Covalent Transfer Research Articles

Presence of heavy chain-hyaluronan/pentraxin 3 (HC-HA/PTX3) complex in human umbilical cord

The heavy chain (HC)-hyaluronan (HA)/pentraxin 3 (HC-HA/PTX3) complex is formed by tumor necrosis factor-stimulated gene-6 (TSG-6) catalyzing the covalent (ester bond) transfer of HC1 from inter-α-trypsin inhibitor (IαI) to HA followed by tight binding of PTX3. The presence of such a complex has been found in human amniotic membrane (AM) and is considered to be a major matrix component responsible for its anti‑inflammatory and anti‑scarring properties to promote regenerative healing. Because the therapeutic potentials of AM and umbilical cord (UC) are similar, we herein evaluated whether human UC also contains HC-HA/PTX3. Immunostaining of UC cross-sections showed abundant PTX3, HC1, HA, TSG-6, and bikunin. Western blot analysis suggested the presence of HC1 complex bound via a NaOH-sensitive bond and tightly bound to PTX3 multimer in UC and AM extracts but not in chorion and placenta extracts. HC-HA/PTX3 was purified from UC extract by successive runs of density gradient ultracentrifugation and verified the presence of HC1 but not HC2 or HC3 based on western blot analysis. These results suggest the presence of HC-HA/PTX3 complex in UC is similar to AM.

Chemical modification of hyaluronan oligosaccharides differentially modulates hyaluronan–hyaladherin interactions

The glycosaminoglycan hyaluronan (HA) is a ubiquitous, nonsulfated polysaccharide with diverse biological roles mediated through its interactions with HA-binding proteins (HABPs). Most HABPs belong to the Link module superfamily, including the major HA receptor, CD44, and secreted protein TSG-6, which catalyzes the covalent transfer of heavy chains from inter-α-inhibitor onto HA. The structures of the HA-binding domains (HABDs) of CD44 (HABD_CD44) and TSG-6 (Link_TSG6) have been determined and their interactions with HA extensively characterized. The mechanisms of binding are different, with Link_TSG6 interacting with HA primarily via ionic and CH-π interactions, whereas HABD_CD44 binds solely via hydrogen bonds and van der Waals forces. Here, we exploit these differences to generate HA oligosaccharides, chemically modified at their reducing ends, that bind specifically and differentially to these target HABPs. Hexasaccharides (HA6AN) modified with 2- or 3-aminobenzoic acid (HA6-2AA, HA6-3AA) or 2-amino-4-methoxybenzoic acid (HA6-2A4MBA), had increased affinities for Link_TSG6 compared to unmodified HA6AN. These modifications did not increase the affinity for CD44_HABD. A model of HA6-2AA (derived from the solution dynamic 3D structure of HA4-2AA) was docked into the Link_TSG6 structure, providing evidence that the 2AA-carboxyl forms a salt bridge with Arginine-81. These modeling results informed a second series of chemical modifications for HA oligosaccharides, which again showed differential binding to the two proteins. Several modifications to HA4 and HA6 were found to convert the oligosaccharide into substrates for heavy chain transfer, whereas unmodified HA4 and HA6 are not. This study has generated valuable research tools to further understand HAbiology.

A novel method to quantify TSG‐6 protein levels in synovial fluid from osteoarthritis patients

AbstractTSG‐6, the protein product of Tumor Necrosis Factor‐stimulated gene‐6, is a potential biomarker of disease activity/progression in osteoarthritis (OA). By ELISA, TSG‐6 is elevated in synovial fluid (SF) from OA patients and its catalytic activity, for the covalent transfer of inter‐α‐inhibitor heavy chains onto hyaluronan (HA), is reported to correlate with progression to total knee replacement. Here, quantitative western blot (qWB) analysis, following sequential digestion with hyaluronidase and chondroitinase enzymes, was developed for the accurate determination of TSG‐6 levels in SFs. qWB analysis of 125 OA SFs yielded values of 1.0–45.2 μg/mL (median: 3.9 μg/mL), 100–1000‐fold higher than those previously reported; TSG‐6 concentration was positively correlated with age and pain/outcome scores in male patients. The values determined, by ELISA or a heavy chain transfer activity assay, following the same digestion conditions (in 23 SFs), were both ≥10‐fold lower. Apparent TSG‐6 catalytic activity was significantly affected by the concentration of HA present in SF or added exogenously. Thus, the interactions of TSG‐6 with components of OA SF prevent accurate determination of its concentration/activity when using existing assays. However, the quantitative western blot method developed here appears more suitable for further evaluation of the utility of TSG‐6 as a biomarker in OA.

