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The common γ-chain (γc) family of cytokine receptors, including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with the common γc receptor in ligand dependent manner. Sharing of γc by the IL receptors (ILRs) is thought to be achieved by concomitant binding of γc and ILR ectodomains to a cytokine. Here, we found that direct interactions between the transmembrane domain (TMD) of γc and those of the ILRs are also required for receptor activation, and remarkably, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences. Heterodimer structures of γc TMD bound to the TMDs of IL-7R and IL-9R, determined in near lipid bilayer environment, reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Functional mutagenesis data indicate the requirement of the heterotypic interactions of TMDs in signaling, which could explain disease mutations within the receptor TMDs.One-Sentence SummaryThe transmembrane anchors of interleukin receptors of the gamma-chain family are critical for receptor sharing and activation.

The S protein of the SARS-CoV-2 is a Type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the nuclear magnetic resonance (NMR) structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeat. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses., Graphical Abstract

HIV-1 envelope glycoprotein (Env) is a transmembrane protein that mediates membrane fusion and viral entry. The membrane-interacting regions of the Env, including the membrane-proximal external region (MPER), the transmembrane domain (TMD), and the cytoplasmic tail (CT), not only are essential for fusion and Env incorporation but also can strongly influence the antigenicity of the Env. Previous studies have incrementally revealed the structures of the MPER, the TMD, and the KS-LLP2 regions of the CT. Here, we determined the NMR structure of the full-length CT using a protein fragment comprising the TMD and the CT in bicelles that mimic a lipid bilayer, and by integrating the new NMR data and those acquired previously on other gp41 fragments, we derived a model of the entire membrane-interacting region of the Env. The structure shows that the CT forms a large trimeric baseplate around the TMD trimer, and by residing in the headgroup region of the lipid bilayer, the baseplate causes severe exclusion of lipid in the cytoleaflet of the bilayer. All-atom molecular dynamics simulations showed that the overall structure of the MPER-TMD-CT can be stable in a viral membrane and that a concerted movement of the KS-LLP2 region compensates for the lipid exclusion in order to maintain both structure and membrane integrity. Our structural and simulation results provide a framework for future research to manipulate the membrane structure to modulate the antigenicity of the Env for vaccine development and for mutagenesis studies for investigating membrane fusion and Env interaction with the matrix proteins.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD(+)-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a β-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high‐resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure‐guided mutations demonstrate the importance of both types of protein–lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria., NMR structures of membrane‐bound Bax core dimer reveal dual interactions with non‐polar tails and charged heads of bilayer lipids contributing directly to apoptotic mitochondrial permeabilization.

The p7 protein encoded by the hepatitis C virus forms a cation-selective viroporin in the membrane. One of the most intriguing findings about the p7 viroporin is its unique hexameric structure in dodecylphosphocholine (DPC) micelles determined by nuclear magnetic resonance (NMR), but the hexameric structure was recently challenged by another NMR study of p7, also in DPC detergent, which claimed that the p7 in this detergent is monomeric. Here, we show that p7 oligomerization is highly sensitive to the detergent:protein ratio used in protein reconstitution and that the 40-fold difference in this ratio between the two studies was the cause of their different conclusions. In addition, we have performed extensive measurements of interchain paramagnetic relaxation enhancements (PREs) for p7 hexamers reconstituted in DPC micelles and in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine bicelles. In both cases, interchain PREs are overall consistent with the hexameric structure determined in micelles. Our data validate the overall architecture of the p7 hexamer while highlighting the importance of the detergent:protein ratio in membrane protein sample preparation.

Hepatitis C Virus (HCV) is the key cause of chronic and severe liver diseases. The recent direct-acting antiviral agents have shown the clinical success on HCV-related diseases, but the rapid HCV mutations of the virus highlight the sustaining necessity to develop new drugs. p7, the viroporin protein from HCV, has been sought after as a potential anti-HCV drug target. Several classes of compounds, such as amantadine and rimantadine have been testified for p7 inhibition. However, the efficacies of these compounds are not high. Here, we screened some novel p7 inhibitors with amantadine scaffold for the inhibitor development. The dissociation constant (Kd) of 42 ARD-series compounds were determined by nuclear magnetic resonance (NMR) titrations. The efficacies of the two best inhibitors, ARD87 and ARD112, were further confirmed using viral production assay. The binding mode analysis and binding stability for the strongest inhibitor were deciphered by molecular dynamics (MD) simulation. These ARD-series compounds together with 49 previously published compounds were further analyzed by molecular docking. Key pharmacophores were identified among the structure-similar compounds. Our studies suggest that different functional groups are highly correlated with the efficacy for inhibiting p7 of HCV, in which hydrophobic interactions are the dominant forces for the inhibition potency. Our findings provide guiding principles for designing higher affinity inhibitors of p7 as potential anti-HCV drug candidates.

