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Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme 期刊论文
NATURE, 2020, 581 (7808) : 323-+
作者:  Nikoo, Mohammad Samizadeh;  Jafari, Armin;  Perera, Nirmana;  Zhu, Minghua;  Santoruvo, Giovanni;  Matioli, Elison
收藏  |  浏览/下载:33/0  |  提交时间:2020/07/03

Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases(1). Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes(2-4), the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 angstrom resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 angstrom shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.


Cryo-electron microscopy structures and functional and mutagenesis studies provide insights into the catalysis of triacylglycerol synthesis by human acyl-CoA diacylglycerol acyltransferase at its intramembrane active site.


  
Processive extrusion of polypeptide loops by a Hsp100 disaggregase 期刊论文
NATURE, 2020, 578 (7794) : 317-+
作者:  Zhao, Peishen;  Liang, Yi-Lynn;  Belousoff, Matthew J.;  Deganutti, Giuseppe;  Fletcher, Madeleine M.;  Willard, Francis S.;  Bell, Michael G.;  Christe, Michael E.;  Sloop, Kyle W.;  Inoue, Asuka;  Truong, Tin T.;  Clydesdale, Lachlan;  Furness, Sebastian G. B.;  Christopoulos, Arthur;  Wang, Ming-Wei;  Miller, Laurence J.;  Reynolds, Christopher A.;  Danev, Radostin;  Sexton, Patrick M.;  Wootten, Denise
收藏  |  浏览/下载:46/0  |  提交时间:2020/07/03

The ability to reverse protein aggregation is vital to cells(1,2). Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore(3,4). This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp70(4,5). However, the model is also controversial, as the necessary motor activity has not been identified(6-8) and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting(9,10). How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore(11,12). Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid  it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)(13) and degradation by the 26S proteasome(14).


A combination of optical tweezers and fluorescent-particle tracking is used to dissect the dynamics of the Hsp100 disaggregase ClpB, and show that the processive extrusion of polypeptide loops is the mechanistic basis of its activity.


  
Reaction mechanisms for barite dissolution and growth 科技报告
来源:US Department of Energy (DOE). 出版年: 2010
作者:  Stack, Andrew G.
收藏  |  浏览/下载:7/0  |  提交时间:2019/04/05
Reaction Mechanisms