Targeted protein degradation via intramolecular bivalent glues.

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

ReCo: automated NGS read-counting of single and combinatorial CRISPR gRNAs.

SUMMARY: CRISPR screens are increasingly performed to associate genotypes with genotypes. So far, however, their analysis required specialized computational knowledge to transform high-throughput next-generation sequencing (NGS) data into sequence formats amenable for downstream analysis. We developed ReCo, a stand-alone and user-friendly analytics tool for generating read-count tables of single and combinatorial CRISPR library and screen-based NGS data. Together with cutadapt and bowtie2 for rapid sequence trimming and alignment, ReCo enables the automated generation of read count tables from staggered NGS reads for the downstream identification of gRNA-induced phenotypes. AVAILABILITY AND IMPLEMENTATION: ReCo is published under the MIT license and available at: https://github.com/KaulichLab/ReCo.

Deciphering the mitophagy receptor network identifies a crucial role for OPTN (optineurin) in acute myeloid leukemia.

The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy.Abbreviations: AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type.

Optimized metrics for orthogonal combinatorial CRISPR screens.

CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.

Protein import motor complex reacts to mitochondrial misfolding by reducing protein import and activating mitophagy.

Mitophagy is essential to maintain mitochondrial function and prevent diseases. It activates upon mitochondria depolarization, which causes PINK1 stabilization on the mitochondrial outer membrane. Strikingly, a number of conditions, including mitochondrial protein misfolding, can induce mitophagy without a loss in membrane potential. The underlying molecular details remain unclear. Here, we report that a loss of mitochondrial protein import, mediated by the pre-sequence translocase-associated motor complex PAM, is sufficient to induce mitophagy in polarized mitochondria. A genome-wide CRISPR/Cas9 screen for mitophagy inducers identifies components of the PAM complex. Protein import defects are able to induce mitophagy without a need for depolarization. Upon mitochondrial protein misfolding, PAM dissociates from the import machinery resulting in decreased protein import and mitophagy induction. Our findings extend the current mitophagy model to explain mitophagy induction upon conditions that do not affect membrane polarization, such as mitochondrial protein misfolding.

Integrative analysis of epigenetics data identifies gene-specific regulatory elements.

Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.

Computational prediction of CRISPR-impaired non-coding regulatory regions.

Genome-wide CRISPR screens are becoming more widespread and allow the simultaneous interrogation of thousands of genomic regions. Although recent progress has been made in the analysis of CRISPR screens, it is still an open problem how to interpret CRISPR mutations in non-coding regions of the genome. Most of the tools concentrate on the interpretation of mutations introduced in gene coding regions. We introduce a computational pipeline that uses epigenomic information about regulatory elements for the interpretation of CRISPR mutations in non-coding regions. We illustrate our analysis protocol on the analysis of a genome-wide CRISPR screen in hTERT-RPE1 cells and reveal novel regulatory elements that mediate chemoresistance against doxorubicin in these cells. We infer links to established and to novel chemoresistance genes. Our analysis protocol is general and can be applied on any cell type and with different CRISPR enzymes.

One-pot synthesis of 6-aminohexanoic acid from cyclohexane using mixed-species cultures.

6-Aminohexanoic acid (6AHA) is a vital polymer building block for Nylon 6 production and an FDA-approved orphan drug. However, its production from cyclohexane is associated with several challenges, including low conversion and yield, and severe environmental issues. We aimed at overcoming these challenges by developing a bioprocess for 6AHA synthesis. A mixed-species approach turned out to be most promising. Thereby, Pseudomonas taiwanensis VLB120 strains harbouring an upstream cascade converting cyclohexane to either є-caprolactone (є-CL) or 6-hydroxyhexanoic acid (6HA) were combined with Escherichia coli JM101 strains containing the corresponding downstream cascade for the further conversion to 6AHA. ε-CL was found to be a better 'shuttle molecule' than 6HA enabling higher 6AHA formation rates and yields. Mixed-species reaction performance with 4 g l-1 biomass, 10 mM cyclohexane, and an air-to-aqueous phase ratio of 23 combined with a repetitive oxygen feeding strategy led to complete substrate conversion with 86% 6AHA yield and an initial specific 6AHA formation rate of 7.7 ± 0.1 U gCDW -1 . The same cascade enabled 49% 7-aminoheptanoic acid yield from cycloheptane. This combination of rationally engineered strains allowed direct 6AHA production from cyclohexane in one pot with high conversion and yield under environmentally benign conditions.

