Structural Basis of Ist1 Function and Ist1–Did2 Interaction in the Multivesicular Body Pathway and Cytokinesis

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.

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Structural basis for UFM1 transfer from UBA5 to UFC1

Nature Communications ◽

10.1038/s41467-021-25994-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Manoj Kumar ◽

Prasanth Padala ◽

Jamal Fahoum ◽

Fouad Hassouna ◽

Tomer Tsaban ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Structural Basis ◽

Adenylation Domain ◽

Post Translational Modification ◽

Target Proteins ◽

Cellular Processes ◽

Enzyme Cascade ◽

Linear Sequence ◽

C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

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VPS37A directs ESCRT recruitment for phagophore closure

The Journal of Cell Biology ◽

10.1083/jcb.201902170 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3336-3354 ◽

Cited By ~ 17

Author(s):

Yoshinori Takahashi ◽

Xinwen Liang ◽

Tatsuya Hattori ◽

Zhenyuan Tang ◽

Haiyan He ◽

...

Keyword(s):

Mammalian Cells ◽

Growth Factor Receptor ◽

Multivesicular Body ◽

Regulatory Mechanisms ◽

Endosomal Sorting ◽

Aaa Atpase ◽

Escrt Machinery ◽

Genome Wide ◽

A Genome ◽

Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

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Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

Biochemical Journal ◽

10.1042/bj20060618 ◽

2006 ◽

Vol 398 (3) ◽

pp. 353-360 ◽

Cited By ~ 32

Author(s):

Jingzhi Li ◽

Yunkun Wu ◽

Xinguo Qian ◽

Bingdong Sha

Keyword(s):

Crystal Structure ◽

Close Contact ◽

Critical Role ◽

Peptide Binding ◽

Structural Basis ◽

Cellular Processes ◽

Terminal Form ◽

Charged Residues ◽

Domain I

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40–Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a β-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge–charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

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Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817752116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 534-539 ◽

Cited By ~ 35

Author(s):

Parijat Majumder ◽

Till Rudack ◽

Florian Beck ◽

Radostin Danev ◽

Günter Pfeifer ◽

...

Keyword(s):

26S Proteasome ◽

Structural Basis ◽

Particle Analysis ◽

Aaa Atpase ◽

Cellular Processes ◽

Domains Of Life ◽

Atpase Subunits ◽

Domain Motions ◽

And Control ◽

Aaa Atpases

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

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Structural Basis of Molecular Recognition between ESCRT-III-like Protein Vps60 and AAA-ATPase Regulator Vta1 in the Multivesicular Body Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m112.390724 ◽

2012 ◽

Vol 287 (52) ◽

pp. 43899-43908 ◽

Cited By ~ 30

Author(s):

Zhongzheng Yang ◽

Cody Vild ◽

Jiaying Ju ◽

Xu Zhang ◽

Jianping Liu ◽

...

Keyword(s):

Molecular Recognition ◽

Multivesicular Body ◽

Structural Basis ◽

Aaa Atpase

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Structural basis of recruitment of RBM39 to DCAF15 by a sulfonamide molecular glue E7820

10.1101/758409 ◽

2019 ◽

Author(s):

Xinlin Du ◽

Oleg Volkov ◽

Robert Czerwinski ◽

HuiLing Tan ◽

Carlos Huerta ◽

...

Keyword(s):

Crystal Structure ◽

Kinetic Analysis ◽

Mechanism Of Action ◽

E3 Ligase ◽

Kinetic Studies ◽

Structural Basis

AbstractE7820 and indisulam are two examples of aryl sulfonamides that recruit RBM39 to Rbx-Cul4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. In order to understand their mechanism of action, we carried out kinetic analysis on the recruitment of RBM39 to DCAF15 and solved a crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39. E7820 packs in a shallow pocket on the surface of DCAF15 and the resulting modified interface binds RBM39 through the α1 helix of RRM2 domain. Our kinetic studies revealed that aryl sulfonamide and RBM39 bind to DCAF15 in a synergistic manner. The structural and kinetic studies confirm aryl sulfonamides as molecular glues in the recruitment of RBM39 and provide a framework for future efforts to utilize DCAF15 to degrade other protein of interests.

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