The Minimal Domain of Adipose Triglyceride Lipase (ATGL) Ranges until Leucine 254 and Can Be Activated and Inhibited by CGI-58 and G0S2, Respectively

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated

Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment

Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jαβ) coupling providing enhanced resolution for the Cα resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42 kDa protein using a single HNCA experiment

The structural determinants of PH domain-mediated regulation of Akt revealed by segmental labeling

Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here, we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties

The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes

Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2Å and in complex with phenylmethylsulfonyl fluoride at 1.8Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism

The Kinetochore-Bound Ska1 Complex Tracks Depolymerizing Microtubules and Binds to Curved Protofilaments

To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule-binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments.Kinship Foundation. Searle Scholars ProgramLeukemia & Lymphoma Society of AmericaNational Institute of General Medical Sciences (U.S.) (Grant GM088313)MIT Faculty Start-up Fun

Inhibiting Fungal Multidrug Resistance by Disrupting an Activator-Mediator Interaction

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a transcription factor-binding site in Mediator as a novel therapeutic strategy in fungal infectious disease