Interaction networks within disease-associated GαS variants characterized by an integrative biophysical approach.

Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein-G protein-coupled receptor complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss of function or constitutive gain of function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed and clinically relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide-binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics simulations, which observed changes in interaction networks between GαS switch regions. Additionally, saturation-transfer difference NMR spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.

A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.

Structural basis of binding the unique N-terminal domain of microtubule-associated protein 2c to proteins regulating kinases of signaling pathways.

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.

Identification of small-molecule ligand-binding sites on and in the ARNT PAS-B domain.

Transcription factors are challenging to target with small-molecule inhibitors due to their structural plasticity and lack of catalytic sites. Notable exceptions include naturally ligand-regulated transcription factors, including our prior work with the hypoxia-inducible factor (HIF)-2 transcription factor, showing that small-molecule binding within an internal pocket of the HIF-2α Per-Aryl hydrocarbon Receptor Nuclear Translocator (ARNT)-Sim (PAS)-B domain can disrupt its interactions with its dimerization partner, ARNT. Here, we explore the feasibility of targeting small molecules to the analogous ARNT PAS-B domain itself, potentially opening a promising route to modulate several ARNT-mediated signaling pathways. Using solution NMR fragment screening, we previously identified several compounds that bind ARNT PAS-B and, in certain cases, antagonize ARNT association with the transforming acidic coiled-coil containing protein 3 transcriptional coactivator. However, these ligands have only modest binding affinities, complicating characterization of their binding sites. We address this challenge by combining NMR, molecular dynamics simulations, and ensemble docking to identify ligand-binding "hotspots" on and within the ARNT PAS-B domain. Our data indicate that the two ARNT/transforming acidic coiled-coil containing protein 3 inhibitors, KG-548 and KG-655, bind to a ß-sheet surface implicated in both HIF-2 dimerization and coactivator recruitment. Furthermore, while KG-548 binds exclusively to the ß-sheet surface, KG-655 can additionally bind within a water-accessible internal cavity in ARNT PAS-B. Finally, KG-279, while not a coactivator inhibitor, exemplifies ligands that preferentially bind only to the internal cavity. All three ligands promoted ARNT PAS-B homodimerization, albeit to varying degrees. Taken together, our findings provide a comprehensive overview of ARNT PAS-B ligand-binding sites and may guide the development of more potent coactivator inhibitors for cellular and functional studies.

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation.

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop.

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.

NMR characterization of an assembling RHIM (RIP homotypic interaction motif) amyloid reveals a cryptic region for self-recognition.

The RIP homotypic interaction motif (RHIM) is an essential protein motif in inflammatory signaling and certain cell death pathways. RHIM signaling occurs following the assembly of functional amyloids, and while the structural biology of such higher-order RHIM complexes has started to emerge, the conformations and dynamics of nonassembled RHIMs remain unknown. Here, using solution NMR spectroscopy, we report the characterization of the monomeric form of the RHIM in receptor-interacting protein kinase 3 (RIPK3), a fundamental protein in human immunity. Our results establish that the RHIM of RIPK3 is an intrinsically disordered protein motif, contrary to prediction, and that exchange dynamics between free monomers and amyloid-bound RIPK3 monomers involve a 20-residue stretch outside the RHIM that is not incorporated within the structured cores of the RIPK3 assemblies determined by cryo-EM or solid-state NMR. Thus, our findings expand on the structural characterization of RHIM-containing proteins, specifically highlighting conformational dynamics involved in assembly processes.

Hemes on a string: insights on the functional mechanisms of PgcA from Geobacter sulfurreducens.

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.

Structural basis of bidirectional allostery across the heme in a cytochrome P450 enzyme.

Cytochromes P450 (CYPs) are heme-containing enzymes that are present in all kingdoms of life and share a structurally homologous, globular protein fold. CYPs utilize structures distal to the heme to recognize and coordinate substrates, while the necessary interactions with redox partner proteins are mediated at the opposite, proximal surface. In the current study, we investigated the functional allostery across the heme for the bacterial enzyme CYP121A1, which utilizes a non-polar distal-to-distal dimer interface for specific binding of its dicyclotyrosine substrate. Fluorine-detected Nuclear Magnetic Resonance (19F-NMR) spectroscopy was combined with site-specific labeling of a distal surface residue (S171C of the FG-loop), one residue of the B-helix (N84C), and two proximal surface residues (T103C and T333C) with a thiol-reactive fluorine label. Adrenodoxin was used as a substitute redox protein and was found to promote a closed arrangement of the FG-loop, similar to the addition of substrate alone. Disruption of the protein-protein interface by mutagenesis of two CYP121 basic surface residues removed the allosteric effect. Moreover, 19F-NMR spectra of the proximal surface indicate that ligand-induced allostery modulates the environment at the C-helix but not the meander region of the enzyme. In light of the high degree of structural homology in this family of enzymes, we interpret the findings from this work to represent a conserved allosteric network in CYPs.

