Enzymic transformations of esterified linoleate (C18:2ω6) in acylceramides help to seal the mammalian skin permeability barrier by promoting Michael addition to protein thiols via the synthesis of 9,10-trans-epoxy-11E-13-oxo and 9,10-erythro-dihydroxy-11E-13-oxo oxidation products. In modeling the conjugation of these oxidized linoleates with cysteine and glutathione, we observed anomalous chromatographic and proton NMR results for the thiol conjugates of the dihydroxy-ketones. Adduction eliminates the 11,12 double bond and allows spontaneous C9-C13 cyclization to hemiketal derivatives. This produces a polar diastereomer that chromatographs as interconverting hemiketal species and a less polar diastereomer favored as a single hemiketal and stereochemically pure as indicated by proton NMR. Structural assignments were aided by precedents from carbohydrate research. For the stable diastereomer, COSY analysis revealed a highly unusual 6-bond J-coupling between the ring H10 proton and the two H14 protons on the omega-side chain, its detection facilitated by the rigidity of the structure and well-dispersed chemical shifts. Hemiketal formation on protein thiol-adducted oxidized acylceramides in the skin barrier should alter the shape and structure and influence access to enzymes or other proteins. Given the common occurrence of hemiketals and hemiacetals in natural products and synthetic chemistry, the current findings should contribute to their secure structural analysis and characterization.

Lipocalin family proteins have been shown to bind a vast array of small molecules and have subsequently been adapted to selectively bind specific ligands. In this study, candesartan, an antihypertension drug, was identified to bind mouse and human siderocalin in biomolecular NMR experiments, allowing for structural insights into the candesartan-siderocalin interaction. The ligand binding site was determined through an integrative structural biology approach using in silico ligand docking guided by NMR experiments. Building on this structurally informed binding model, we used rational protein design to modulate the binding pocket for increased or decreased ligand binding affinity. The predicted mutations were evaluated in vitro using isothermal titration calorimetry. This resulted in a mutant with a 50-fold increase in binding affinity in addition to a second mutant with a five-fold decrease in binding affinity. Thus, siderocalins have potential as a scaffold for creation of various ligand binding-based tools, including drug scavengers.

The lipocalin protein family is a structurally conserved group of proteins with a variety of biological functions defined by their ability to bind small molecule ligands and interact with partner proteins. One member of this family is siderocalin, a protein found in mammals. Its role is discussed in inflammatory processes, iron trafficking, protection against bacterial infections and oxidative stress, cell migration, induction of apoptosis, and cancer. Though it seems to be involved in numerous essential pathways, the exact mechanisms are often not fully understood. The NMR backbone assignments for the human siderocalin and its rat ortholog have been published before. In this work we describe the backbone NMR assignments of siderocalin for another important model organism, the mouse - data that might become important for structure-based drug discovery. Secondary structure elements were predicted based on the assigned backbone chemical shifts using TALOS-N and CSI 3.0, revealing a high content of beta strands and one prominent alpha helical region. Our findings correlate well with the known crystal structure and the overall conserved fold of the lipocalin family.

Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.

In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially due to the lack of comprehensive datasets including a structural readout. In addition, the recent breakthrough in structure prediction through deep learning demands an update of computational deletion mutation prediction. In this study, we deleted individually every residue of a small α-helical sterile alpha motif domain and investigated the structural and thermodynamic changes using 2D NMR spectroscopy and differential scanning fluorimetry. Then, we tested computational protocols to model and classify observed deletion mutants. We show a method using AlphaFold2 followed by RosettaRelax performs the best overall. In addition, a metric containing pLDDT values and Rosetta ΔΔG is most reliable in classifying tolerated deletion mutations. We further test this method on other datasets and show they hold for proteins known to harbor disease-causing deletion mutations.

Davidson's plum is a native Australian fruit used traditionally as food and medicine. The fruit contains anthocyanins as glucosides and sambubiosides of cyanidin and peonidin (691 mg/100 g of dried pulp) with rutin and quercetin glycosides (193 mg/100 g). Dietary supplementation of Davidson's plum at approximately 8 mg anthocyanins/kg/day in rats attenuated the signs of metabolic syndrome induced by high-carbohydrate, high-fat diet. Davidson's plum reduced visceral fat accumulation, total abdominal fat weight, size of retroperitoneal adipocytes, and plasma triglycerides and non-esterified fatty acids, normalised blood pressure, reduced left ventricular stiffness, decreased infiltration of inflammatory cells in both left ventricle and liver, decreased collagen deposition in heart, and reduced both fat vacuoles in liver and obesity-induced degeneration of knee cartilage. There were no changes in glucose tolerance with treatment. Davidson's plum reduced colonic Clostridiaceae spp, and increased Turicibacter spp. and Akkermansia muciniphila. Our findings indicate that Davidson's plum is a potential complementary treatment for metabolic syndrome.

The N-terminus of S. aureus staphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SC·ProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1→R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D with K D ~50 - 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1→R7) bound Frag D with K D ~7 - 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1→R7) for Frag D binding, with a 1:3 stoichiometry and K D ~7 - 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1→R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.

Dietary exposure to aflatoxin B1 (AFB1) is a significant risk factor for developing hepatocellular carcinomas (HCCs). Following ingestion and bioactivation by microsomal P450s, AFB1 reacts with the N7‐position of guanine, leading to formation of highly genotoxic AFB1‐Fapy‐dG adducts. AFB1 5′‐interface intercalation stabilizes DNA duplex significantly through strong base‐stacking interactions with neighbor base‐pairs. Also being sterically bulky DNA lesion, it was unexpectedly found to be excised by DNA glycosylase NEIL1, base‐excision repair enzyme, from DNA in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. We hypothesized that the DNA sequence context in which the AFB1‐Fapy‐dG adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1‐initiated repair, ultimately contributing towards AFB1associated mutational spectrum. To test this, site‐specific AFB1‐Fapy‐dG adducts were synthesized in three sequence contexts where 5′ neighbor base was varied. We observed differential DNA thermal stability specific to 5′‐neighbor base‐pair using UV absorbance and NMR‐based melting studies. Furthermore, sequence‐dependent stability differences also guided NEIL1‐mediated base removal tendency where single turnover kinetic analyses showed an inverse correlation between the modified duplex stability and the NEIL1‐catalyzed excision rates.

The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates. NMR studies identified residues involved specifically in interactions with G4 DNA. Three of the amino acids in the WRN RQC domain that exhibited the largest G4-specific changes in NMR signal were then mutated alone or in combination. Mutating individual residues implicated in G4 binding had a modest effect on WRN binding to DNA, decreasing the preference for G4 substrates by ∼25%. Mutating two G4-interacting residues (T1024G and T1086G) abrogated preferential binding of WRN to G4 DNA. Very modest decreases in G4 DNA-stimulated ATPase activity were observed for the mutant enzymes. Most strikingly, G4 unwinding by WRN was inhibited ∼50% for all three point mutants and >90% for the WRN double mutant (T1024G/T1086G) relative to normal B-form dsDNA substrates. Our work has helped to identify residues in the WRN RQC domain that are involved specifically in the interaction with G4 DNA.

The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the 13 C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum.

