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1.  Incorporation of the Rpn12 Subunit Couples Completion of Proteasome Regulatory Particle Lid Assembly to Lid-Base Joining 
Molecular cell  2011;44(6):907-917.
Summary
The 26S proteasome, the central eukaryotic protease, comprises a core particle capped by a 19S regulatory particle (RP). The RP is divisible into base and lid subcomplexes. Lid biogenesis and incorporation into the RP remain poorly understood. We report several lid intermediates, including the free Rpn12 subunit and a lid particle (LP) containing the remaining eight subunits, LP2. Rpn12 binds LP2 in vitro, and each requires the other for assembly into 26S proteasomes. Stable Rpn12 incorporation depends on all other lid subunits, indicating Rpn12 distinguishes LP2 from smaller lid subcomplexes. The highly conserved C-terminus of Rpn12 bridges the lid and base, mediating both stable binding to LP2 and lid-base joining. Our data suggest a hierarchical assembly mechanism where Rpn12 binds LP2 only upon correct assembly of all other lid subunits, and the Rpn12 tail then helps drive lid-base joining. Rpn12 incorporation thus links proper lid assembly to subsequent assembly steps.
doi:10.1016/j.molcel.2011.11.020
PMCID: PMC3251515  PMID: 22195964
2.  Complete subunit architecture of the proteasome regulatory particle 
Nature  2012;482(7384):186-191.
The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes.
doi:10.1038/nature10774
PMCID: PMC3285539  PMID: 22237024
26S proteasome; 19S regulatory particle; ubiquitin recognition; deubiquitination; AAA+ ATPase; cryoEM; UPS; PCI domain
3.  Multiple Assembly Chaperones Govern Biogenesis of the Proteasome Regulatory Particle Base 
Cell  2009;137(5):887-899.
Summary
The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.
doi:10.1016/j.cell.2009.04.061
PMCID: PMC2718848  PMID: 19446322
4.  The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome 
Molecular Biology of the Cell  2007;18(2):569-580.
The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ΔN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ΔN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.
doi:10.1091/mbc.E06-07-0635
PMCID: PMC1783769  PMID: 17135287
5.  Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly 
Cell Biochemistry and Biophysics  2011;60(1-2):13-20.
The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.
doi:10.1007/s12013-011-9178-4
PMCID: PMC3256250  PMID: 21461838
Proteasome; ATPase; multiprotein complex assembly; proteolysis; ubiquitin; RP assembly chaperone (RAC)
6.  Proteasomal Proteomics: Identification of Nucleotide-sensitive Proteasome-interacting Proteins by Mass Spectrometric Analysis of Affinity-purified Proteasomes 
Molecular Biology of the Cell  2000;11(10):3425-3439.
Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex. Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells. Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-γ-S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap. To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry. Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex). Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction. Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies.
PMCID: PMC15004  PMID: 11029046
7.  Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes 
A combinatorial depletion strategy is combined with biochemistry, quantitative proteomics and computational approaches to elucidate the structure of the SAGA/ADA complexes. The analysis reveals five connected functional modules capable of independent assembly.
A combinatorial approach of gene depletions with multiple bait proteins coupled with biochemical, proteomic and computational approaches can experimentally determine modules of stable multi-protein complexes.SAGA is a 19-subunit complex consisting of five connected modules with Spt20 being particularly important for the assembly of the intact complex.One of the modules, the HAT/Core module, is also shared with the distinct six-subunit complex ADA.Architectural models of large multi-protein complexes can be assembled using our approach, which is an alternative method to generate novel insight into the organization and architecture of multi-protein complexes.
Determining the architectures of protein complexes improves our understanding of protein cellular functions. In order to efficiently characterize the subunits of protein complexes assembled in vivo, affinity purification followed by proteomics mass spectrometry (APMS) strategies have been devised. Partial or whole protein complexes are first biochemically isolated using tagged components of the complex, followed by an identification of all co-purified proteins using mass spectrometry. However, those approaches are insufficient to provide information about the spatial arrangement and the interrelationship of the proteins of the respective complex.
In this study, we developed and applied a novel method utilizing biochemistry, quantitative proteomics and computational approaches in order to characterize the organization of proteins in a complex. The key of our method is the systematic purification of several tagged components of the protein complex in multiple genetic deletion strains, which serve to compromise the integrity of the complex. Using a series of computational methods, these raw quantitative values are next interpreted in order to determine the modular organization of the complex as well as the interrelationships between its subunits, which in turn can be used to predict a macromolecular model of the complex.
We tested this approach to obtain novel insights into the architecture of multi-protein complexes on the Saccharomyces cerevisiae Spt–Ada–Gcn5 histone acetyltransferase (HAT) (SAGA) and ADA complexes, which are conserved complexes involved in chromatin remodeling (Koutelou et al, 2010). Regular quantitative APMS strategies in wild-type backgrounds were not sufficient to separate tight protein complexes like SAGA/ADA into its distinct modules. However, after perturbing the system using genetic deletions of several subunits located in different topological parts of SAGA, hierarchical cluster analysis performed on 34 purifications (generated using 10 different TAP-tagged baits) resulted in a dissociation of the Gcn5 HAT complexes into five modules: (1) the SA_TAF module, (2) the SA_SPT module, (3) the DUB module, (4) the HAT/Core module and (5) the ADA module (Figure 2A and B).
