New insights into the human 26S proteasome function and regulation

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.

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Conserved prolines in the coiled coil-OB domain linkers of proteasomal ATPases facilitate eukaryotic proteasome base assembly

10.1101/2020.11.13.381962 ◽

2020 ◽

Author(s):

Chin Leng Cheng ◽

Michael K Wong ◽

Yanjie Li ◽

Mark Hochstrasser

Keyword(s):

Protein Quality ◽

Coiled Coil ◽

26S Proteasome ◽

Single Type ◽

Mutant Form ◽

Peptide Bonds ◽

Atpase Subunit ◽

Aaa Atpase ◽

Growth Defects ◽

Atpase Subunits

AbstractThe proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.

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Structure of the human 26S proteasome at a resolution of 3.9 Å

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608050113 ◽

2016 ◽

Vol 113 (28) ◽

pp. 7816-7821 ◽

Cited By ~ 123

Author(s):

Andreas Schweitzer ◽

Antje Aufderheide ◽

Till Rudack ◽

Florian Beck ◽

Günter Pfeifer ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Structural Features ◽

26S Proteasome ◽

The Other ◽

Aaa Atpase ◽

Loop Region ◽

C Terminus ◽

Proteasome Subunits ◽

Atpase Subunits ◽

Substrate Processing

Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.

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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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Tubulin structure at intermediate resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140634 ◽

1995 ◽

Vol 53 ◽

pp. 852-853

Author(s):

K. H. Downing ◽

S. G. Wolf ◽

E. Nogales

Keyword(s):

High Resolution ◽

Structural Data ◽

Therapeutic Drug ◽

Zinc Ions ◽

Two Dimensional ◽

X Ray Diffraction ◽

X Ray ◽

Negative Stain ◽

Functional Mechanisms ◽

Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

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HRTEM study of valleriite, a hydroxide-bearing copper sulfide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100175442 ◽

1990 ◽

Vol 48 (4) ◽

pp. 462-463

Author(s):

S. Wang ◽

P. R. Buseck

Keyword(s):

Electron Microscope ◽

High Resolution ◽

Copper Sulfide ◽

Carbonaceous Chondrite ◽

Structural Data ◽

Basic Structure ◽

Long Period ◽

Selected Area Electron Diffraction ◽

Transmission Electron ◽

Wavy Structure

Valleriite is an unusual mineral, consisting of intergrowths of sulfide layers (corresponding in structure to the mineral smythite - Fe9S11) and hydroxide layers (corresponding to brucite - Mg(OH2)). It has a composition of approximately 1.526[Mg.68Al.32(OH)2].[Fe1.07Cu.93S2] and consists of two interpenetrating lattices, each of which retains its individual structural and diffraction characteristics parallel to the layering. The valleriite structure is related to that of tochilinite, an unusual iron-rich mineral that is of considerable interest for the origin of certain carbonaceous chondrite meteorites and to those of franckeite and cylindrite, two minerals that are of interest because of their unique morphological and crystallographic properties, e.g., the distinctive curved form of cylindrite and the perfect mica-like cleavage with unusual striations and the long-period wavy structure of franckeite.Our selected-area electron diffraction (SAED) patterns and high-resolution transmission electron microscope (HRTEM) images of valleriite provide new structural data. A basic structure and a new superstructure have been observed.

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Studies of Conformational Changes of Tubulin Induced by Interaction with Kinesin Using Atomistic Molecular Dynamics Simulations

International Journal of Molecular Sciences ◽

10.3390/ijms22136709 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6709

Author(s):

Xiao-Xuan Shi ◽

Peng-Ye Wang ◽

Hong Chen ◽

Ping Xie

Keyword(s):

Molecular Dynamics ◽

High Resolution ◽

Binding Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Weak Interactions ◽

Molecular Motor ◽

Structural Data ◽

Calculated Binding Energy ◽

Dynamics Simulations

The transition between strong and weak interactions of the kinesin head with the microtubule, which is regulated by the change of the nucleotide state of the head, is indispensable for the processive motion of the kinesin molecular motor on the microtubule. Here, using all-atom molecular dynamics simulations, the interactions between the kinesin head and tubulin are studied on the basis of the available high-resolution structural data. We found that the strong interaction can induce rapid large conformational changes of the tubulin, whereas the weak interaction cannot. Furthermore, we found that the large conformational changes of the tubulin have a significant effect on the interaction of the tubulin with the head in the weak-microtubule-binding ADP state. The calculated binding energy of the ADP-bound head to the tubulin with the large conformational changes is only about half that of the tubulin without the conformational changes.