G‐quadruplex (G4) DNA is a specialized DNA structure that is now well established to occur in vivo . G4‐DNA structures present a significant block for replication, and must be overcome to complete the process efficiently. G4 motifs have been shown to occur at functional regions of prokaryotic and eukaryotic genomes, like promoters of oncogenes, replication origins and telomeres. G4 sequences are also enriched at chromosomal breakpoints in multiple types of cancer. The Werner's syndrome protein (WRN) is a RecQ‐family helicase that has been implicated in efficiently unwinding G4‐DNA structures during replication in humans. The aim of our study was to identify the residues of WRN that were involved in this role. Using a c‐Myc G4‐DNA model sequence and recombinant WRN, we determined that the RecQ‐C‐terminal (RQC) domain of WRN imparted a 2‐fold preference for binding to G4‐DNA relative to non‐G4 DNA substrates. NMR spectroscopic studies were performed using 15 N‐labeled RQC‐domain in DNA titration experiments where both non‐structured and G4‐DNA substrates were used to identify residues that are involved specifically in interacting with G4‐DNA. Residues that showed the largest G4‐specific changes in NMR signal were chosen for further validation. Point mutations were made in these RQC‐domain residues in the WRN construct 500–1092, to generate single‐mutant proteins WRN T1024G , WRN L1063G , WRN T1086G , and a double‐mutant WRN T1024G/T1086G . The mutant proteins were studied in detail for their ATPase activity, ability to unwind G4‐DNA and their relative affinities towards non‐structured or G4‐DNA substrates. ATPase activity of all mutant proteins was reduced compared to wild‐type. The effect was moderate when nonG4‐DNA was used, and most pronounced for the double mutant when G4‐ssDNA was used to stimulate ATPase activity (activity was only 40% of wild‐type). The helicase/DNA‐unwinding activity of all mutant proteins was relatively unaffected on a non‐G4‐DNA substrate. However, for the WRN T1024G mutant, this activity was reduced by ~50% on a G4‐DNA substrate, while for the double mutant, it was drastically reduced (by 90%) on G4‐DNA as compared to wild‐type. DNA‐binding affinity of all mutant proteins except WRN T1024G/T1086G was relatively unaffected for both control and G4‐DNA substrates. Binding preference towards G4‐DNA was reduced only moderately for the single mutants. In the case of the double mutant, the preference for G4‐DNA was reduced from ~2‐fold (in wild‐type) to ~1.1‐fold. Our work has, thus, for the first time, helped identify the residues of WRN that are involved specifically in its interaction with G4‐DNA, and has laid the groundwork for future studies on properties of RecQ‐helicases, which enable several of them to unwind G‐quadruplex DNA. Support or Funding Information Grants GM084460, CA183895

The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non‐clathrin coat on vesicles departing the trans‐Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para‐ or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 β4 C‐terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C‐terminus, which binds directly and specifically to the C‐terminal β4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4‐coated vesicles in cell biology, and especially in neuronal function.

(DDD) duplexes with incorporated 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC), we inadvertently failed to reference two publications by others who had reported structures of the DDD with single 5hmC residues inserted per strand. Spingler and co-workers 1 reported the structures of DDDs with 5hmC replacing either C3 (5′-dCG-5hmC-GAATTCGCG-3′; two crystal forms) or C9 (5′-dCGCGAATT-5hmC-GCG-3′) at resolutions of between 1.66 and 1.99 Å. Schofield and co-workers 2 subsequently published a structure of the DDD with 5hmC replacing C9 at greater resolution (1.3 Å) and assessed the water structure around and conformational variations of the 5hmC hydroxyl moiety. Our structure of the modified DDD 5′-dCGCGAATT-5hmC-GCG-3′ had a resolution of 1.02 Å. Similar to the structures published before, the presence of 5hmC had negligible effects on the …

5-Hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) form during active demethylation of 5-methylcytosine (5mC) and are implicated in epigenetic regulation of the genome. They are differentially processed by thymine DNA glycosylase (TDG), an enzyme involved in active demethylation of 5mC. Three modified Dickerson-Drew dodecamer (DDD) sequences, amenable to crystallographic and spectroscopic analyses and containing the 5'-CG-3' sequence associated with genomic cytosine methylation, containing 5hmC, 5fC, or 5caC placed site-specifically into the 5'-T(8)X(9)G(10)-3' sequence of the DDD, were compared. The presence of 5caC at the X(9) base increased the stability of the DDD, whereas 5hmC or 5fC did not. Both 5hmC and 5fC increased imino proton exchange rates and calculated rate constants for base pair opening at the neighboring base pair A(5):T(8), whereas 5caC did not. At the oxidized base pair G(4):X(9), 5fC exhibited an increase in the imino proton exchange rate and the calculated kop. In all cases, minimal effects to imino proton exchange rates occurred at the neighboring base pair C(3):G(10). No evidence was observed for imino tautomerization, accompanied by wobble base pairing, for 5hmC, 5fC, or 5caC when positioned at base pair G(4):X(9); each favored Watson-Crick base pairing. However, both 5fC and 5caC exhibited intranucleobase hydrogen bonding between their formyl or carboxyl oxygens, respectively, and the adjacent cytosine N(4) exocyclic amines. The lesion-specific differences observed in the DDD may be implicated in recognition of 5hmC, 5fC, or 5caC in DNA by TDG. However, they do not correlate with differential excision of 5hmC, 5fC, or 5caC by TDG, which may be mediated by differences in transition states of the enzyme-bound complexes.