The approach of purifying a protein in a deletion strain furthermore provides valuable information about the influence of the deleted subunit on the association and interdependency of the bait and the remaining preys. In order to quantify these associations, we calculated a probability between every prey and bait in the deletion strain purifications based on Bayes' theorem (Sardiu et al, 2008). In conjunction with preexisting interaction data obtained from yeast two-hybrid and genetic complementation assays, we finally used these probabilities to predict a low-resolution model for the architecture of the SAGA and ADA complexes (Figure 4).
This novel approach revealed that the SAGA/ADA complexes are composed of five distinct functional modules, of which two were not previously described (SA_SPT and SA_TAF). These modules, which are responsible for different functions of the SAGA complex, are capable of assembling independently from the remaining modules of the complex. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 HAT complexes. Compared with other structural studies, which mapped 9 of the 19 known SAGA subunits using single EM reconstruction (Wu et al, 2004) or resolved the structure of the 4 subunits of the DUB module using X-ray crystallography (Kohler et al, 2010; Samara et al, 2010), our approach is not limited to a maximum number of complex subunits. Consequently, we were able to construct a macromolecular model consisting of all 21 SAGA/ADA subunits, which bridges the gap between the previous limited EM analysis and focused X-ray crystallography analysis.
Despite the availability of several large-scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild-type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well-studied complexes, Spt–Ada–Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high-resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.
doi:10.1038/msb.2011.40
PMCID: PMC3159981  PMID: 21734642
ADA; architecture; protein interaction network; quantitative proteomics; SAGA
8.  Structure of Rpn10 and Its Interactions with Polyubiquitin Chains and the Proteasome Subunit Rpn12* 
The Journal of Biological Chemistry  2010;285(44):33992-34003.
Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys48-linked diUb, which it binds selectively over monoUb and Lys63-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys48-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor.
doi:10.1074/jbc.M110.134510
PMCID: PMC2962499  PMID: 20739285
Biophysics; NMR; Proteasome; Signal Transduction; Ubiquitin; X-ray Crystallography
9.  Simulating Molecular Mechanisms of the MDM2-Mediated Regulatory Interactions: A Conformational Selection Model of the MDM2 Lid Dynamics 
PLoS ONE  2012;7(7):e40897.
Diversity and complexity of MDM2 mechanisms govern its principal function as the cellular antagonist of the p53 tumor suppressor. Structural and biophysical studies have demonstrated that MDM2 binding could be regulated by the dynamics of a pseudo-substrate lid motif. However, these experiments and subsequent computational studies have produced conflicting mechanistic models of MDM2 function and dynamics. We propose a unifying conformational selection model that can reconcile experimental findings and reveal a fundamental role of the lid as a dynamic regulator of MDM2-mediated binding. In this work, structure, dynamics and energetics of apo-MDM2 are studied as a function of posttranslational modifications and length of the lid. We found that the dynamic equilibrium between “closed” and “semi-closed” lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size. Our results revealed that these factors may regulate p53-MDM2 binding by fine-tuning the thermodynamic equilibrium between preexisting conformational states of apo-MDM2. In agreement with NMR studies, the effect of phosphorylation on MDM2 interactions was more pronounced with the truncated lid variant that favored the thermodynamically dominant closed form. The phosphomimetic mutation S17D may alter the lid dynamics by shifting the thermodynamic equilibrium towards the ensemble of “semi-closed” conformations. The dominant “semi-closed” lid form and weakened dependence on the phosphorylation seen in simulations with the complete lid can provide a rationale for binding of small p53-based mimetics and inhibitors without a direct competition with the lid dynamics. The results suggested that a conformational selection model of preexisting MDM2 states may provide a robust theoretical framework for understanding MDM2 dynamics. Probing biological functions and mechanisms of MDM2 regulation would require further integration of computational and experimental studies and may help to guide drug design of novel anti-cancer therapeutics.
doi:10.1371/journal.pone.0040897
PMCID: PMC3397965  PMID: 22815859
10.  Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase 
PLoS ONE  2013;8(8):e70357.
Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.
doi:10.1371/journal.pone.0070357
PMCID: PMC3735599  PMID: 23936414
11.  Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex 
Cell  2014;158(5):1123-1135.
Summary
Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.
Graphical Abstract
Highlights
•X-ray structures of major yeast eIF3 components and subcomplexes•Crosslinking coupled to mass-spectrometry analysis of 40S⋅eIF1⋅eIF3 complex•Integrative modeling reveals architecture of 40S⋅eIF1⋅eIF3 complex
A hybrid approach drawing on X-ray structures, crosslinking coupled to mass spectrometry, electron microscopy, and integrative modeling yields mechanistic insights into how eIF3 coordinates translation initiation.
doi:10.1016/j.cell.2014.07.044
PMCID: PMC4151992  PMID: 25171412
12.  Chemical cross-linking, mass spectrometry and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii 
Proteomics  2012;12(11):1806-1814.