Discovery and validation of a novel inhibitor of HYPE-mediated AMPylation.

Adenosyl monophosphate (AMP)ylation (the covalent transfer of an AMP from Adenosine Triphosphate (ATP) onto a target protein) is catalyzed by the human enzyme Huntingtin Yeast Interacting Partner E (HYPE)/FicD to regulate its substrate, the heat shock chaperone binding immunoglobulin protein (BiP). HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the endoplasmic reticulum and mounting a unfolded protein response in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy towardthe treatment of various protein misfolding diseases, including neuropathy and early-onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE's AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE's enzymatic activity at scale, and provided the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.

Static charge is an ionic molecular fragment

What is static charge? Despite the long history of research, the identity of static charge and mechanism by which static is generated by contact electrification are still unknown. Investigations are challenging due to the complexity of surfaces. This study involves the molecular-scale analysis of contact electrification using highly well-defined surfaces functionalized with a self-assembled monolayer of alkylsilanes. Analyses show the elementary molecular steps of contact electrification: the exact location of heterolytic cleavage of covalent bonds (i.e., Si-C bond), exact charged species generated (i.e., alkyl carbocation), and transfer of molecular fragments. The strong correlation between charge generation and molecular fragments due to their signature odd-even effects further shows that contact electrification is based on cleavage of covalent bonds and transfer of ionic molecular fragments. Static charge is thus an alkyl carbocation; in general, it is an ionic molecular fragment. This mechanism based on cleavage of covalent bonds is applicable to general types of insulating materials, such as covalently bonded polymers. The odd-even effect of charging caused by the difference of only one atom explains the highly sensitive nature of contact electrification.

Analysis of Lysine Acetylation and Acetylation-like Acylation In Vitro and In Vivo.

Protein lysine acetylation refers to the covalent transfer of an acetyl moiety from acetyl coenzyme A to the epsilon-amino group of a lysine residue and is critical for regulating protein functions in almost all living cells or organisms. Studies in the past decade have demonstrated the unexpected finding that acetylation-like acylation, such as succinylation, propionylation, butyrylation, crotonylation, and lactylation, is also present in histones and many non-histone proteins. Acetylation and acetylation-like acylation serve as reversible on/off switches for regulating protein function while interplaying with other post-translational modifications (such as phosphorylation and methylation) in a codified manner. Lysine acetylation and acetylation-like acylation are important for regulating different cellular and developmental processes in normal and pathological states. Thus, the detection of such modifications is important for related basic research and molecular diagnostics. Traditionally, lysine acetylation is detected by autoradiography, but recent decades have seen great improvement in the quality of site-specific antibodies against acetylation (or acetylation-like acylation), thereby providing competitive alternatives to the use of radioactive acetate and acetyl-coenzyme A for in vivo and in vitro labeling, respectively. This article describes protocols for the detection of lysine acetylation and acetylation-like acylation with site-specific antibodies to complement extant autoradiography-based methods (Pelletier etal., 2017). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acylation assays in vitro Basic Protocol 2: Determination of in vivo acylation.

In-depth understanding of catalytic and adsorbing effects in polysulfides conversion and rationally designing coaxial nanofibers for Li-S batteries

Lithium‐sulfur (Li‐S) batteries are plagued by shuttling effects and slow kinetics of polysulfides conversion. Although various additives have been widely developed to adsorb polysulfides or catalyze their conversion, molecular-level insights into the correlation between catalytic activity and adsorption strength are still lacking. Therefore, a rational design of Li‐S battery cathodes is limited by the deficiency of a fundamental understanding of the interacting mechanism. Here, we report a RuSe2 nanocatalyst that has excellent catalytic activity for polysulfide conversion and unfortunately low adsorbing capability as compared to TiO2. Theoretic and experimental analyses reveal that covalent interaction and electron transfer between RuSe2 and Li2S4 weakens S–S bonds whereas strong ionic interaction of TiO2 strengthens only the adsorption of polysulfides. To reconcile their individual drawbacks and make full use of their advantages, we electrospun coaxial nanofibers with RuSe2 catalytic core and TiO2 adsorbing shell as the S cathode. The resultant RuSe2/TiO2 nanofibers can rationally maximize their individual functions of RuSe2 and TiO2 and realize an ultrahigh capacity of 1402.8 mAh g−1 at 0.1 C and an areal capacity of 7.45 mAh cm−2 under a sulfur loading of 5 mg cm−2. This work demonstrates an integrated approach to improving Li–S battery performance by strengthening catalysis via an increased covalency between active sites and polysulfides and by electrospinning catalysts and adsorbents into coaxial nano-reactors.

