Simulation-predicted and -explained inheritance model of pathogenicity confirmed by transgenic mice models.

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In this study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal "plugs" aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictive ability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homo-hexameric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable hetero-hexameric channels did not. From the perspective of the conformational energetics of WT and variant Cx26 hexamers, we propose that unaffected carriers could result from a conformational predominance of the WT and pore-shrinkage-incapable hetero-hexamers, while mice with homozygous variants can only harbor an unstable and dysfunctional N-termini-blocking V37M homo-hexamer. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

Full-Privacy Secured Search Engine Empowered by Efficient Genome-Mapping Algorithms.

Since the 90s, keyword-based search engines have been the only option for people to locate relevant web content through a simple query comprising one to a few keywords. These engines, whether free or paid, retained users' search queries and preferences, often to deliver targeted ads. Additionally, user-uploaded articles for plagiarism detection can further be stored as part of service providers' expanding databases for profit. Essentially, users could not search without exposing their queries to these providers. We present a new solution here: a method for searching the internet using a full article as a query without disclosing the content. Our Sapiens Aperio Veritas Engine (S.A.V.E.) uses an encoding scheme and an FM-index search, borrowed from next-generation human genome sequencing. Each word in a user's query is transformed into one of 12 "amino acids" to create a pseudo-biological sequence (PBS) on the user's device. Plagiarism checks are done by users submitting their locally created PBSs to our cloud service. This detects identical content in our database, which includes all English and Chinese Wikipedia articles and Open Access journals up to April 2021. PBSs, longer than 12 "amino acids", show accurate results with less than 0.8% false positives. Performance-wise, S.A.V.E. runs at a similar genome-mapping speed as Bowtie and is >5 orders faster than BLAST. With both standard and private modes, S.A.V.E. offers a revolutionary, privacy-first search and plagiarism check system. We believe this sets an exciting precedent for future search engines prioritizing user confidentiality. S.A.V.E. can be accessed at https://dyn.life.nthu.edu.tw/SAVE/.

3D printing for customized carbon electrodes.

Traditional carbon electrodes are made of glassy carbon or carbon fibers and have limited shapes. 3D printing offers many advantages for manufacturing carbon electrodes, such as complete customization of the shape and the ability to fabricate devices and electrodes simultaneously. Additive manufacturing is the most common 3D printing method, where carbon materials are added to the material to make it conductive, and treatments applied to enhance electrochemical activity. A newer form of 3D printing is 2-photon lithography, where electrodes are printed in photoresist via laser lithography and then annealed to carbon by pyrolysis. Applications of 3D printed carbon electrodes include nanoelectrode measurements of neurotransmitters, arrays of biosensors, and integrated electrodes in microfluidic devices.

MPCVD-Grown Nanodiamond Microelectrodes with Oxygen Plasma Activation for Neurochemical Applications.

Nanodiamonds (NDs) are a carbon nanomaterial that has a diamond core with heteroatoms and defects at the surface. The large surface area, defect sites, and functional groups on NDs make them a promising material for electrochemical sensing. Previously, we dip-coated ND onto carbon-fiber microelectrodes (CFMEs) and found increases in sensitivity, but the coating was sparse. Here, we directly grew thin films of ND on niobium wires using microwave plasma chemical vapor deposition (MP-CVD) to provide full surface coverage. ND microelectrodes show a reliable performance in neurotransmitter detection with good antifouling properties. To improve sensitivity, we oxygen plasma etched ND films to activate the surface and intentionally add defects and oxygen surface functional groups. For fast-scan cyclic voltammetry detection of dopamine, oxygen plasma-etching increases the sensitivity from 21 nA/µM to 90 nA/µM after treatment. Fouling was tested by repeated injections of serotonin or tyramine, and both ND and plasma oxidized nanodiamond (NDO) microelectrodes maintain their currents better compared to CFMEs and therefore are more antifouling. A biofouling test in brain slices shows that ND microelectrodes barely have any current drop, while the more hydrophilic NDO microelectrodes decrease more, but still not as much as CFMEs. Overall, grown ND microelectrodes are promising in neurotransmitter detection with excellent fouling resistance, whereas oxygen plasma etching slightly lowers the fouling resistance but dramatically increases sensitivity.