SummaryBax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. They may embed in the cytosolic leaflet of the lipid bilayer generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, they may comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determine a high-resolution structure of the Bax core region that forms a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

Roxadustat is a novel, small-molecule, first-in-class therapeutic that stimulates erythropoiesis by inhibiting hypoxia-inducible factor prolyl hydroxylase enzymes. This agent (roxadustat) is in clinical development for the treatment of anemia in patients with non-dialysis-dependent (NDD) and dialysis-dependent (DD) chronic kidney disease. A population pharmacokinetic analysis was undertaken to evaluate the effect of intrinsic and extrinsic factors on roxadustat pharmacokinetics. Non-linear mixed-effects models implemented in NONMEM software were fitted to 8209 pharmacokinetic samples from 2855 DD and NDD subjects enrolled in four phase III studies with roxadustat dose concentrations of 20–400 mg as orally administered tablets. Effects of intrinsic and extrinsic factors were evaluated using a stepwise covariate modeling procedure in combination with the full covariate approach, and defined no-effect boundaries for exposure were based on the difference in exposure between 70 and 100 mg of roxadustat (i.e., − 30%, + 43%). A two-compartment model with first-order absorption adequately described roxadustat pharmacokinetics, with parameter estimates (relative standard error) for apparent clearance of 1.1 (0.0223) L/h in NDD subjects, and apparent central and peripheral volumes of distribution of 14.9 (0.0278) L and 9.5 (0.0872) L, respectively. Stepwise covariate modeling identified bodyweight, dialysis status, race, and dose as statistically significant covariates on apparent clearance, and bodyweight, sex, and albumin as statistically significant covariates on apparent central volume of distribution. However, the effects of these covariates did not result in roxadustat area under the curve or maximum plasma concentration changes outside of the defined no-effect boundaries. The effects of concomitant oral iron, clopidogrel, and staggered sevelamer, calcium carbonate, or calcium acetate were investigated using a full covariate approach but did not result in roxadustat area under the curve or maximum plasma concentration changes outside of the defined no-effect boundaries. A population pharmacokinetic model was developed for the pharmacokinetics of roxadustat in the target population. None of the investigated intrinsic or extrinsic factors resulted in a significant change in roxadustat exposure outside of the defined no-effect boundaries.

Receptors in the tumor necrosis factor receptor superfamily (TNFRSF) regulate proliferation of immune cells or induce programmed cell death, and many of them are candidates for antibody-based immunotherapy. Previous studies on several death receptors in the TNFRSF including Fas, p75NTR, and DR5 showed that the transmembrane helix (TMH) of these receptors can specifically oligomerize and their oligomeric states have direct consequences on receptor activation, suggesting a much more active role of TMH in receptor signaling than previously appreciated. Here, we report the structure of the TMH of TNFR1, another well studied member of the TNFRSF, in neutral bicelles that mimic a lipid bilayer. We find that TNFR1 TMH forms a defined trimeric complex in bicelles, and no evidences of higher-order clustering of trimers have been detected. Unexpectedly, a conserved proline, which is critical for Fas TMH trimerization, does not appear to play an important role in TNFR1 TMH trimerization, which is instead mediated by a glycine near the middle of the TMH. Further, TNFR1 TMH trimer shows a larger hydrophobic core than that of Fas or DR5, with four layers of hydrophobic interaction along the threefold axis. Comparison of the TNFR1 TMH structure with that of Fas and DR5 reveals reassuring similarities that have functional implications but also significant structural diversity that warrants systematic investigation of TMH oligomerization property for other members of the TNFRSF.

Targeting T‐cells against cancer cells is a direct means of treating cancer, and has already shown great responses in clinical treatment of B‐cell malignancies. A simple way to redirect T‐cells to cancer cells is by using multispecific antibody (MsAb) that contains different arms for specifically “grabbing” the T‐cells and cancer cells; as such, the T‐cells are activated upon target engagement and the killing begins. Here, a nucleic acid mediated protein–protein assembly (NAPPA) approach is implemented to construct a MsAb for T‐cell engaging and tumor killing. Anti ‐CD19 and ‐CD3 single‐chain variable fragments (scFvs) are conjugated to different l‐DNAs with sequences that form the Holliday junction, thus allowing spontaneous assembly of homogeneous protein–DNA oligomers containing two anti‐CD19 and one anti‐CD3 scFvs. The new MsAb shows strong efficacy in inducing Raji tumor cell cytotoxicity in the presence of T‐cells with EC50 ≈ 0.2 × 10−9 m; it also suppresses tumor growth in a Raji xenograft mouse model. The data indicates that MsAbs assembled from protein–DNA conjugates are effective macromolecules for directing T‐cells for tumor killing. The modular nature of the NAPPA platform allows rapid generation of complex MsAbs from simple antibody fragments, while offering a general solution for preparing antibodies with high‐order specificity., Herein, a general solution for generating multispecific antibodies with N degree of specificity is reported. The method is used to generate a multispecific antibody containing two anti‐CD19 and one anti‐CD3 antibody fragments (scFvs). The new multispecific antibody shows strong efficacy in mediating Raji tumor cell cytotoxicity in the presence of human T‐cells both in vitro and in vivo.

HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. Recent studies showed that the transmembrane domain (TMD) of the Env forms a trimer in lipid bilayer and that disruption of the TMD could significantly alter the antigenic properties of the Env. The TMD structure has several peculiar features that remain difficult to explain. One is the presence of an arginine R696 (three in the trimer) in the middle of the TM helix. Additionally, the N- and C-terminal halves of the TM helix form trimeric cores of opposite nature (hydrophobic for the N half and hydrophilic for the C half). Here we determined the membrane partition and solvent accessibility of the TMD in bicelles that mimic a lipid bilayer. Solvent paramagnetic relaxation enhancement analysis showed that the R696 is indeed positioned close to the center of the bilayer, but, surprisingly, can exchange rapidly with water as indicated by hydrogen-deuterium exchange measurements. The solvent accessibility of R696 is likely mediated by the hydrophilic core, which also showed fast water exchange. In contrast, the N-terminal hydrophobic core showed extremely slow solvent exchange, suggesting the trimer formed by this region is extraordinarily stable. Our data explain how R696 is accommodated in the middle of the membrane while reporting the overall stability of the Env TMD trimer in lipid bilayer.

Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of protein transmembrane partition in lipid bilayer. Here, we address the above question from the perspective of protein residing in the bicelles, through systematic measurement of protein chemical shift and transmembrane partition at different lipid:detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when the q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q = 0.5 and 0.7 are very similar, but at q = 0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of protein position in lipid bilayer.

The mitochondrial calcium uniporter (MCU) plays a critical role in mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multicomponent system, including the pore-forming subunit MCU and several regulators (MICU1, MICU2, and Essential MCU REgulator, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as high-order oligomers. Mutagenesis showed that the acidic residues Asp60, Glu72, and Glu74, which appeared to mediate the interface II, as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex was disrupted in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

The mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE.Dictyostelium discoideum(Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

Targeting T-cells against cancer cells is a direct means of treating cancer, and already showed great responses in clinical treatment of B-cell malignancies. A simple way to redirect T-cells to cancer cells is using multi-specific antibody (MsAb) that contains different arms for specifically “grabbing” the T-cells and cancer cells; as such, the T-cells are activated upon target engagement and the killing begins. Here, a Nucleic Acid mediated Protein-Protein Assembly (NAPPA) approach is implemented to construct a MsAb for T-cell engaging and tumor killing. Anti -CD19 and -CD3 single-chain variable fragments (scFvs) each are conjugated to different L-DNAs with sequences that form the Holliday junction, thus allowing spontaneous assembly of homogeneous protein-DNA oligomers containing two anti-CD19 and one anti-CD3 scFvs. The new MsAb shows strong efficacy in inducing Raji tumor cell cytotoxicity in the presence of T-cells with EC50~ 0.2 nM; it also suppresses tumor growth in the Raji xenograft mouse model. The data indicate that MsAbs assembled from protein-DNA conjugates are effective macromolecules for directing T-cells for tumor killing. The modular nature of the NAPPA platform allows rapid generation of complex MsAbs from simple antibody fragments, while offering a general solution for preparing antibodies with high-order specificity.