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Structure, function and dynamics of alphavirus capping pores.

Positive-sense single stranded RNA viruses such as coronaviruses, flaviviruses or alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells. Remodelling of the host cell membranes for formation of these organelles is coupled to the membrane association of the viral replication complexes and RNA synthesis. These viral niches allow for concentration of metabolites and proteins for the synthesis of viral RNA, also preventing its detection by the cellular innate immune system. I will present the cryo-EM structural characterization of the chikungunya virus non-structural protein 1, the viral protein responsible for RNA capping and membrane binding of the viral replication machinery, and the capping mechanism. The structures show the enzyme in its active form assembled in a monotopic membrane-associated dodecameric rings. It provides the structural basis for the coupling between membrane binding and allosteric activation of the capping enzyme. We also show different stages of the reaction pathway carried out by the enzyme, showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and SAM, how it reaches a metastable post-methylation state with SAH and m7GTP in the active site, the subsequent covalent transfer of m7GMP to nsP1 and post-reaction conformational changes triggering the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of intermediates of the reaction. Our data identify the molecular determinants allowing each transition, provide explanation for the need for the SAM methyl donor all along the pathway and new clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together our results set new ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.

Functional Analysis of Histone ADP-Ribosylation In Vitro and in Cells.

Gene regulation in the nucleus requires precise control of the molecular processes that dictate how, when, and which genes are transcribed. The posttranslational modification (PTM) of histones in chromatin is an effective means to link cellular signaling to gene expression outcomes. The repertoire of histone PTMs includes phosphorylation, acetylation, methylation, ubiquitylation, and ADP-ribosylation (ADPRylation). ADPRylation is a reversible PTM that results in the covalent transfer of ADP-ribose units derived from NAD+ to substrate proteins on glutamate, aspartate, serine, and other amino acids. Histones were the first substrate proteins identified for ADPRylation, over five decades ago. Since that time, histone ADPRylation has been shown to be a widespread and critical regulator of chromatin structure and function during transcription, DNA repair, and replication. Here, we describe a set of protocols that allow the user to investigate site-specific histone ADPRylation and its functional consequences in biochemical assays and in cells in a variety of biological systems. With the recent discovery that some cancer-causing histone mutations (i.e., oncohistone mutations) occur at functional sites of regulatory ADPRylation, these protocols may have additional utility in studies of oncology.

Covalent transfer of chemical gradients onto a graphenic surface with 2D and 3D control

Control over the functionalization of graphenic materials is key to enable their full application in electronic and optical technologies. Covalent functionalization strategies have been proposed as an approach to tailor the interfaces’ structure and properties. However, to date, none of the proposed methods allow for a covalent functionalization with control over the grafting density, layer thickness and/or morphology, which are key aspects for fine-tuning the processability and performance of graphenic materials. Here, we show that the no-slip boundary condition at the walls of a continuous flow microfluidic device offers a way to generate controlled chemical gradients onto a graphenic material with 2D and 3D control, a possibility that will allow the sophisticated functionalization of these technologically-relevant materials.

