are accessible in the context of IgM BCR. By contrast, pentameric IgM can recruit four FcµR molecules to bind on the same side and thereby facilitate the formation of an FcµR oligomer. One of these FcµR molecules occupies the binding site of the secretory component. Nevertheless, four FcµR molecules bind to the other side of secretory component-containing secretory IgM, consistent with the function of FcµR in the retrotransport of secretory IgM. These results reveal intricate mechanisms of IgM perception by FcµR.

Identification of nonhistone substrates of the lysine methyltransferase PRDM9.

Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.

Structures of BIRC6-client complexes provide a mechanism of SMAC-mediated release of caspases.

Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis, and inhibitor of apoptosis proteins (IAPs) are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived proapoptotic factors such as SMAC and HTRA2. Through a series of cryo-electron microscopy structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to SMAC, caspases, and HTRA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by SMAC. The architecture of BIRC6, together with near-irreversible binding of SMAC, elucidates how the IAP inhibitor SMAC can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.

Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B, which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures showed that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. On the basis of these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.

Characterizing the consensus residue specificity and surface of BCL-2 binding to BH3 ligands using the Knob-Socket model.

Cancer cells bypass cell death by changing the expression of the BCL-2 family of proteins, which are apoptotic pathway regulators. Upregulation of pro-survival BCL-2 proteins or downregulation of cell death effectors BAX and BAK interferes with the initiation of the intrinsic apoptotic pathway. In normal cells, apoptosis can occur through pro-apoptotic BH3-only proteins interacting and inhibiting pro-survival BCL-2 proteins. When cancer cells over-express pro-survival BCL-2 proteins, a potential remedy is the sequestration of these pro-survival proteins through a class of anti-cancer drugs called BH3 mimetics that bind in the hydrophobic groove of pro-survival BCL-2 proteins. To improve the design of these BH3 mimetics, the packing interface between BH3 domain ligands and pro-survival BCL-2 proteins was analyzed using the Knob-Socket model to identify the amino acid residues responsible for interaction affinity and specificity. A Knob-Socket analysis organizes all the residues in a binding interface into simple 4 residue units: 3-residue sockets defining surfaces on a protein that pack a 4th residue knob from the other protein. In this way, the position and composition of the knobs packing into sockets across the BH3/BCL-2 interface can be classified. A Knob-Socket analysis of 19 BCL-2 protein and BH3 helix co-crystals reveal multiple conserved binding patterns across protein paralogs. Conserved knob residues such as a Gly, Leu, Ala and Glu most likely define binding specificity in the BH3/BCL-2 interface, whereas other residues such as Asp, Asn, and Val are important for forming surface sockets that bind these knobs. These findings can be used to inform the design of BH3 mimetics that are specific to pro-survival BCL-2 proteins for cancer therapeutics.

Peptides from human BNIP5 and PXT1 and non-native binders of pro-apoptotic BAK can directly activate or inhibit BAK-mediated membrane permeabilization.

Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes conformational changes that promote mitochondrial outer membrane disruption, leading to cell death. This is termed "activation" and can be induced by peptides from the human proteins BID, BIM, and PUMA. To identify additional peptides that can regulate BAK, we used computational protein design, yeast surface display screening, and structure-based energy scoring to identify 10 diverse new binders. We discovered peptides from the human proteins BNIP5 and PXT1 and three non-native peptides that activate BAK in liposome assays and induce cytochrome c release from mitochondria. Crystal structures and binding studies reveal a high degree of similarity among peptide activators and inhibitors, ruling out a simple function-determining property. Our results shed light on the vast peptide sequence space that can regulate BAK function and will guide the design of BAK-modulating tools and therapeutics.

Anti-apoptotic Bcl-2 protein in apo and holo conformation anchored to the membrane: comparative molecular dynamics simulations.

The interaction between the anti-apoptotic Bcl-2 protein and its antagonist Bax is essential to the regulation of the mitochondrial pathway of apoptosis. For this work, we built models by homology of Bcl-2 full-sequence length in monomeric form (apo-Bcl-2) and in complex with the BH3 domain of Bax (holo-Bcl-2). The Bcl-2 protein was analyzed with its transmembrane domain anchored to a lipidic bilayer of DPPC, imitating physiological conditions. We performed molecular dynamics (MD) simulations using the GROMACS program. Conformational changes showed that the flexible loop domain (FLD) tends to fold on itself and move towards the main core. Furthermore, the BH3 peptide of pro-apoptotic protein Bax, showed an allosteric stabilizing effect on FLD upon being bound to the hydrophobic cleft of the anti-apoptotic protein Bcl-2, causing a reduction in its structural flexibility. However, FLD is distal from the main core of Bcl-2. Principal component analysis (PCA) showed a weak correlation between FLD residues and BH3 peptide from Bax. Upon MD simulations, several new contacts appeared between FLD and some α-helices of the core of Bcl-2, which contribute to maintaining the stability of Bcl-2. This knowledge sheds light on the behavior of Bcl-2 in the cell's native environment.Communicated by Ramaswamy H. Sarma.

Synthesis of Scyllatoxin-Based BH3 Domain Mimetics with Diverse Patterns of Native Disulfide Bonds.

