A study on the influence of fast amide exchange on the accuracy of 15N relaxation rate constants

15N relaxation rates of amide moieties provide insight both into global as well as local backbone dynamics of peptides and proteins. As the differences in the relaxation rates in general are small, their accurate determination is of prime importance. One potential source of error is fast amide exchange. It is well known that in its presence the effects of saturation transfer and H/D exchange may result in erroneous apparent relaxation rates R 1 and R 2. Here, the extent of these errors is rigorously examined. Theoretical considerations reveal that even when saturation effects are absent, H/D exchange will easily result in significant deviations from the true values. In particular overestimations of up to 10 % in R 1 and up to 5 % in R 2 are observed. An alternative scheme for fitting the relaxation data to the corresponding exponentials is presented that in the best cases not only delivers more accurate relaxation rates but also allows extracting estimates for the exchange rates. The theoretical computations were tested and verified for the case of ubiquiti

A study on the influence of fast amide exchange on the accuracy of 15N relaxation rate constants

15N relaxation rates of amide moieties provide insight both into global as well as local backbone dynamics of peptides and proteins. As the differences in the relaxation rates in general are small, their accurate determination is of prime importance. One potential source of error is fast amide exchange. It is well known that in its presence the effects of saturation transfer and H/D exchange may result in erroneous apparent relaxation rates R1 and R2. Here, the extent of these errors is rigorously examined. Theoretical considerations reveal that even when saturation effects are absent, H/D exchange will easily result in significant deviations from the true values. In particular overestimations of up to 10% in R1 and up to 5% in R2 are observed. Alternative scheme for fitting the relaxation data to the corresponding exponentials is presented that in the best cases not only delivers more accurate relaxation rates but also allows extracting estimates for the exchange rates. The theoretical computations were tested and verified for the case of ubiquitin

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution NMR

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin, the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well-ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10% residual proton pumping activity, are less well ordered suggesting a link between side chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR

Xanthones and Xanthone O\textit{O}‐β\textit{β}‐D‐Glucosides from the Roots of Polygala azizsancarii\textit{Polygala azizsancarii} Dönmez

Nine xanthone derivatives (1–9) were isolated from the roots of Polygala azizsancarii, which is a narrow endemic species for the flora of Türkiye. Based on all of the evidence, the structures of 1–9 were established as two previously undescribed xanthone O-glucosides, 3-O-β-D-glucopyranosyloxy-1,6-dihydroxy-2,5,7-trimethoxyxanthone (1), 3-O-β-D-glucopyranosyloxy-1,6-dihydroxy-2,7-dimethoxyxanthone (2), and seven previously described xanthones, 1,3,6-trihydroxy-2,5,7-trimethoxyxanthone (3), 1,3,6-trihydroxy-2,7-dimethoxyxanthone (4), 1,2,3,4,7-pentamethoxyxanthone (5), 1,3-dihydroxy-2,5,6,7-tetramethoxyxanthone (6), 1,3-dihydroxy-4,7-dimethoxyxanthone (7), 1,7-dihydroxy-3-methoxyxanthone (8), and 1,7-dihydroxy-2,3-methylenedioxyxanthone (9). The structures of the compounds were determined by spectroscopic methods, including 1D-NMR (1H-NMR, 13C-NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC, INADEQUATE), and HR-MS. The solid-state structures of 1–4, including the absolute configurations of the stereogenic carbons of the sugar moiety in 1 and 2, were established by X-ray crystal-structure analyses. For the newly described compounds, the trivial names sancarosides A (1) and B (2) are proposed

Unexpected dynamics in femtomolar complexes of binding proteins with peptides

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders

Side‐chain dynamics of the <scp>α_1B‐</scp>adrenergic receptor determined by <scp>NMR</scp> via methyl relaxation

G protein‐coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~μs–ms). On a faster timescale (~ps–ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side‐chain dynamics for GPCRs. Here, we investigate the rapid side‐chain dynamics of a thermostabilized α$_{1B}$‐adrenergic receptor (α$_{1B}$‐AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ‐TIA). Despite the differences in the ligands, the receptor's overall side‐chain dynamics are very similar, including those of the apo form. However, ρ‐TIA increases the flexibility of Ile176$^{4×56}$ and possibly of Ile214$^{5×49}$, adjacent to Pro215$^{5×50}$ of the highly conserved P$^{5×50}$I$^{3×40}$F$^{6×44}$ motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side‐chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α$_{1B}$‐AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein‐surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor‐wide side‐chain dynamics in GPCRs to gain functional insights

Phenolic Substitution in Fidaxomicin: A Semisynthetic Approach to Antibiotic Activity Across Species

Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50 years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M. tuberculosis, C. difficile, and the Gram-negative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho-methyl Fdx also exhibited activity against the Gram-negative C. crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics

Discovery of a small molecule ligand of FRS2 that inhibits invasion and tumor growth

Purpose: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. Methods: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. Results: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. Conclusions: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors. Keywords: Bioactive small molecule compound; Cell invasion; FGFR; FRS2; Protein–protein interaction interference; Thermal proteome profilin

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond (‘H-latch’), altering the flexibility of the α2–α3 and β2–α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity