RESUMEN
Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.
Asunto(s)
Proteoma , Espectrometría de Masas , Proteoma/química , Reactivos de Enlaces Cruzados/químicaRESUMEN
In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.
Asunto(s)
Adenosina Trifosfatasas/química , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Microscopía por Crioelectrón , Nucleótidos/química , Complejo de la Endopetidasa Proteasomal/ultraestructura , Conformación ProteicaRESUMEN
The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP). Here, we probe the dynamic architecture of the proteasome by applying quantitative proteomics and mass spectrometry (MS) of intact complexes to provide a detailed characterization of how Ubp6 assists this assembly process. Our MS data demonstrate stoichiometric binding of chaperones and Ubp6 to the basal part of the RP. Genetic interactions of Ubp6 with Hsm3, but not with the other chaperones, indicate a functional overlay with Hsm3. Our biochemical data identified Ubp6 as an additional member of the Hsm3 module. Deletions of ubp6 with hsm3 perturb 26S proteasome assembly, which we attribute to an accumulation of ubiquitylated substrates on these assembly precursors. We therefore propose that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity.
Asunto(s)
Endopeptidasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Endopeptidasas/química , Endopeptidasas/metabolismo , Eliminación de Gen , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Mapeo de Interacción de Proteínas , Proteómica/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Humanos , Microscopía Electrónica de Transmisión , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , LevadurasRESUMEN
In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.
Asunto(s)
Endopeptidasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dominio Catalítico , Microscopía por Crioelectrón , Endopeptidasas/química , Complejo de la Endopetidasa Proteasomal/química , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Three recent Molecular Cell papers from Zhang et al. (2009a, 2009b) and Djuranovic et al. (2009) provide new insights into how proteasomal ATPases recognize, unfold, and translocate their substrates, thus enhancing our understanding of regulated proteolysis.
Asunto(s)
Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/fisiología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Complejo de la Endopetidasa Proteasomal/química , Pliegue de Proteína , Proteínas/metabolismoRESUMEN
Two canonical subunits of the 26S proteasome, Rpn10 and Rpn13, function as ubiquitin (Ub) receptors. The mutual arrangement of these subunits--and all other non-ATPase subunits--in the regulatory particle is unknown. Using electron cryomicroscopy, we calculated difference maps between wild-type 26S proteasome from Saccharomyces cerevisiae and deletion mutants (rpn10Δ, rpn13Δ, and rpn10Δrpn13Δ). These maps allowed us to localize the two Ub receptors unambiguously. Rpn10 and Rpn13 mapped to the apical part of the 26S proteasome, above the N-terminal coiled coils of the AAA-ATPase heterodimers Rpt4/Rpt5 and Rpt1/Rpt2, respectively. On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome.
Asunto(s)
Microscopía por Crioelectrón/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Drosophila melanogaster , Espectrometría de Masas , Modelos MolecularesRESUMEN
The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.
Asunto(s)
Endopeptidasas/química , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Estructura Terciaria de ProteínaRESUMEN
Parkinson's disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network. Budding yeast Saccharomyces cerevisiae was used as eukaryotic reference organism to study the consequences of α-syn expression on protein dynamics. To address this, we investigated the impact of overexpression of α-syn and S129A variant on the abundance and stability of most yeast proteins using a genome-wide yeast library and a tandem fluorescent protein timer (tFT) reporter as a measure for protein stability. This revealed that the stability of in total 377 cellular proteins was altered by α-syn expression, and that the impact on protein stability was significantly enhanced by phosphorylation at Ser129 (pS129). The proteasome assembly chaperone Rpn14 was identified as one of the top candidates for increased protein stability by expression of pS129 α-syn. Elevated levels of Rpn14 enhanced the growth inhibition by α-syn and the accumulation of ubiquitin conjugates in the cell. We found that Rpn14 interacts physically with α-syn and stabilizes pS129 α-syn. The expression of α-syn along with elevated levels of Rpn14 or its human counterpart PAAF1 reduced the proteasome activity in yeast and in human cells, supporting that pS129 α-syn negatively affects the 26S proteasome through Rpn14. This comprehensive study into the alternations of protein homeostasis highlights the critical role of the Rpn14/PAAF1 in α-syn-mediated proteasome dysfunction.
Asunto(s)
Chaperonas Moleculares , Complejo de la Endopetidasa Proteasomal , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Chaperonas Moleculares/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.
Asunto(s)
Adenosina Trifosfatasas , Adenosina Trifosfato , Humanos , Adenosina Trifosfato/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteína que Contiene Valosina , Simulación de Dinámica MolecularRESUMEN
The ubiquitin-proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Elementos Reguladores de la Transcripción , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Subunidades de Proteína/químicaRESUMEN
A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and ß subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four ß subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Ratones , Péptidos/química , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ProteolisisRESUMEN
The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 Å by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the α-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high "activity" surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Schizosaccharomyces/química , Microscopía por Crioelectrón/métodos , Espectrometría de Masas/métodos , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas/metabolismoRESUMEN
Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Cullin/química , Proteínas Ligasas SKP Cullina F-box/química , Ubiquitinas/química , Proteínas Portadoras/química , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Proteína NEDD8 , Poliubiquitina/química , Unión Proteica , Ubiquitinas/genética , Regulación hacia ArribaRESUMEN
The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome.
Asunto(s)
Endopeptidasas/química , Endopeptidasas/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Dominio Catalítico , Microscopía por Crioelectrón , Citoplasma , Endopeptidasas/genética , Complejo de la Endopetidasa Proteasomal/genética , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMEN
In eukaryotic cells, the ubiquitin-proteasome system serves to remove proteins that are either dysfunctional or no longer needed. The 26S proteasome is a 2.5 MDa multisubunit complex comprising the 20S core particle, where degradation is executed, and one or two regulatory particles which prepare substrates for degradation. Whereas the 20S core particles of several species had been studied extensively by X-ray crystallography, the 26S holocomplex structure had remained elusive for a long time. Recent advances in single-particle cryo-electron microscopy have changed the situation and provided atomic resolution models of this intriguing molecular machine and its dynamics. Besides, cryo-electron tomography enables structural studies in situ, providing molecular resolution images of macromolecules inside pristinely preserved cellular environments. This has greatly contributed to our understanding of proteasome dynamics in the context of cells.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Complejo de la Endopetidasa Proteasomal/química , Conformación Proteica , Fracciones Subcelulares/metabolismoRESUMEN
Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85A resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.
Asunto(s)
Lisina/química , Poliubiquitina/química , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/química , Conformación ProteicaRESUMEN
The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution and report two conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.
Asunto(s)
Simulación de Dinámica Molecular , Complejo de la Endopetidasa Proteasomal/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The investigation of common structural motifs provides additional information on why proteins conserve similar topologies yet may have non-conserved amino acid sequences. Proteins containing the ubiquitin superfold have similar topologies, although the sequence conservation is rather poor. Here, we present novel similarities and differences between the proteins ubiquitin and NEDD8. They have 57% identical sequence, almost identical backbone topology and similar functional strategy, although their physiological functions are mutually different. Using variable pressure NMR spectroscopy, we found that the two proteins have similar conformational fluctuation in the evolutionary conserved enzyme-binding region and contain a structurally similar locally disordered conformer (I) in equilibrium with the basic folded conformer (N). A notable difference between the two proteins is that the equilibrium population of I is far greater for NEDD8 (DeltaG(0)(NI)<5 kJ/mol) than for ubiquitin (DeltaG(0)(NI)=15.2(+/-1.0) kJ/mol), and that the tendency for overall unfolding (U) is also far higher for NEDD8 (DeltaG(0)(NU)=11.0(+/-1.5) kJ/mol) than for ubiquitin (DeltaG(0)(NU)=31.3(+/-4.7) kJ/mol). These results suggest that the marked differences in thermodynamic stabilities of the locally disordered conformer (I) and the overall unfolding species (U) are a key to determine the functional differences of the two structurally similar proteins in physiology.