Unbiased and Tailored CRISPR/Cas gRNA Libraries by SynthesizingCovalently-closed-circular (3Cs) DNA.

Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning techniques, thus coupling library diversity with distribution. Here, we provide additional technical expertise to apply our covalently-closed-circular synthesized (3Cs) gRNA library generation technology for the generation of high-quality CRISPR/Cas gRNA libraries. F1-origin of replication-containing plasmid DNA is transformed into CJ236 bacteria for single colony outgrow followed by M13KO7 bacteriophage superinfection for the production and preparation of circular dU-containing ssDNA. dU-ssDNA is annealed with homology- and gRNA-encoding DNA oligonucleotides for their T7 DNA polymerase-mediated extension to form hetero-duplexed CCC-dsDNA (3Cs-dsDNA). 3Cs-dsDNA is electroporated for the selected amplification of the newly synthesized, gRNA-containing strand. To remove wild-type plasmid remnants, the purified plasmid DNA is digested with restriction enzymes targeting the gRNA-placeholder sequence in the template DNA. Undigested plasmid is electroporated for the extraction of the final 3Cs gRNA library. Due to the absence of PCR amplification and conventional cloning steps, the 3Cs technology uncouples sequence diversity from sequence distribution, thereby generating gRNA libraries with near-uniform distribution in diversities being only limited by electroporation efficiencies.

Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.

Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.

UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells.

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.

Surface as a motion surrogate for gated re-scanned pencil beam proton therapy.

This simulation study investigated the dosimetric effectiveness and treatment efficiency of surface motion guided gating of pencil beam scanning (PBS) proton therapy for liver tumour treatments. Dedicated 4D dose calculations were performed for simulating gated treatments using 4DCT data for six patients derived from 4DMRI (4DCT(MRI)). Surface motion as a surrogate for tumour motion was extracted from the 4DMRI images and a linear internal-external correlation model applied to derive amplitude-based gating windows (GWs) of 10 and 5 mm. 4D treatments were simulated using gating and layered/volumetric rescanning (either alone or combined) and four assumed system latencies (50/100/200/500 ms) for the response time of the beam gating to the surrogate. Resulting 4D plans were compared using D5-D95 and V95 in the CTV as the primary metrics, as well as dose to the healthy liver and total treatment time. With no motion mitigation, interplay effects deteriorate the dose homogeneity by more than 30% with respect to the static reference plan, whereas with surface motion guided gating alone, this could be reduced to 12/20% and 5/10% (mean/max over all cases) for 10 mm and 5 mm GWs, respectively. Furthermore, by combining ×5 layered rescans with 5 mm GW, plan homogeneities to within 1/5% of the static references could be achieved. Dose inhomogeneities were however still pronounced for latencies ⩾200 ms but limited when ⩽100 ms. ITV volumes could be decreased by 19/25% when 10/5 mm GW was employed, leading to reductions in mean dose to the healthy liver tissue of 2.6/3.3%. Our results confirm the potential of combining gating and re-scanning (re-gating) for mitigating large tumor motions, and the potential of surface motion monitoring as a gating signal.

APP-A Novel Player within the Presynaptic Active Zone Proteome.

The amyloid precursor protein (APP) was discovered in the 1980s as the precursor protein of the amyloid A4 peptide. The amyloid A4 peptide, also known as A-beta (Aß), is the main constituent of senile plaques implicated in Alzheimer's disease (AD). In association with the amyloid deposits, increasing impairments in learning and memory as well as the degeneration of neurons especially in the hippocampus formation are hallmarks of the pathogenesis of AD. Within the last decades much effort has been expended into understanding the pathogenesis of AD. However, little is known about the physiological role of APP within the central nervous system (CNS). Allocating APP to the proteome of the highly dynamic presynaptic active zone (PAZ) identified APP as a novel player within this neuronal communication and signaling network. The analysis of the hippocampal PAZ proteome derived from APP-mutant mice demonstrates that APP is tightly embedded in the underlying protein network. Strikingly, APP deletion accounts for major dysregulation within the PAZ proteome network. Ca2+-homeostasis, neurotransmitter release and mitochondrial function are affected and resemble the outcome during the pathogenesis of AD. The observed changes in protein abundance that occur in the absence of APP as well as in AD suggest that APP is a structural and functional regulator within the hippocampal PAZ proteome. Within this review article, we intend to introduce APP as an important player within the hippocampal PAZ proteome and to outline the impact of APP deletion on individual PAZ proteome subcommunities.