The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize envelope proteins in viruses. Through a series of studies of the TM domains (TMDs) of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic-resolution structures of TM oligomers by NMR in bicelles that closely mimic a lipid bilayer. Our protocol overcomes hurdles typically encountered by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) when studying small TMDs. Here, we provide the details of the protocol, covering five major technical aspects: (i) a general method for producing isotopically labeled TM or membrane-proximal (MP) protein fragments that involves expression of the protein (which is fused to TrpLE) into inclusion bodies and releasing the target protein by cyanogen bromide (CNBr) cleavage; (ii) determination of the oligomeric state of TMDs in bicelles; (iii) detection of intermolecular contacts using nuclear Overhauser effect (NOE) experiments; (iv) structure determination; and (v) paramagnetic probe titration (PPT) to characterize the membrane partition of the TM oligomers. This protocol is broadly applicable for filling structural gaps of many type I/II membrane proteins. The procedures may take 3-6 months to complete, depending on the complexity and stability of the protein sample.

Presentation of membrane proteins to host immune systems has been a challenging problem due to complexity arising from the poor in vivo stability of the membrane-mimetic media often used for solubilizing the membrane proteins. The Inventors report the use of functionalized, biocompatible nanoparticles as substrates to guide the formation of proteoliposomes that can present many copies of membrane proteins in a unidirectional manner. The approach was demonstrated to present the membrane-proximal region of the HIV-1 envelope glycoprotein. These nanoparticle-supported liposomes are broadly applicable as membrane antigen vehicles for inducing host immune responses. In some instances, the technology supports generation of antibodies that do not generate an immunogenic response in comparison to conventional protein presentation (i.e., liposome).

Conference: Proceedings of the conference MOL2NET International Conference on Multidisciplinary Sciences (4th edition), 2018 is part of a year-round worldwide conference series hosted by MDPI Sciforum, Basel, Switzerland. This conference series has had organized more than 20 associated workshop series in universities worldwide: USA, France, Portugal, Spain, China, Chile, Brazil, India, etc. These workshop series run in person and/or online. Some of these workshops are the SRI-10 St Thomas University (STU)- Miami Dade College (MDC), Miami, USA; USINEWS-02 University of Minnesota, USA; BIOCHEMPHYS-01 CNAM, Paris, France; WCUCW, West Coast University, Miami, USA; IWMEDIC UDC, Coruna, Spain, LAWSCI-02, UPV/EHU, Bilbao, Spain, etc. Workshops allow both in person and/or online only publication of papers, research highlights of previous papers, letters, short reviews, etc. Topics: The topics are multidisciplinary covering, but not limited to, Chemistry (All areas), Physics, Biology, Ecology, Statistics, Bioinformatics, Education, Nanotechnology, Materials, Computational, Complex Networks, Legal, and Social sciences, etc. Statistics: This edition hosted >10 workshops that attracted >300 communications submitted by >700 authors. We organized 7 special issues published in JCR journals (MDPI editorial) such as Molecules, Entropy, and Appl. Sci. We also organized 3 bootcamps, hand-training, or capstone courses in MDC, Miami, WCU Miami, and UPV/EHU Bilbao. Proceedings Book: The present book of proceedings have been released in two versions. The first is a short version without communications including links to online versions of all communications (only 94 pages). The second one is the long version including full text of all communications and abstracts (2985 pages). Download short version from MDPI AG Sciforum publisher link: https://sciforum.net/paper/view/conference/6143. We released long versiong to Researchgate public repository: https://www.researchgate.net/project/Mol2Net-conf-series. Thank you very much to all colleagues for your kind support.

HIV-1 envelope spike (Env) is a heavily glycosylated, type I membrane protein that mediates fusion of viral and cell membranes to initiate infection. It is also a primary target of neutralizing antibodies and thus an important candidate for vaccine development. We have recently reported a nuclear magnetic resonance structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in a membrane-like environment. Taking HIV-1 as an example, we discuss here how a TM domain can anchor, stabilize, and modulate a viral envelope spike and how its high-resolution structure can contribute to understanding viral membrane fusion and to immunogen design.

Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand-induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.

Fas (CD95, Apo-1, TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. Here, we determined the nuclear magnetic resonance (NMR) structures of mouse and human Fas-TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas-TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas-TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization.

Nucleic acid–mediated protein–protein assembly (NAPPA) represents a true modular means for preparing multispecific antibodies (MsAbs). In article number https://doi.org/10.1002/advs.201900973, James J. Chou and co‐workers prepare MsAbs displaying T‐cell engaging and tumor killing properties in vivo. NAPPA allows for the preparation of individual antibody modules separately, stored in an antibody library (middle cubic grid), which can be used to assemble a large variety of MsAbs immediately before use.