A conserved, noncanonical insert in FIS1 mediates TBC1D15 and DRP1 recruitment for mitochondrial fission.

Mitochondrial fission protein 1 (FIS1) is conserved in all eukaryotes, yet its function in metazoans is thought divergent. Structure-based sequence alignments of FIS1 revealed a conserved, but noncanonical, three-residue insert in its first tetratricopeptide repeat (TPR) suggesting a conserved function. In vertebrates, this insert is serine (S45), lysine (K46), and tyrosine (Y47). To determine the biological role of the "SKY insert," three variants were tested in HCT116 cells for altered mitochondrial morphology and recruitment of fission mechanoenzyme DRP1 and mitophagic adaptor TBC1D15. Similar to ectopically expressed wildtype FIS1, substitution of the SKY insert with alanine (AAA) fragmented mitochondria into perinuclear clumps associated with increased mitochondrial DRP1. In contrast, deletion variants (either ∆SKY or ∆SKYD49G) elongated mitochondrial networks with reduced mitochondrial recruitment of DRP1, despite DRP1 coimmunoprecipitates being highly enriched with ΔSKY variants. Ectopic wildtype FIS1 drove co-expressed YFP-TBC1D15 entirely from the cytoplasm to mitochondria as punctate structures concomitant with enhanced mitochondrial DRP1 recruitment. YFP-TBC1D15 co-expressed with the AAA variant further enhanced mitochondrial DRP1 recruitment, indicating a gain of function. In contrast, YFP-TBC1D15 co-expressed with deletion variants impaired mitochondrial DRP1 and YFP-TBC1D15 recruitment; however, mitochondrial fragmentation was restored. These phenotypes were not due to misfolding or poor expression of FIS1 variants, although ∆SKYD49G induced conformational heterogeneity that is lost upon deletion of the regulatory Fis1 arm, indicating SKY-arm interactions. Collectively, these results support a unifying model whereby FIS1 activity is effectively governed by intramolecular interactions between its regulatory arm and a noncanonical TPR insert that is conserved across eukaryotes.

Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

NMR structure of emfourin, a novel protein metalloprotease inhibitor: Insights into the mechanism of action.

Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission.

Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in Saccharomyces cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 (KD = 12-68 µM), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss-of-function and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A), demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S. cerevisiae. These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.

Identification and Biosynthesis of an N-Glucuronide Metabolite of Camonsertib.

Camonsertib is a novel ATR kinase inhibitor in clinical development for advanced cancers targeting sensitizing mutations. This article describes the identification and biosynthesis of an N-glucuronide metabolite of camonsertib. This metabolite was first observed in human hepatocyte incubations and was subsequently isolated to determine the structure, evaluate its stability as part of bioanalytical method development and for use as a standard for estimating its concentration in Phase I samples. The N-glucuronide was scaled-up using a purified bacterial culture preparation and was subsequently isolated using preparative chromatography. The bacterial culture generated sufficient material of the glucuronide to allow for one- and two-dimensional 1H and 13C NMR structural elucidation and further bioanalytical characterization. The NOE data combined with the gradient HMBC experiment and molecular modeling, strongly suggests that the point of attachment of the glucuronide results in the formation of (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(5-(4-((1R,3r,5S)-3-hydroxy-8-oxabicyclo[3.2.1]octan-3-yl)-6-((R)-3-methylmorpholino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-1H-pyrazol-1-yl)tetrahydro-2H-pyran-2-carboxylic acid. Significance Statement This is the first report of a glucuronide metabolite of camonsertib formed by human hepatocyte incubations. This study reveals the structure of an N-glucuronide metabolite of camonsertib using detailed elucidation by one- and two-dimensional NMR after scale-up using a novel bacterial culture approach yielding significant quantities of a purified metabolite.

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions.

Nuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in the context of their applications to structural studies in biology with specific focus on protein interactions and evaluate their limitations. We provide specific examples in which these techniques can complement each other, providing new information on the same scientific case. We discuss how the field may develop and what challenges are expected in the future. Overall, the combination of NMR and MS plays an increasingly important role in integrative structural biology, assisting scientists in deciphering the three-dimensional structure of composite macromolecular assemblies.

The role of magnesium ion in the interactions between humic acid and tetracycline in solution.