Oxygen-vacancy-induced charge localization and atomic site activation in ultrathin Bi4O5Br2 nanotubes for boosted CO2 photoreduction

Solar CO2 reduction into value-added carbon fuels emerges as a sustainable and significant approach for CO2 conversion. However, poor photoabsorption, sluggish dynamics of multi-electrons transfer and high CO2 activation barrier substantially hamper CO2 photoreduction efficiency. Herein, freestanding ultrathin Bi4O5Br2 nanotubes with abundant oxygen vacancies were initially fabricated for optimizing these crucial processes. The ultrathin-shelled topologies of open tubular scaffolds and abundant surface oxygen vacancies endow the Bi4O5Br2 nanotubes with extended photo-response region and boosted charge separation. Furthermore, the presence of oxygen vacancies alters charge density distribution and affords abundant localized electrons on the catalysts’ surface. These not only reinforce covalent interactions, electron exchange and transfer between CO2 and oxygen vacancies, but also lower the CO2 reaction energy barriers and stabilize the rate-limiting COOH* intermediate. As a result, the OV-rich Bi4O5Br2 ultrathin nanotubes exhibit an outstanding CO production rate of 19.56 µmol g−1h−1 without using any cocatalyst or sacrificial agent, approximately 11.2 times higher than the Bi4O5Br2 nanoplates counterpart. This work provides insights into the future design of ultrathin hollow scaffolds for solar fuel photosynthesis and other applications.

Insights on α-Glucose Biosensors/Carriers Based on Boron-Nitride Nanomaterials from an Atomistic and Electronic Point of View.

The interaction of α-glucose with a BN-nanosheet, BN-nanotube, and BN-fullerene, was analyzed from an atomistic and electronic point of view, to evaluate such nanostructures as possible carriers and/or biosensors of the α-glucose molecule. Adsorption energies are in the range of physisorption (-0.79 eV to -0.91 eV) for the BN-nanosheet and -nanotube, and chemisorption (-2.24 eV to -2.35 eV), for the BN-fullerene. All systems, exhibit semiconductor-like behavior and great stability according to |LUMO-HOMO| energy gap [GapLH ] and chemical potential values, respectively. For the BN-nanosheet and -nanotube, the stabilization of the complexes is through hydrogen bonds, while for BN-fullerene is through a covalent bond and charge transfer. Furthermore, the BN-fullerene is able to dissociate the α-glucose molecule, which could help to decomposer such a compound, and be used for biological applications. The data taking into consideration solvent effects have no significant impact with respect to gas phase, except in the dipole moment (Md ) where we noticed an increase up to ∼45 %. Our results suggest that BN-nanosheet and -nanotube, may act as biosensors, while BN-fullerene, may serve as a carrier or degrader of the α-glucose molecule.

Genome-Wide DNA Methylation Protocol for Epigenetics Studies.

Epigenetic modification are heritable changes in gene expression not encoded by the DNA sequence therefore playing a significant role in a broad range of biological processes and diseases.A key player of the epigenetic modifications is the DNA methylation, a process involving the covalent transfer of a methyl group to the C-5 position of the cytosine ring of DNA forming 5-methylcytosine (5mC), catalyzed by DNA methyltransferases. Altering the structure of the chromatin, DNA methylation has the potential to down-regulate gene expression.The here presented protocol shows a method to obtain DNA samples ready for NGS sequencing for genome-wide methylation analysis.

Deletion of mFICD AMPylase alters cytokine secretion and affects visual short-term learning in vivo

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial short-term learning is stronger in old mFICD−/− mice than in wild-type controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.

High-Efficient Electrochemiluminescence of Au Nanoclusters Induced by the Electrosensitizer Cu2O: The Mechanism Insights from the Electrogenerated Process.

Herein, a novel Au nanoclusters/Cu2O (Au NCs/Cu2O) heterostructure exhibited exceptionally strong electrochemiluminescence (ECL) emission, in which the p-type semiconductor Cu2O was defined as the electrosensitizer to provide the electrogenerated holes for rapidly transferring the electrogenerated hot electrons of Au NCs. Thus, the fast charge transfer of Au NCs/Cu2O was achieved by the electrosensitizer compared to the sluggish one via intramolecular covalent bond charge transfer of traditional Au NCs, resulting in a greatly higher ECL efficiency (63.8%) than that of pure Au NCs (2.7%) versus the standard [Ru(bpy)3]2+. It solved one main challenge of electrochemiluminophore-based metal NCs: high efficiency with energic charge-transport kinetics. As a proof of concept, Au NCs/Cu2O was successfully employed in an ultrasensitive ECL biosensing platform for determining the biological antioxidant glutathione with a limit of detection (LOD) as low as 6.3 pM. The heterostructure as an ECL emitter is a very promising start for guiding the rational design of efficient electrochemiluminophores in intense light-emitting devices and high-definition ECL imaging.