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Structural and functional analysis of the Na+/H+ exchanger

Biochemical Journal ◽

10.1042/bj20061062 ◽

2007 ◽

Vol 401 (3) ◽

pp. 623-633 ◽

Cited By ~ 165

Author(s):

Emily R. Slepkov ◽

Jan K. Rainey ◽

Brian D. Sykes ◽

Larry Fliegel

Keyword(s):

Intracellular Ph ◽

Sequence Similarity ◽

Structural Data ◽

Structural Similarity ◽

Integral Membrane Protein ◽

Volume Control ◽

Amino Acid Residues ◽

Physiological Processes ◽

High Resolution Structure ◽

Extracellular Sodium

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.

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Assembly of the Drosophila 26 S proteasome is accompanied by extensive subunit rearrangements

Biochemical Journal ◽

10.1042/bj20011520 ◽

2002 ◽

Vol 365 (2) ◽

pp. 527-536 ◽

Cited By ~ 26

Author(s):

Éva KURUCZ ◽

István ANDÓ ◽

Máté SÜMEGI ◽

Harald HÖLZL ◽

Barbara KAPELARI ◽

...

Keyword(s):

Monoclonal Antibodies ◽

26S Proteasome ◽

Cross Linking ◽

20S Proteasome ◽

Electron Microscopic ◽

Immunoblot Assay ◽

Optimal Conformation ◽

Regulatory Complex ◽

Atpase Subunits ◽

The Cross

The subunit contacts in the regulatory complex of the Drosophila 26 S proteasome were studied through the cross-linking of closely spaced subunits of the complex, and analysis of the cross-linking pattern in an immunoblot assay with the use of subunit-specific monoclonal antibodies. The cross-linking pattern of the purified 26 S proteasome exhibits significant differences as compared with that of the purified free regulatory complex. It is shown that the observed differences are due to extensive rearrangement of the subunit contacts accompanying the assembly of the 26 S proteasome from the regulatory complex and the 20S proteasome. Cross-linking studies and electron microscopic examinations revealed that these changes are reversible and follow the assembly or the disassembly of the 26 S proteasome. Although the majority of the changes observed in the subunit contacts affected the hexameric ring of the ATPase subunits, the alterations extended over the whole of the regulatory complex, affecting subunit contacts even in the lid subcomplex. Changes in the subunit contacts, similar to those in the regulatory complex, were detected in the 20S proteasome. These observations indicate that the assembly of the 26 S proteasome is not simply a passive docking of two rigid subcomplexes. In the course of the assembly, the interacting subcomplexes mutually rearrange their structures so as to create the optimal conformation required for the assembly and the proper functioning of the 26S proteasome.

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Cooperative subunit dynamics modulate p97 function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815495116 ◽

2018 ◽

Vol 116 (1) ◽

pp. 158-167 ◽

Cited By ~ 14

Author(s):

Rui Huang ◽

Zev A. Ripstein ◽

John L. Rubinstein ◽

Lewis E. Kay

Keyword(s):

Bound State ◽

Nmr Studies ◽

Biologically Relevant ◽

Aaa Atpase ◽

Cellular Processes ◽

Allosteric Communication ◽

Functional Dynamics ◽

Nmr Study ◽

Wide Range

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

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The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix

Journal of Biological Chemistry ◽

10.1074/jbc.m412043200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22356-22364 ◽

Cited By ~ 50

Author(s):

Ruby H. P. Law ◽

James A. Irving ◽

Ashley M. Buckle ◽

Katya Ruzyla ◽

Marguerite Buzza ◽

...

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Tumor Suppressor ◽

Physiological Role ◽

Human Tumor ◽

Salt Bridge ◽

Structural Data ◽

Crystal Forms ◽

Reactive Center ◽

Α Helix

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-Å resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A β-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E α-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G α-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor.

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