APP Deletion Accounts for Age-Dependent Changes in the Bioenergetic Metabolism and in Hyperphosphorylated CaMKII at Stimulated Hippocampal Presynaptic Active Zones.

Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.

An evaluation of rescanning technique for liver tumour treatments using a commercial PBS proton therapy system.

BACKGROUND AND PURPOSE: The treatment quality of pencil beam scanned (PBS) proton therapy to mobile tumour treatments can be compromised due to interplay effects. The aim of this work is to systematically evaluate the effectiveness of rescanning for liver tumour treatments for a commercial PBS delivery system. MATERIALS AND METHODS: Plans were calculated to patient specific ITV's (2GyRBE), using spot spacings of 4 and 8mm for 1- and 3-field plans. 4D dose calculations were performed using regular and irregular motion extracted from nine 4DCT(MRI) liver datasets with 4 different starting phases. Up to 19 times adaptive-scaled layered and volumetric rescanning were simulated using beam profiles and delivery dynamics of a commercial proton therapy system. RESULTS: For small (∼10mm) motions, 3-field plans achieved CTV HI's (D5-D95) to within 8.5% (80th percentile) of the static case without rescanning. For larger motions, volumetric rescanning resulted in 4.5% improved HI in comparison to layered, but requires 5 times longer treatment times and is more sensitive to detailed plan characteristics and delivery dynamics. Increased spot spacings were found to reduce sensitivity to interplay and reduce delivery times by 60%, whilst reduced energy switching times decreased treatment time by up to 75% for volumetric rescanning without however improving plan quality. CONCLUSION: For the investigated proton therapy system, rescanning can help recover dose homogeneity under conditions of motion but, particularly for motions over 10mm, should be combined with additional motion mitigation techniques.

APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone.

The hallmarks of Alzheimer's disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aß-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.

The Amyloid Precursor Protein-A Novel Player within the Molecular Array of Presynaptic Nanomachines.

More than 20 years ago the amyloid precursor protein (APP) was identified as the precursor protein of the Aß peptide, the main component of senile plaques in brains affected by Alzheimer's disease (AD). The pathophysiology of AD, characterized by a massive loss of synapses, cognitive decline, and behavioral changes was in principle attributed to the accumulation of Aß. Within the last decades, much effort has gone into understanding the molecular basis of the progression of AD. However, little is known about the actual physiological function of APPs. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone (PAZ) highlights APP as a hitherto unknown player within the setting of the presynapse. The molecular array of presynaptic nanomachines comprising the life cycle of synaptic vesicles, exo- and endocytosis, cytoskeletal rearrangements, and mitochondrial activity provides a balance between structural and functional maintenance and diversity. The generation of genetically designed mouse models further deciphered APP as an essential player in synapse formation and plasticity. Deletion of APP causes an age-dependent phenotype: while younger mice revealed almost no physiological impairments, this condition was changed in the elderly mice. Interestingly, the proteomic composition of neurotransmitter release sites already revealed substantial changes at young age. These changes point to a network that incorporates APP into a cluster of nanomachines. Currently, the underlying mechanism of how APP acts within these machines is still elusive. Within the scope of this review, we shall construct a network of APP interaction partners within the PAZ. Furthermore, we intend to outline how deletion of APP affects this network during space and time leading to impairments in learning and memory. These alterations may provide a molecular link to the pathogenesis of AD and the physiological function of APP in the central nervous system.

Local disorder in hydrogen storage compounds: the case of lithium amide/imide.

Amides and imides of alkali metals are a very promising class of materials for use as a hydrogen-storage system, as they are able to store and release hydrogen via a chemical route at controllable temperatures and pressures. We critically revise the present picture of the atomic structure of the lightest member (LiNH(2)/Li(2)NH) by using a combined computational and experimental approach. Specifically, ab initio path integral molecular dynamics simulations and solid-state (1)H NMR techniques are combined. The results show that the presently assumed local structure might be inconsistent or at least incomplete and needs considerable revision. In particular, the Li atoms turn out to be more mobile and more disordered than suggested by structural data obtained from X-ray scattering. Also, the configuration of the hydrogen atoms, which is accessible via the NMR experiment and the corresponding first-principles calculations, is different from the previously assumed data. The computed and experimentally observed (1)H NMR parameters are in very good mutual agreement and illustrate the unusual chemical environment of the hydrogen atoms in this system. Incorporating our results on the new lithium data, we show that the effect of nuclear quantum delocalization for the hydrogen atoms is considerably reduced compared to the perfect crystal structure.