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Small noncoding RNAs (sncRNAs) regulate many genes in eukaryotic cells. Hua enhancer 1 (Hen1) is a 2′-O-methyltransferase that adds a methyl group to the 2′-OH of the 3′-terminal nucleotide of sncRNAs. The types and properties of sncRNAs may vary among different species, and the domain composition, structure, and function of Hen1 proteins differ accordingly. In mammals, Hen1 specifically methylates sncRNAs called P-element–induced wimpy testis-interacting RNAs (piRNAs). However, other types of sncRNAs that are methylated by Hen1 have not yet been reported, and the structures and the substrates of mammalian Hen1 remain unknown. Here, we report that mouse Hen1 (mHen1) performs 3′-end methylation of classical piRNAs, as well as those of most noncanonical piRNAs derived from rRNAs, small nuclear RNAs and tRNAs in murine spermatogonial stem cells. Moreover, we found that a distinct class of tRNA-derived sncRNAs are mHen1 substrates. We further determined the crystal structure of the putative methyltransferase domain of human Hen1 (HsHen1) in complex with its cofactor AdoMet at 2.0 Å resolution. We observed that HsHen1 has an active site similar to that of plant Hen1. We further found that the putative catalytic domain of HsHen1 alone exhibits no activity. However, an FXPP motif at its N terminus conferred full activity to this domain, and additional binding assays suggested that the FXPP motif is important for substrate binding. Our findings shed light on its methylation substrates in mouse spermatogonial stem cells and the substrate-recognition mechanism of mammalian Hen1.

We are glad to invite all colleagues worldwide to participate on a new edition of this International Conference Series. The official title of this conference series is MOL2NET International Conference Series on Multidisciplinary Sciences. MOL2NET (the conference running title) is the acronym of the lemma of the conference: From Molecules to Networks. This running title is inspired by the possibility of multidisciplinary collaborations in science. The topics of interest include, but are not limited to, Chemistry (All areas), Mathematics (Applied), Physics (Applied), Materials Science, Nanotechnology, Biology and Life Sciences (All areas), Medicine, Biomedical Engineering, Education, along with Computer Sciences, Data Analysis, Statistics, Artificial Intelligence, Deep Learning, Bioinformatics, Systems Biology, and Complex Networks Sciences. See the following note to authors on topics outside the scope of the conference and associated workshops . The Scientific Headquarters (HQs) of this conference series are in the Faculty of Science and Technology, University of Basque Country (UPV/EHU), Biscay. However, the idea of this multidisciplinary conference emerged from the melting pot formed as the result of multiple collaborations of professors from many centers worldwide. Locally, the founders and strongest supporters of the conference are professors endowed by IKERBASQUE, Basque Foundation for Sciences, professors from the two departments Department of Organic Chemistry I and Department of Organic Chemistry II of the University of Basque Country (UPV/EHU), and professors from the Department of Computer Sciences of the University of Coruna (UDC). In addition, professors / researchers from the Center for the Study of Biological Complexity of the Virginia Commonwealth University (VCU), USA, the Natural Resources Research Institute, of the University of Minnesota, USA, and many other institutions are also founders and supporters of this conference, please see full committees lists.

Proceedings of the conference MOL2NET International Conference on Multidisciplinary Sciences (2nd edition), 2016. Year-Round conferences hosted by MDPI Sciforum, Basel, Switzerland with > 10 associated in person workshops in USA, Spain, China, Chile, Brazil, etc. Some of the workshops are tSRI-08 St Thomas University (STU)- Miami Dade College (MDC), Miami, USA; IWMEDIC-04 UDC, Coruna, Spain, etc. This edition attracted >200 communications submitted by >400 authors. Thank you very much to all colleagues for your kind support.

Viruses have developed a large variety of transmembrane proteins to carry out their infectious cycles. Some of these proteins are simply anchored to membrane via transmembrane helices. Others, however, adopt more interesting structures to perform tasks such as mediating membrane fusion and forming ion-permeating channels. Due to the dynamic or plastic nature shown by many of the viral membrane proteins, structural and mechanistic understanding of these proteins has lagged behind their counterparts in prokaryotes and eukaryotes. This chapter provides an overview of the use of NMR spectroscopy to unveil the transmembrane and membrane-proximal regions of viral membrane proteins, as well as their interactions with potential therapeutics.

The mitochondrial matrix is the supplier of cellular adenosine triphosphate (ATP). The short Ca2+-binding mitochondrial carrier (SCaMC) is one of the two mitochondrial carriers responsible for transporting ATP across the mitochondrial inner membrane. While the ADP/ATP carrier (AAC) accounts for the bulk ADP/ATP recycling in the matrix, the function of SCaMC is important for mitochondrial activities that depend on adenine nucleotides, such as gluconeogenesis and mitochondrial biogenesis. A key difference between SCaMC and AAC is that SCaMC selectively transport MgATP whereas AAC only transport free nucleotides. Here we use a combination of nuclear magnetic resonance (NMR) experiments and functional mutagenesis to investigate the structural basis of the MgATP selectivity in SCaMC. Our data revealed an MgATP binding site inside the transporter cavity, while identifying an aspartic acid residue that plays an important role in the higher selectivity for MgATP over free ATP.

The 63 amino acid polytopic membrane protein, p7, encoded by hepatitis C virus (HCV) is involved in the modulation of electrochemical gradients across membranes within infected cells. Structural information relating to p7 from multiple genotypes has been generated in silico (e.g. genotype (GT) 1a), as well as obtained from experiments in form of monomeric and hexameric structures (GTs 1b and 5a, respectively). However, sequence diversity and structural differences mean that comparison of their channel gating behaviour has not thus far been simulated. Here, a molecular model of the monomeric GT 1a protein is optimized and assembled into a hexameric bundle for comparison with both the 5a hexamer structure and another hexameric bundle generated using the GT 1b monomer structure. All bundles tend to turn into a compact structure during molecular dynamics (MD) simulations (Gromos96 (ffG45a3)) in hydrated lipid bilayers, as well as when simulated at ‘low pH’, which may trigger channel opening according to some functional studies. Both GT 1a and 1b channel models are gated via movement of the parallel aligned helices, yet the scenario for the GT 5a protein is more complex, with a short N-terminal helix being involved. However, all bundles display pulsatile dynamics identified by monitoring water dynamics within the pore., Graphical abstract, Highlights • Genotype specific structural features of hexameric bundles of p7 of HCV • In silico built models of genotype 1a bundle compared with models of genotypes 1b and 5a from NMR • Genotype 1a and 1b channels are gated via movement of the parallel aligned helices. • Complex scenario for genotype 5a bundle model • All bundles show pulsatile dynamics.

Nuclear magnetic resonance (NMR) has been an important source of structural restraints for solving structures of oligomeric transmembrane domains (TMDs) of cell surface receptors and viral membrane proteins. In NMR studies, oligomers are assembled using inter-protomer distance restraints. But, for oligomers that are higher than dimer, these distance restraints all have two-fold directional ambiguity, and resolving such ambiguity often requires time-consuming trial-and-error calculations using restrained molecular dynamics (MD) with simulated annealing (SA). We report an Exhaustive Search algorithm for Symmetric Oligomer (ExSSO), which can perform near-complete search of the symmetric conformational space in a very short time. In this approach, the predetermined protomer model is subject to full angular and spatial search within the symmetry space. This approach, which can be applied to any rotationally symmetric oligomers, was validated using the structures of the Fas death receptor, the HIV-1 gp41 fusion protein, the influenza proton channel, and the MCU pore. The algorithm is able to generate approximate oligomer solutions quickly as initial inputs for further refinement using the MD/SA method.

The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.

SummaryModulation of cellular energy expenditure is fundamental to normal and pathological cell growth and differentiation. Mitochondria stores energy as a proton gradient across their inner membrane. Uncoupling proteins (UCPs) can dissipate the gradient to produce heat or regulate metabolite fluxes. UCP-mediated proton currents require fatty acids (FAs) and are blocked by nucleotides, but the molecular basis of these processes is unknown. We find, by nuclear magnetic resonance and functional mutagenesis, that UCP2 can bind FAs laterally through its peripheral site, and this intramembrane molecular recognition is essential for UCP2-catalyzed FA flipping across the membrane, which in turn is essential for proton translocation. The antagonist GDP binds inside the UCP2 cavity and perturbs its conformation, which can displace FA from the peripheral site as a mean of inhibiting proton currents. Our data provide a biophysical perspective of the intricate interplay of UCPs, FA, and nucleotides in determining proton fluxes in mitochondria.