This article outlines the design and development of scyllatoxin (ScTx)-based BH3 domain mimetics with diverse patterns of native disulfide bonds. More specifically, this method summarizes the total chemical synthesis of ScTx-based peptides that contain zero, one, two, or three disulfide linkages, including techniques to generate variants with any combination of native disulfides. Each peptide reported herein is generated on solid-phase support using microwave-assisted coupling procedures, and all reaction parameters related to the peptide synthesis are described in detail. The various disulfide patterns of the ScTx-based constructs are established during peptide synthesis and are ultimately verified by mass analysis of trypsin-digested fragments. The BH3 domain mimetics developed herein were generated by transposing residues from the helical BH3 domain of the pro-apoptotic BCL2 protein Bax to the α-helix of wild-type ScTx. Interestingly, we found that the relative binding affinities of ScTx-Bax peptides for the anti-apoptotic BCL2 protein Bcl-2 (proper) were heavily influenced by the number and position of disulfide linkages within the ScTx-Bax sequence. As a consequence, we were able to utilize ScTx-Bax BH3 domain mimetics with varied patterns of disulfide bonds to survey how structural rigidity within the helical Bax BH3 domain affects binding to promiscuous anti-apoptotic BCL2 proteins. More broadly, the ability to generate ScTx-based molecules that contain any combination of native disulfide bonds expands the utility of such constructs as tools to study the molecular nature of protein-protein interactions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of ScTx-based Bax BH3 domain mimetics Basic Protocol 2: Oxidation of ScTx-Bax BH3 domain mimetics containing one, two, or three disulfide linkages Support Protocol: Mapping of disulfide linkages in oxidized ScTx-Bax BH3 domain mimetics.

In silico SELEX screening and statistical analysis of newly designed 5mer peptide-aptamers as Bcl-xl inhibitors using the Taguchi method.

Drug development for cancer treatment is a complex process that requires special efforts. Targeting crucial proteins is the most essential purpose of drug design in cancers. Bcl-xl is an anti-apoptotic protein that binds to pro-apoptotic proteins and interrupts their signals. Pro-apoptotic Bcl-xl effectors are short BH3 sequences that form an alpha helix and bind to anti-apoptotic proteins to inhibit their activity. Computational systematic evolution of ligands by exponential enrichment (SELEX) is an exclusive approach for developing peptide aptamers as potential effectors. Here, the amino acids with a high tendency for constructing an alpha-helical structure were selected. Due to the enormous number of pentapeptides, Taguchi method was used to study a selected number of peptides. The binding affinity of the peptides to Bcl-xl was assessed using molecular docking, and after analysis of the obtained results, a final set of optimized peptides was arranged and constructed. For a better comparison, three chemical compounds with approved anti-Bcl-xl activity were selected for comparison with the top-ranked 5mer peptides. The optimized peptides showed considerable binding affinity to Bcl-xl. The molecular dynamics (MD) simulation indicated that the designed peptide (PO5) could create considerable interactions with the BH3 domain of Bcl-xl. The MM/GBSA calculations revealed that these interactions were even stronger than those created by chemical compounds. In silico SELEX is a novel approach to design and evaluate peptide-aptamers. The experimental design improves the SELEX process considerably. Finally, PO5 could be considered a potential inhibitor of Bcl-xl and a potential candidate for cancer treatment.

PEA-15 engages in allosteric interactions using a common scaffold in a phosphorylation-dependent manner.

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein. We delineated that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED. Upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly.

Bacterial gasdermins reveal an ancient mechanism of cell death.

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.

The crystal structure of the FAM134B-GABARAP complex provides mechanistic insights into the selective binding of FAM134 to the GABARAP subfamily.

The mammalian Atg8 family (Atg8s proteins) consists of two subfamilies: GABARAP and LC3. All members can bind to the LC3-interacting region (LIR) or Atg8-interacting motif and participate in multiple steps of autophagy. The endoplasmic reticulum (ER) autophagy receptor FAM134B contains an LIR motif that can bind to Atg8s, but whether it can differentially bind to the two subfamilies and, if so, the structural basis for this preference remains unknown. Here, we found that FAM134B bound to the GABARAP subfamily more strongly than to the LC3 subfamily. We then solved the crystal structure of the FAM134B-GABARAP complex and demonstrated that FAM134B used both its LIR core and the C-terminal helix to bind to GABARAP. We further showed that these properties might be conserved in FAM134A or FAM134C. The structure also allowed us to identify the structural determinants for the binding selectivity. Our work may be valuable for studying the differential functions of GABARAP and LC3 subfamilies in ER phagy in future.

In silico structure evaluation of BAG3 and elucidating its association with bacterial infections through protein-protein and host-pathogen interaction analysis.

BAG3, a co-chaperone protein with a Bcl-2-associated athanogene (BAG) domain, has diverse functionalities in protein-folding, apoptosis, inflammation, and cell cycle regulatory cross-talks. It has been well characterised in cardiac diseases, cancers, and viral pathogenesis. The multiple roles of BAG3 are attributed to its functional regions like BAG, Tryptophan-rich (WW), isoleucine-proline-valine-rich (IPV), and proline-rich (PXXP) domains. However, to study its structural impact on various functions, the experimental 3D structure of BAG3 protein was not available. Hence, the structure was predicted through in silico modelling and validated through computational tools and molecular dynamics simulation studies. To the best of our knowledge, the role of BAG3 in bacterial infections is not explicitly reported. We attempted to study them through an in-silico protein-protein interaction network and host-pathogen interaction analysis. From structure-function relationships, it was identified that the WW and PXXP domains were associated with cellular cytoskeleton rearrangement and adhesion-mediated response, which might be involved in BAG3-related intracellular bacterial proliferation. From functional enrichment analysis, Gene Ontology terms and topological matrices, 18 host proteins and 29 pathogen proteins were identified in the BAG3 interactome pertaining to Legionellosis, Tuberculosis, Salmonellosis, Shigellosis, and Pertussis through differential phosphorylation events associated with serine metabolism. Furthermore, it was evident that direct (MAPK8, MAPK14) and associated (MAPK1, HSPD1, NFKBIA, TLR2, RHOA) interactors of BAG3 could be considered as therapeutic markers to curb down intracellular bacterial propagation in humans.