Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-i...Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo- dimer of Uch37, each of the catalytic domains was blocking the other＇s ubiquitin （Ub）-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combi- nation of mutagenesis, biochemical, NMR, and small- angle X-ray scattering （SAXS） techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubi- quitin-7-amino-4-methylcoumarin （Ub-AMC） of de-ubiq- uitinating enzymes. Uch37AHb＇Hc＇KEKE, a truncation removal of the C-terminal extension region （residues 256- 329） converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C （Rpn13 residues 270- 407） could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 com- plex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS （TR-SAXS） and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1：1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.展开更多
TRAF4 is a unique member of TRAF family,which is es-sential for innate immune response,nervous system and other systems.In addition to being an adaptor protein,TRAF4 was identifi ed as a regulator protein in recent st...TRAF4 is a unique member of TRAF family,which is es-sential for innate immune response,nervous system and other systems.In addition to being an adaptor protein,TRAF4 was identifi ed as a regulator protein in recent studies.We have determined the crystal structure of TRAF domain of TRAF4(residues 292-466)at 2.60Åresolution by X-ray crystallography method.The trimericly assembled TRAF4 resembles a mushroom shape,containing a super helical“stalk”which is made of three right-handed intertwinedαhelixes and a C-terminal“cap”,which is divided at resi-due L302 as a boundary.Similar to other TRAFs,both intermolecular hydrophobic interaction in super helical“stalk”and hydrogen bonds in“cap”regions contribute directly to the formation of TRAF4 trimer.However,differ-ing from other TRAFs,there is an additional fl exible loop(residues 421-426),which contains a previously identifi ed phosphorylated site S426 exposing on the surface.This S426 was reported to be phosphorylated by IKKαwhich is the pre-requisite for TRAF4-NOD2 complex formation and thus to inhibit NOD2-induced NF-κB activation.Therefore,the crystal structure of TRAF4-TRAF is valuable for under-standing its molecular basis for its special function and provides structural information for further studies.展开更多
基金This work was supported by the National Basic Research Program （973 Program） （Nos. 2014CB910400 and 2013CB911103）, the Ministry of Health of China （Grant No. 2013ZX10004-602）, National Key Technology Research and Development Program of the Ministry of Science and Technology of China （Grant No. 2014BAI07B02） and the National Natural Science Foundation of China （Grant Nos. 31330019, 31200559）. We would like to thank Dr. Li-Qin Li from the Institute of High Energy Physics, CAS and Professor Robert E. Cohen for the gen- erous gift of the hUch37 （C88A） plasmid and Xiaoxia Yu and Yu- anyuan Chen at the Protein Science Core Facility of IBP for their technical help with the AUC and SPR experiments. The authors would also like to thank the staff at beamline BL13.3.1 at ALS for their technical support with the SAXS data collection. BL13.3.1 is supported in part by the DOE program Inte- grated Diffraction Analysis Technologies （IDAT） and the DOE pro- gram Molecular Assemblies Genes and Genomics Integrated Efficiently （MAGGIE） under Contract Number DE-AC02-05CH11231 with the DOE. The ALS is supported by the Director, Office of Sci- ence, Office of Basic Energy Sciences of the DOE under Contract No. DE-AC02-05CH11231. Use of the Advanced Photon Source, an Office of the Science User Facility operated for the U.S. Department of Energy （DOE） Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02- 06CHl1357. BioCAT was supported by grants from the National Center for Research Resources （2P41RR008630-17） and the National Institute of General Medical Sciences （9 P41 GM103622- 17） from the National Institutes of Health. The authors would like to thank the staff at 121D and 181D for the setup support.
文摘Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo- dimer of Uch37, each of the catalytic domains was blocking the other＇s ubiquitin （Ub）-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combi- nation of mutagenesis, biochemical, NMR, and small- angle X-ray scattering （SAXS） techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubi- quitin-7-amino-4-methylcoumarin （Ub-AMC） of de-ubiq- uitinating enzymes. Uch37AHb＇Hc＇KEKE, a truncation removal of the C-terminal extension region （residues 256- 329） converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C （Rpn13 residues 270- 407） could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 com- plex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS （TR-SAXS） and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1：1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
基金the Ministry of Health of China(grant 2013ZX10004-602)the National Basic Research Program(973 Program)(Nos.2013CB911103,2009DFB30310,2009CB918803 and 2011CB911103)the National Natural Science Foundation of China(Grant Nos.31270795,31200559,31070660 and 31021062).
文摘TRAF4 is a unique member of TRAF family,which is es-sential for innate immune response,nervous system and other systems.In addition to being an adaptor protein,TRAF4 was identifi ed as a regulator protein in recent studies.We have determined the crystal structure of TRAF domain of TRAF4(residues 292-466)at 2.60Åresolution by X-ray crystallography method.The trimericly assembled TRAF4 resembles a mushroom shape,containing a super helical“stalk”which is made of three right-handed intertwinedαhelixes and a C-terminal“cap”,which is divided at resi-due L302 as a boundary.Similar to other TRAFs,both intermolecular hydrophobic interaction in super helical“stalk”and hydrogen bonds in“cap”regions contribute directly to the formation of TRAF4 trimer.However,differ-ing from other TRAFs,there is an additional fl exible loop(residues 421-426),which contains a previously identifi ed phosphorylated site S426 exposing on the surface.This S426 was reported to be phosphorylated by IKKαwhich is the pre-requisite for TRAF4-NOD2 complex formation and thus to inhibit NOD2-induced NF-κB activation.Therefore,the crystal structure of TRAF4-TRAF is valuable for under-standing its molecular basis for its special function and provides structural information for further studies.
