Stuckey JA

References (2)

Title : Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2) - Won_2016_ACS.Chem.Biol_11_3374
Author(s) : Won SJ , Davda D , Labby KJ , Hwang SY , Pricer R , Majmudar JD , Armacost KA , Rodriguez LA , Rodriguez CL , Chong FS , Torossian KA , Palakurthi J , Hur ES , Meagher JL , Brooks CL, 3rd , Stuckey JA , Martin BR
Ref : ACS Chemical Biology , 11 :3374 , 2016
Abstract : Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively redistribute S-palmitoylated proteins toward different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The depalmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein, we report the high-resolution structure of human APT2 in complex with ML349 (1.64 A), as well as the complementary structure of human APT1 bound to ML348 (1.55 A). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, the trifluoromethyl group of ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.
ESTHER : Won_2016_ACS.Chem.Biol_11_3374
PubMedSearch : Won_2016_ACS.Chem.Biol_11_3374
PubMedID: 27748579
Gene_locus related to this paper: human-LYPLA1 , human-LYPLA2

Title : Crystal Structure of Patatin-17 in Complex with Aged and Non-Aged Organophosphorus Compounds - Wijeyesakere_2014_PLoS.One_9_e108245
Author(s) : Wijeyesakere SJ , Richardson RJ , Stuckey JA
Ref : PLoS ONE , 9 :e108245 , 2014
Abstract : Patatin is a non-specific plant lipase and the eponymous member of a broad class of serine hydrolases termed the patatin-like phospholipase domain containing proteins (PNPLAs). Certain PNPLA family members can be inhibited by organophosphorus (OP) compounds. Currently, no structural data are available on the modes of interaction between the PNPLAs and OP compounds or their native substrates. To this end, we present the crystal structure of patatin-17 (pat17) in its native state as well as following inhibition with methyl arachidonyl fluorophosphonate (MAFP) and inhibition/aging with diisopropylphosphorofluoridate (DFP). The native pat17 structure revealed the existence of two portals (portal1 and portal2) that lead to its active-site chamber. The DFP-inhibited enzyme underwent the aging process with the negatively charged phosphoryl oxygen, resulting from the loss of an isopropyl group, being within hydrogen-binding distance to the oxyanion hole. The MAFP-inhibited pat17 structure showed that MAFP did not age following its interaction with the nucleophilic serine residue (Ser77) of pat17 since its O-methyl group was intact. The MAFP moiety is oriented with its phosphoryl oxygen in close proximity to the oxyanion hole of pat17 and its O-methyl group located farther away from the oxyanion hole of pat17 relative to the DFP-bound state. The orientation of the alkoxy oxygens within the two OP compounds suggests a role for the oxyanion hole in stabilizing the emerging negative charge on the oxygen during the aging reaction. The arachidonic acid side chain of MAFP could be contained within portals 1 or 2. Comparisons of pat17 in the native, inhibited, and aged states showed no significant global conformational changes with respect to their Calpha backbones, consistent with observations from other alpha/beta hydrolases such as group VIIA phospholipase A2.
ESTHER : Wijeyesakere_2014_PLoS.One_9_e108245
PubMedSearch : Wijeyesakere_2014_PLoS.One_9_e108245
PubMedID: 25248161