Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions,itisindispensable to observe their threedimensional (3D) structures at the atomic level in aphysiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/ baculovirus system using NMR data from living cells exclusively.The method was applied to five proteins,rat calmodulin, human HRas,h uman ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein GB 1d omain. In all cases,w ec ould obtain structural information from wellresolved in-cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be astandard tool for protein structure determinations in living eukaryotic cells.F or three proteins,w ea chieved well-converged 3D structures.A mong these,t he in-cell structure of protein GB 1 domain was most accurately determined, demonstrating that ah elix-loop region is tilted away from a b-sheet compared to the conformation in diluted solution.Biomacromolecules occupy as ignificant fraction of the intracellular volume (resulting in molecular crowding) [1] in which proteins are exposed to the excluded-volume effect, specific and non-specific interactions,a nd various dynamic intracellular processes. [2] Their biophysical properties under these effects,p articularly their molecular structures at the atomic level, are not fully understood. Therefore,i ti s indispensable to elucidate their native structures and dynamics in the physiologically natural environment inside cells,and to determine whether there are differences in the threedimensional (3D) structures of the biomacromolecules in cells compared to their diluted solution state.I n-cell NMR [3] is currently the only tool with which to observe proteins and deoxyribonucleic acid (DNA) at atomic resolution in living biological systems.I ta lso provides direct observations of protein behaviors in conjunction with chemical compounds that are potential targets for drug screening inside cells. [2a,4] Although in-cell NMR studies in various eukaryotic cells have become possible by either expressing target proteins inside cells [4] or by introducing stable isotope-enriched proteins from outside, [2a, 5] high-resolution protein 3D structures have been determined only in Escherichia coli cells. [6] To date,t he achievable target-protein concentration in eukaryotic cells was too low to obtain as ufficient number of nuclear Overhauser effect (NOE)-derived distance restraints.I nt he meantime,in-cell NMR studies of human-cultured cells have revealed that the intracellular environment does indeed influence the protein folding stability [5f] and reduces the volume occupied by intrinsically disordered proteins. [7] Recently,p rotein global folds in cells were obtained by exploiting NMR chemical shifts and paramagnetic NMR effects induced by intracellularly stable lanthanoid-binding t...
By using in-cell NMR experiments, we have demonstrated that the protein folding state in cells is significantly influenced by the cellular health conditions. hAK1 was denatured in cells under stressful culture conditions, while it remained functional and properly folded in cells continuously supplied with a fresh medium.
Cyclorasins 9A5 and 9A54 are 11‐mer cyclic peptides that inhibit the Ras‐Raf protein interaction. The peptides share a cell‐penetrating peptide (CPP)‐like motif; however, only cyclorasin 9A5 can permeabilize cells to exhibit strong cell‐based activity. To unveil the structural origin underlying their distinct cellular permeabilization activities, we compared the three‐dimensional structures of cyclorasins 9A5 and 9A54 in water and in the less polar solvent dimethyl sulfoxide (DMSO) by solution NMR. We found that cyclorasin 9A5 changes its extended conformation in water to a compact amphipathic structure with converged aromatic residues surrounded by Arg residues in DMSO, which might contribute to its cell permeabilization activity. However, cyclorasin 9A54 cannot adopt this amphipathic structure, due to the steric hindrance between two neighboring bulky amino‐acid sidechains, Tle‐2 and dVal‐3. We also found that the bulkiness of the sidechains at positions 2 and 3 negatively affects the cell permeabilization activities, indicating that the conformational plasticity that allows the peptides to form the amphipathic structure is important for their cell permeabilization activities.
Cryptic ligand binding sites, which are not evident in the unligated structures, are beneficial in tackling with difficult but attractive drug targets, such as protein-protein interactions (PPIs). However, cryptic sites have thus far not been rationally pursued in the early stages of drug development. Here, we demonstrated by nuclear magnetic resonance that the cryptic site in Bcl-xL exists in a conformational equilibrium between the open and closed conformations under the unligated condition. While the fraction of the open conformation in the unligated wild-type Bcl-xL is estimated to be low, F143W mutation that is distal from the ligand binding site can substantially elevate the population. The F143W mutant showed a higher hit rate in a phage-display peptide screening, and the hit peptide bound to the cryptic site of the wild-type Bcl-xL. Therefore, by controlling the conformational equilibrium in the cryptic site, the opportunity to identify a PPI inhibitor could be improved.
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