Carbon microelectrodes with customized shapes for neurotransmitter detection: A review.

Carbon is a popular electrode material for neurotransmitter detection due to its good electrochemical properties, high biocompatibility, and inert chemistry. Traditional carbon electrodes, such as carbon fibers, have smooth surfaces and fixed shapes. However, newer studies customize the shape and nanostructure the surface to enhance electrochemistry for different applications. In this review, we show how changing the structure of carbon electrodes with methods such as chemical vapor deposition (CVD), wet-etching, direct laser writing (DLW), and 3D printing leads to different electrochemical properties. The customized shapes include nanotips, complex 3D structures, porous structures, arrays, and flexible sensors with patterns. Nanostructuring enhances sensitivity and selectivity, depending on the carbon nanomaterial used. Carbon nanoparticle modifications enhance electron transfer kinetics and prevent fouling for neurochemicals that are easily polymerized. Porous electrodes trap analyte momentarily on the scale of an electrochemistry experiment, leading to thin layer electrochemical behavior that enhances secondary peaks from chemical reactions. Similar thin layer cell behavior is observed at cavity carbon nanopipette electrodes. Nanotip electrodes facilitate implantation closer to the synapse with reduced tissue damage. Carbon electrode arrays are used to measure from multiple neurotransmitter release sites simultaneously. Custom-shaped carbon electrodes are enabling new applications in neuroscience, such as distinguishing different catecholamines by secondary peaks, detection of vesicular release in single cells, and multi-region measurements in vivo.

Pannexin1 channels regulate mechanically stimulated but not spontaneous adenosine release.

Fast-scan cyclic voltammetry (FSCV) is a rapid technique to measure neuromodulators, and using FSCV, two modes of rapid adenosine have been discovered. Spontaneous transients occur randomly in the brain, while mechanical stimulation also causes a rapid adenosine event. Pannexin1 channels are membrane channels that transport ions, including ATP, out of the cell where it is rapidly broken down into adenosine. Pannexin 1 channels (Panx1) have a flickering mode of rapid opening and are also mechanically stimulated. Here, we test the extent to which pannexin channels, specifically pannexin1 (Panx1) channels, are responsible for rapid adenosine events. Spontaneous adenosine release or mechanosensitive adenosine release were measured using fast-scan cyclic voltammetry in hippocampal (CA1) brain slices. In global Panx1KO mice, there is no significant difference in the frequency or concentration of spontaneous adenosine release, indicating Panx1 is not a release mechanism for spontaneous adenosine. Spontaneous adenosine frequency decreased slightly after administration of a large (100 µM) dose of carbenoxolone, a nonspecific inhibitor of many pannexin and connexin channels, suggesting other hemichannels only play a small role at most. For mechanically stimulated adenosine release, the concentration of each adenosine event significantly decreased 30% in Panx1KO mice and the frequency of stimulations that evoked adenosine also decreased. The response was similar in WT mice with carbenoxolone. Thus, Panx1 is a release mechanism for mechanically stimulated adenosine release, but not the only mechanism. These results demonstrate that pannexin channels differentially regulate rapid adenosine release and could be targeted to differentially affect mechanically stimulated adenosine due to brain damage.

Helical structure motifs made searchable for functional peptide design.

