A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site

Abstract

The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150–180° orients the side chain β-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a >2,000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.

Targeting ovarian cancer and endothelium with an allosteric PTP4A3 phosphatase inhibitor

“…The three-dimensional (3D) coordinates of PTP4A3 were extracted from PDB entry 5TSR [ 22 ]; the 3D coordinates of inhibitor JMS-053 were obtained from the Cambridge Crystallographic Data Centre (CCDC number 1476250) [ 19 ]. The structure of PTP4A3 was energy refined in Insight 2005 using a previously reported strategy starting with hydrogens only minimization, followed by side-chain relaxation, and finally, all atoms optimization until the norm of the gradient was <0.001 kcal/Å [ 51 ]. The RMSD of the energy refined structure versus the starting X-ray structure was 1.29 Å, which was well within crystallographic resolution.…”

“…This cavity was chosen for docking studies given that its location was removed from the active site, yet in contact with the critical WPD loop. Docking of the inhibitor in this site was guided by the constant scoring of favorable and unfavorable intermolecular, atom-atom contacts using the HINT program (implemented in Discovery Studio) as described previously [ 51 ]. Briefly, an systematic optimization of the binding pose applied iterative steps and manual adjustments to the inhibitor in Cartesian space, as well as to inhibitor bond torsions, in conjunction with adjustments to amino acid side chain torsions.…”

Modified carbazoles destabilize microtubules and kill glioblastoma multiform cells

“…Carbazole structures were built and initially optimized with the MM2 force field and transferred into the cff91/cvff force fields for potential assignment [35]. For molecular docking studies, we used methodology employed previously with added refinements [36]. Briefly, the carbazoles were docked into the energy refined 5JCB X-ray co-crystal structure to examine all translation and rotational steric complementarity between the most potent ligand of the carbazole series, 20, its conformational isomers, and the side chains of the energy-refined podophyllotoxin binding site.…”

Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy Structure

“…Cryo-EM samples prepared from nonfouling mixed-affinity monolayers of this type have been shown to be appropriate for either purified proteins or target proteins present within clarified lysates. 5 Two conjugates that are closely related analogs of UPCDC30245 19,32,33 were used as p97 bait constructs. These derivatives possess the same phenyl indole motif as UPCDC30245 that engages the D1−D2 hinge region; however, they contain a phenyl azido group in the extended side chain that is capable of undergoing a 1,3-dipolar cycloaddition reaction with a 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-poly(ethylene glycol[5000])-N-amidosuccinyldibenzocyclooctyne (DSPE−PEG5000−DBCO) to produce ligand−PEG−DSPE conjugates (Figure 1B).…”