(Invited) Mechanistic Study of the Covalent Bond-Mediated Charge Transfer between Carbon Nanotubes and Molecular Probes

The noncovalent interaction between carbon nanotubes and molecular groups leads to hybrid systems with low stability and poorly defined structure in terms of the density of functional groups, their relative positions and orientation. Since these factors are affected by microenvironmental parameters, studying the charge transfer of such dynamic systems is challenging. Covalent attachment of molecular probes, on the other hand, disturb the π-conjugated integrity of carbon nanotubes and suppress their light emission1,2. In 2017, we explored a nondestructive covalent functionalization (NCF) that opens up new avenues to overcome the aforementioned challenges3. NCF is an effective strategy for the preparation of stable carbon nanotube hybrids with defined functionality and preserved photoluminescence property4. In this work, we have used NCF for the mechanistic study of covalent bond-mediated charge transfer between molecular probes and carbon nanotubes by manipulating the structure of the probes. Different molecular probes with the same backbone but different functionality were synthesized by reaction between aniline derivatives and cyanuric chloride (Figure 1a). The conjugation of the molecular probes to the surface of carbon nanotubes by nitrene [2+1] cycloaddition reaction resulted in hybrid systems with the preserved physicochemical properties. π conjugated system of carbon nanotubes was integrated outwards into the backbone of molecular probes upon opening the aziridine ring in the bridge position and resulted in charge transfer between these two components (Figure 1b). Due to the same chemical backbone but different number of electron donating groups, variation in the optical properties of carbon nanotubes was assigned to the contribution of these groups in doping through which a mechanism for the covalent bond-mediated charge transfer was proposed. As a proof of concept, we have demonstrated a new way to forward the π conjugated system of carbon nanotubes outwards into other (macro)molecules which results in new systems with the unique optoelectronic properties. References Cognet, L. et al. Science 2007, 316, 1465.Piao, Y. et al. Chem. 2013, 5, 840.Setaro et al. Nat Commun. 2017, 8, 14281.Godin et al. Science Advances, 2019, 5, eaax116. Figure 1

Assessing the Covalent Attachment and Energy Transfer Capabilities of Upconverting Phosphors With Cofactor Containing Bioactive Enzymes.

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Examples of biosensors utilizing the apparent energy transfer of UCPs and nanophosphors (UCNPs) with biomolecules have started to appear in the literature but very few exploit the covalent anchoring of the biomolecule to the surface of the UCP to improve the sensitivity of the systems. Here, we demonstrate a robust and versatile method for the covalent attachment of biomolecules to the surface of a variety of UCPs and UCNPs in which the UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate biomolecules with surface-accessible cysteines. Variants of BM3Heme, cytochrome C, glucose oxidase, and glutathione reductase were then attached via maleimide-thiol coupling. BM3Heme, glucose oxidase, and glutathione reductase were shown to retain their activity when coupled to the UCPs potentially opening up opportunities for biosensing applications.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein—termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Reproducibility of potential energy surfaces of organic/metal interfaces on the example of PTCDA on Ag(111).

Molecular adsorption at organic/metal interfaces depends on a range of mechanisms: covalent bonds, charge transfer, Pauli repulsion, and van der Waals (vdW) interactions shape the potential energy surface (PES), making it key to understanding organic/metal interfaces. Describing such interfaces with density functional theory requires carefully selecting the exchange correlation (XC) functional and vdW correction scheme. To explore the reproducibility of the PES with respect to the choice of method, we present a benchmark of common local, semi-local, and non-local XC functionals in combination with various vdW corrections. We benchmark these methods using perylene-tetracarboxylic dianhydride on Ag(111), one of the most frequently studied organic/metal interfaces. For each method, we determine the PES using a Gaussian process regression algorithm, which requires only about 50 density functional theory calculations as input. This allows a detailed analysis of the PESs' features, such as the positions and energies of minima and saddle points. Comparing the results from different combinations of XC functionals and vdW corrections enables us to identify trends and differences between the approaches. PESs for different computation methods are in qualitative agreement but also display significant quantitative differences. In particular, the lateral positions of adsorption geometries agree well with experiment, while adsorption heights, energies, and barriers show larger discrepancies.