The systematic design of functional peptides has technological and therapeutic applications. However, there is a need for pattern-based search engines that help locate desired functional motifs in primary sequences regardless of their evolutionary conservation. Existing databases such as The Protein Secondary Structure database (PSS) no longer serves the community, while the Dictionary of Protein Secondary Structure (DSSP) annotates the secondary structures when tertiary structures of proteins are provided. Here, we extract 1.7 million helices from the PDB and compile them into a database (Therapeutic Peptide Design database; TP-DB) that allows queries of compounded patterns to facilitate the identification of sequence motifs of helical structures. We show how TP-DB helps us identify a known purification-tag-specific antibody that can be repurposed into a diagnostic kit for Helicobacter pylori. We also show how the database can be used to design a new antimicrobial peptide that shows better Candida albicans clearance and lower hemolysis than its template homologs. Finally, we demonstrate how TP-DB can suggest point mutations in helical peptide blockers to prevent a targeted tumorigenic protein-protein interaction. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/ .

Dual-Channel Electrochemical Measurements Reveal Rapid Adenosine is Localized in Brain Slices.

Rapid adenosine signaling has been detected spontaneously or after mechanical stimulation in the brain, providing rapid neuromodulation in a local area. To measure rapid adenosine signaling, a single carbon-fiber microelectrode has traditionally been used, which limits spatial resolution and an understanding of regional coordination. In this study, we utilized dual-channel fast-scan cyclic voltammetry to measure the spontaneous or mechanically stimulated adenosine release at two electrodes placed at different spacings in hippocampal CA1 mouse brain slices. For mechanically stimulated adenosine release, adenosine can be detected up to 150 µm away from where it was stimulated, although the signal is smaller and delayed. While spontaneous adenosine transients were detected at both electrodes, only 10 percent of the events were detected concurrently, and that number was similar at 50 and 200 µm electrode spacings. Thus, most adenosine transients were not caused by the widespread coordination of release. There was no evidence of diffusion of spontaneous transients to a second electrode 50-200 µm away. This study shows that spontaneous adenosine events are very localized and thus provide only local neuromodulation. Injury, such as mechanical stimulation, allows adenosine to diffuse farther, but the neuroprotective effects are still regional. These results provide a better understanding of the spatial and temporal profiles of adenosine available to act at receptors, which is crucial for future studies that design neuroprotective treatments based on rapid adenosine signaling.

Electrochemical treatment in KOH renews and activates carbon fiber microelectrode surfaces.

Carbon fiber microelectrodes (CFMEs) are the standard electrodes for fast-scan cyclic voltammetry (FSCV) detection of neurotransmitters. CFMEs are generally used untreated but the surface can be activated with different treatments to improve electrochemical performance. In this work, we explored electrochemical treatments to clean and activate the CFME surface. We used different solution conditions for electrochemical treatment and found that electrochemical pretreatment in KOH outperforms treatment in KCl, H2O2, or HCl by accelerating the surface renewal process. The etching rate of carbon with electrochemical treatment in KOH is 37 nm/min, which is 10 times faster than that in the other solutions. Electrochemical treatment in KOH for several minutes regenerates a new carbon surface, which introduces more oxygen functional groups beneficial for adsorption and electron transfer. The KOH-treated CFMEs improved the limit of detection (LOD) to 9 ± 2 nM from 14 ± 4 nM for untreated CFMEs, and they successfully detected stimulated dopamine release in rat brain slices, demonstrating that they are stable and sensitive enough to use in biological systems. Electrochemical treatment in KOH completely restores the electrode sensitivity after biofouling. The proposed electrochemical treatment is simple and fast and can be applied prior to using CFMEs or after use to restore the surface. Thus, the method has potential to be a standard step to clean the carbon surface, or restore the sensitivity of electrodes from biofouling.

An Effective and Safe Enkephalin Analog for Antinociception.

Opioids account for 69,000 overdose deaths per annum worldwide and cause serious side effects. Safer analgesics are urgently needed. The endogenous opioid peptide Leu-Enkephalin (Leu-ENK) is ineffective when introduced peripherally due to poor stability and limited membrane permeability. We developed a focused library of Leu-ENK analogs containing small hydrophobic modifications. N-pivaloyl analog KK-103 showed the highest binding affinity to the delta opioid receptor (68% relative to Leu-ENK) and an extended plasma half-life of 37 h. In the murine hot-plate model, subcutaneous KK-103 showed 10-fold improved anticonception (142%MPE·h) compared to Leu-ENK (14%MPE·h). In the formalin model, KK-103 reduced the licking and biting time to ~50% relative to the vehicle group. KK-103 was shown to act through the opioid receptors in the central nervous system. In contrast to morphine, KK-103 was longer-lasting and did not induce breathing depression, physical dependence, and tolerance, showing potential as a safe and effective analgesic.

A1 and A2A Receptors Modulate Spontaneous Adenosine but Not Mechanically Stimulated Adenosine in the Caudate.

Adenosine is a neuromodulator, and rapid increases in adenosine in the brain occur spontaneously or after mechanical stimulation. However, the regulation of rapid adenosine by adenosine receptors is unclear, and understanding it would allow better manipulation of neuromodulation. The two main adenosine receptors in the brain are A1 receptors, which are inhibitory, and A2A receptors, which are excitatory. Here, we investigated the regulation of spontaneous adenosine and mechanically stimulated adenosine by adenosine receptors, using global A1 or A2A knockout mice. Results were compared in vivo and in brain slices' models. A1 KO mice have increased frequency of spontaneous adenosine events, but no change in the average concentration of an event, while A2A KO mice had no change in frequency but increased average event concentration. Thus, both A1 and A2A self-regulate spontaneous adenosine release; however, A1 acts on the frequency of events, while A2A receptors regulate concentration. The trends are similar both in vivo and slices, so brain slices are a good model system to study spontaneous adenosine release. For mechanically stimulated adenosine, there was no effect of A1 or A2A KO in vivo, but in brain slices, there was a significant increase in concentration evoked in A1KO mice. Mechanically stimulated release was largely unregulated by A1 and A2A receptors, likely because of a different release mechanism than spontaneous adenosine. Thus, A1 receptors affect the frequency of spontaneous adenosine transients, and A2A receptors affect the concentration. Therefore, future studies could probe drug treatments targeting A1 and A2A receptors to increase rapid adenosine neuromodulation.

Structural Similarity Image Analysis for Detection of Adenosine and Dopamine in Fast-Scan Cyclic Voltammetry Color Plots.

Fast-scan cyclic voltammetry (FSCV) is widely used for in vivo detection of neurotransmitters, but identifying analytes, particularly mixtures, is difficult. Data analysis has focused on identifying dopamine from cyclic voltammograms, but it would be better to analyze all the data in the three-dimensional FSCV color plot. Here, the goal was to use image analysis-based analysis of FSCV color plots for the first time, specifically the structural similarity index (SSIM), to identify rapid neurochemical events. Initially, we focused on identifying spontaneous adenosine events, as adenosine cyclic voltammograms have a primary oxidation at 1.3 V and a secondary oxidation peak that grows in over time. Using SSIM, sample FSCV color plots were compared with reference color plots, and the SSIM cutoff score was optimized to distinguish adenosine. High-pass digital filtering was also applied to remove the background drift and lower the noise, which produced a better LOD. The SSIM algorithm detected more adenosine events than a previous algorithm based on current versus time traces, with 99.5 ± 0.6% precision, 95 ± 3% recall, and 97 ± 2% F1 score (n = 15 experiments from three researchers). For selectivity, it successfully rejected signals from pH changes, histamine, and H2O2. To prove it is a broad strategy useful beyond adenosine, SSIM analysis was optimized for dopamine detection and is able to detect simultaneous events with dopamine and adenosine. Thus, SSIM is a general strategy for FSCV data analysis that uses three-dimensional data to detect multiple analytes in an efficient, automated analysis.

Optimization of graphene oxide-modified carbon-fiber microelectrode for dopamine detection.

Graphene oxide (GO) is a carbon-based material that is easily obtained from graphite or graphite oxide. GO has been used broadly for electrochemistry applications and our hypothesis is that GO coating a carbon-fiber microelectrode (CFME) will increase the sensitivity for dopamine by providing more adsorption sites due to the enhancement of oxygen functional groups. Here, we compared drop casting, dip coating, and electrodeposition methods to directly coat commercial GO on CFME surfaces. Dip coating did not result in much GO coating and drop casting resulted in large agglomerations that produced noisy signals and slow rise times. Electrodeposition method with cyclic voltammetry increase the current for dopamine and this method was the most reproducible and had the least noise compared to the other two coating methods. The optimized method used a triangular waveform scanned from -1.2 V to 1.5 V at 100 mV/s for 5 cycles in 0.2 mg/mL GO in water. With fast-scan cyclic voltammetry (FSCV), the optimized GO/CFME enhanced the dopamine oxidation peak two-fold. The sensitivity of the modified electrode is 41±2 nA/µM with a linear range from 25 nM to 1 µM, and a limit of detection of 11 nM. The optimized electrodes were used to detect electrically-stimulated dopamine in brain slices to demonstrate their performance in tissue. Thus, GO can be used to enhance the sensitivity of electrodes for dopamine and improve biological measurements.

CD73 or CD39 Deletion Reveals Different Mechanisms of Formation for Spontaneous and Mechanically Stimulated Adenosine and Sex Specific Compensations in ATP Degradation.

Adenosine is important for local neuromodulation, and rapid adenosine signaling can occur spontaneously or after mechanical stimulation, but little is known about how adenosine is formed in the extracellular space for those stimulations. Here, we studied mechanically stimulated and spontaneous adenosine to determine if rapid adenosine is formed by extracellular breakdown of adenosine triphosphate (ATP) using mice globally deficient in extracellular breakdown enzymes, either CD39 (nucleoside triphosphate diphosphohydrolase 1, NTPDase1) or CD73 (ecto-5'-nucleotidase). CD39 knockout (KO) mice have a lower frequency of spontaneous adenosine events than wild-type (WT, C57BL/6). Surprisingly, CD73KO mice demonstrate sex differences in spontaneous adenosine; males maintain similar event frequencies as WT, but females have significantly fewer events and lower concentrations. Examining the mRNA expression of other enzymes that metabolize ATP revealed tissue nonspecific alkaline phosphatase (TNAP) was upregulated in male CD73KO mice, but not secreted prostatic acid phosphatase (PAP) or transmembrane PAP. Thus, TNAP upregulation compensates for CD73 loss in males but not in females. These sex differences highlight that spontaneous adenosine is formed by metabolism of extracellular ATP by many enzymes. For mechanically stimulated adenosine, CD39KO or CD73KO did not change stimulation frequency, concentration, or t1/2. Thus, the mechanism of formation for mechanically stimulated adenosine is likely direct release of adenosine, different than spontaneous adenosine. Understanding these different mechanisms of rapid adenosine formation will help to develop pharmacological treatments that differentially target modes of rapid adenosine signaling, and all treatments should be studied in both sexes, given possible differences in extracellular ATP degradation.

An Efficient Timer and Sizer of Biomacromolecular Motions.

Life ticks as fast as how proteins move. Computationally expensive molecular dynamics simulation has been the only theoretical tool to gauge the time and sizes of these motions, though barely to their slowest ends. Here, we convert a computationally cheap elastic network model (ENM) into a molecular timer and sizer to gauge the slowest functional motions of structured biomolecules. Quasi-harmonic analysis, fluctuation profile matching, and the Wiener-Khintchine theorem are used to define the "time periods," t, for anharmonic principal components (PCs), which are validated by nuclear magnetic resonance (NMR) order parameters. The PCs with their respective "time periods" are mapped to the eigenvalues (λENM) of the corresponding ENM modes. Thus, the power laws t(ns) = 56.1λENM-1.6 and σ2(Å2) = 32.7λENM-3.0 can be established allowing the characterization of the timescales of NMR-resolved conformers, crystallographic anisotropic displacement parameters, and important ribosomal motions, as well as motional sizes of the latter.

Resolution-exchanged structural modeling and simulations jointly unravel that subunit rolling underlies the mechanism of programmed ribosomal frameshifting.