Coexisting tetracycline (TC), dissolved organic matter (DOM), and metal cations in aqueous environments might form complexes and consequently affect the environmental fate of TC. In this study, the interactions among coexisting humic acid (HA), TC, and Mg(II) in solutions were investigated by equilibrium dialysis batch experiments and nuclear magnetic resonance hydrogen spectroscopy (1H NMR) characterization. In the binary systems, the dimethylamine (4Me2NH+) functional group on the A-ring of TC bound to the oxygen-containing functional groups of HA via hydrogen bond. The solution pH affected the agglomeration morphology and dissociation of the oxygen-containing functional groups of HA as well as protonation and spatial conformation of TC, which in turn affected the HA-TC interactions. The complexation sites and ratio of Mg(II) on TC affect the binding mode in the ternary system. When the TC-Mg(II) complexation ratio is 1:1, the B, C, and D rings of TC preferentially complex with Mg(II), resulting in the change of TC from an extended to a twisted conformation. At this time, Mg(II) had a weaker inhibitory effect on binding affinity between HA and TC. When the complexation ratio was 1:2, the second Mg(II) complexation deactivated the 4Me2NH + on the A ring and further stabilized TC twisted conformation, resulting in a stronger inhibitory effect on the binding of TC to HA. Under acidic conditions, the solution pH mainly caused the difficulty in forming TC-Mg(II) complexes. The inhibitory effect of Mg(II) on the binding between HA and TC is weaker than that under alkaline conditions.

Structural determinants of the interactions of catechins with Aß oligomers and lipid membranes.

The aberrant self-assembly of intrinsically disordered proteins (IDPs) into soluble oligomers and their interactions with biological membranes underlie the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease. Catechins have emerged as useful tools to reduce the toxicity of IDP oligomers by modulating their interactions with membranes. However, the structural determinants of catechin binding to IDP oligomers and membranes remain largely elusive. Here, we assemble a catechin library by combining several naturally occurring chemical modifications and, using a coupled NMR-statistical approach, we map at atomic resolution the interactions of such library with the Alzheimer's-associated amyloid-beta (Aß) oligomers and model membranes. Our results reveal multiple catechin affinity drivers and show that the combination of affinity-reducing covalent changes may lead to unexpected net gains in affinity. Interestingly, we find that the positive cooperativity is more prevalent for Aß oligomers than membrane binding, and that the determinants underlying catechin recognition by membranes are markedly different from those dissected for Aß oligomers. Notably, we find that the unanticipated positive cooperativity arises from the critical regulatory role of the gallate catechin moiety, which recruits previously disengaged substituents into the binding interface and leads to an overall greater compaction of the receptor-bound conformation. Overall, the previously elusive structural attributes mapped here provide an unprecedented foundation to establish structure-activity relationships of catechins.

The ASCC2 CUE domain in the ALKBH3-ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin.

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.

A dedicated C-6 ß-hydroxyacyltransferase required for biosynthesis of the glycolipid anchor for Vi antigen capsule in typhoidal Salmonella.

Vi antigen is an extracellular polysaccharide produced by Salmonella enterica Typhi, Citrobacter freundii, and some soil bacteria belonging to the Burkholderiales. In Salmonella Typhi, Vi-antigen capsule protects the bacterium against host defenses, and the glycan is used in a current glycoconjugate vaccine to protect against typhoid. Vi antigen is a glycolipid assembled in the cytoplasm and translocated to the cell surface by an export complex driven by an ABC transporter. In Salmonella Typhi, efficient export and cell-surface retention of the capsule layer depend on a reducing terminal acylated-HexNAc moiety. Although the precise structure and biosynthesis of the acylated terminus has not been resolved, it distinguishes Vi antigen from other known glycolipid substrates for bacterial ABC transporters. The genetic locus for Vi antigen-biosynthesis encodes a single acyltransferase candidate (VexE), which is implicated in the acylation process. Here, we determined the structure of the VexE in vitro reaction product by mass spectrometry and NMR spectroscopy to reveal that VexE catalyzes ß-hydroxyacyl-ACP dependent acylation of the activated sugar precursor, uridine-5'-diphospho-GlcNAc, at C-6 to form UDP-6-O-[ß-hydroxymyristoyl]-α-d-GlcNAc. VexE belongs to the lysophosphatidyl acyltransferase family, and comparison of an Alphafold VexE model to solved lysophosphatidyl acyltransferase structures, together with modeling enzyme:substrate complexes, led us to predict an enzyme mechanism. This study provides new insight into Vi terminal structure, offers a new model substrate to investigate the mechanism of glycolipid ABC transporters, and adds biochemical understanding for a novel reaction used in the synthesis of an important bacterial virulence factor.