MOTIVATION: Programmed ribosomal frameshifting (PRF) is widely used by viruses and bacteria to produce different proteins from a single mRNA template. How steric hindrance of a PRF-stimulatory mRNA structure transiently modifies the conformational dynamics of the ribosome, and thereby allows tRNA slippage, remains elusive. RESULTS: Here, we leverage linear response theories and resolution-exchanged simulations to construct a structural/dynamics model that connects and rationalizes existing structural, single-molecule and mutagenesis data by resolution-exchanged structural modelling and simulations. Our combined theoretical techniques provide a temporal and spatial description of PRF with unprecedented mechanistic details. We discover that ribosomal unfolding of the PRF-stimulating pseudoknot exerts resistant forces on the mRNA entrance of the ribosome, and thereby drives 30S subunit rolling. Such motion distorts tRNAs, leads to tRNA slippage, and in turn serves as a delicate control of cis-element's unwinding forces over PRF. AVAILABILITY AND IMPLEMENTATION: All the simulation scripts and computational implementations of our methods/analyses (including linear response theory) are included in the bioStructureM suite, provided through GitHub at https://github.com/Yuan-Yu/bioStructureM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exploring the Steric and Electronic Factors Governing the Regio- and Enantioselectivity of the Pd-Catalyzed Decarboxylative Generation and Allylation of 2-Azaallyl Anions.

The impact of the steric and electronic factors in both the para-substituted benzaldimine and 2,2-diarylglycine components on the regioselectivity and enantioselectivity of the palladium-catalyzed decarboxylative allylation of allyl 2,2-diarylglycinate aryl imines was explored. These studies revealed that using 2,2-di(2-methoxyphenyl)glycine as the amino acid linchpin allowed for the exclusive synthesis of the desired homoallylic benzophenone imine regioisomers, independent of the nature of the imine moiety, in typically high yields. The resulting enantiomeric ratios, however, are slightly decreased in comparison to the transformations involving the corresponding allyl 2,2-diphenylglycinate imines, but this is more than balanced out by the increases in yield and regioselectivity. Overall, these studies suggest a general strategy for the highly regioselective functionalization of 2-azaallyl anions.

DynOmics: dynamics of structural proteome and beyond.

DynOmics (dynomics.pitt.edu) is a portal developed to leverage rapidly growing structural proteomics data by efficiently and accurately evaluating the dynamics of structurally resolved systems, from individual molecules to large complexes and assemblies, in the context of their physiological environment. At the core of the portal is a newly developed server, ENM 1.0, which permits users to efficiently generate information on the collective dynamics of any structure in PDB format, user-uploaded or database-retrieved. ENM 1.0 integrates two widely used elastic network models (ENMs)-the Gaussian Network Model (GNM) and the Anisotropic Network Model (ANM), extended to take account of molecular environment. It enables users to assess potentially functional sites, signal transduction or allosteric communication mechanisms, and protein-protein and protein-DNA interaction poses, in addition to delivering ensembles of accessible conformers reconstructed at atomic details based on the global modes of motions predicted by the ANM. The 'environment' is defined in a flexible manner, from lipid bilayer and crystal contacts, to substrate or ligands bound to a protein, or surrounding subunits in a multimeric structure or assembly. User-friendly interactive features permit users to easily visualize how the environment alter the intrinsic dynamics of the query systems. ENM 1.0 can be accessed at http://enm.pitt.edu/ or http://dyn.life.nthu.edu.tw/oENM/.

Structural Insights into Substrate Recognition by Clostridium difficile Sortase.

Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26-PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB.

Structure and function of chicken interleukin-1 beta mutants: uncoupling of receptor binding and in vivo biological activity.

Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1ß) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1ß signaling in vitro but can antagonize wild-type (WT) IL-1ß. Crystal structures of T117A, E118A, and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1ß. Molecular dynamics simulations of IL-1ß bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1ß/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1ß that are essential to immune responses and elucidate a mechanism by which IL-1ß activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.