A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrial barrel-like structures of four-stacked homoheptameric rings of α- and β-type subunits organized in α7β7β7α7 stoichiometry. The CPs of H. volcanii are formed from a single type of β subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α17β7β7α17 and α27β7β7α27 CPs from H. volcanii. Chemically cross-linked peptides of the H. volcanii CPs were analyzed by high-performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross-linking mass spectrometry (CXMS), together with the available structural data of non-halophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1-, α2-, and β-homoheptameric rings from among several different possiblePDB structures.
doi:10.1002/pmic.201100260
PMCID: PMC3517063  PMID: 22623373
cross-linking; haloarchaea; modeling; proteasomes; tandem mass spectrometry
13.  Structural Insights into a Unique Legionella pneumophila Effector LidA Recognizing Both GDP and GTP Bound Rab1 in Their Active State 
PLoS Pathogens  2012;8(3):e1002528.
The intracellular pathogen Legionella pneumophila hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated Legionella-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.
Author Summary
Legionella pneumophila delivers 275 validated substrates into the host cytosol by its Dot/Icm type IV secretion system. Several substrates including SidM/DrrA and LidA directly interact with the host Rab GTPases and interfere with the vesicle secretion pathway. SidM/DrrA is necessary for Rab1 recruitment, function as a Rab1 specific GDI displacement factor and guanine nucleotide exchange factor. LidA has the auxiliary activity for Rab1 recruitment, whereas it is more important for the formation of the replication vacuole compared with SidM/DrrA. LidA is predicted to be the first substrate secreted by the Dot/Icm system and is critical for maintaining the integrity of the bacterial cell. Moreover, it expresses throughout the intracellular growth phase, localizes to early secretory compartments, and interacts with several members of Rab family. Here we present the crystal structures of LidA coiled-coil domain in complex with two different states of Rab1, GDP- and GTP-bound. The GDP-bound Rab1 in the complex surprisingly has the same conformation with the GTP-bound Rab1, revealing that LidA can retain Rab1 persistently in its active state. Our structures add a new insight into the regulation of the host Rab1 membrane cycle by pathogen-secreted coiled-coil effector.
doi:10.1371/journal.ppat.1002528
PMCID: PMC3295573  PMID: 22416225
14.  Assembly of the 20S Proteasome 
Biochimica et biophysica acta  2013;1843(1):10.1016/j.bbamcr.2013.03.008.
The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation.
doi:10.1016/j.bbamcr.2013.03.008
PMCID: PMC3752329  PMID: 23507199
15.  Assembly manual for the proteasome regulatory particle: the first draft 
Biochemical Society transactions  2010;38(Pt 1):6-13.
The proteasome is the most complex protease known, with a molecular mass of approximately 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the core particle (CP) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP-CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails but proximal to them. Some but probably not all of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel, CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.
doi:10.1042/BST0380006
PMCID: PMC3431156  PMID: 20074027
proteasome; assembly; regulatory particle; AAA protein; chaperone
16.  Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome 
Molecular Biology of the Cell  2011;22(7):911-920.
Functional redundancy of Rpn5 in budding yeast allows its participation and function in two distinct but structurally related PCI complexes: the proteasome lid and the CSN. As a lid subunit, Rpn5 stabilizes proteasome integrity; as a CSN subunit, Rpn5 is required for enzymatic hydrolysis of Rub1/Nedd8 from cullins.
Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.
doi:10.1091/mbc.E10-08-0655
PMCID: PMC3069016  PMID: 21289098
17.  Modeling of solvent-dependent conformational transitions in Burkholderia cepacia lipase 
Background
The characteristic of most lipases is the interfacial activation at a lipid interface or in non-polar solvents. Interfacial activation is linked to a large conformational change of a lid, from a closed to an open conformation which makes the active site accessible for substrates. While for many lipases crystal structures of the closed and open conformation have been determined, the pathway of the conformational transition and possible bottlenecks are unknown. Therefore, molecular dynamics simulations of a closed homology model and an open crystal structure of Burkholderia cepacia lipase in water and toluene were performed to investigate the influence of solvents on structure, dynamics, and the conformational transition of the lid.
Results
The conformational transition of B. cepacia lipase was dependent on the solvent. In simulations of closed B. cepacia lipase in water no conformational transition was observed, while in three independent simulations of the closed lipase in toluene the lid gradually opened during the first 10–15 ns. The pathway of conformational transition was accessible and a barrier was identified, where a helix prevented the lid from opening to the completely open conformation. The open structure in toluene was stabilized by the formation of hydrogen bonds.
In simulations of open lipase in water, the lid closed slowly during 30 ns nearly reaching its position in the closed crystal structure, while a further lid opening compared to the crystal structure was observed in toluene. While the helical structure of the lid was intact during opening in toluene, it partially unfolded upon closing in water. The closing of the lid in water was also observed, when with eight intermediate structures between the closed and the open conformation as derived from the simulations in toluene were taken as starting structures. A hydrophobic β-hairpin was moving away from the lid in all simulations in water, which was not observed in simulations in toluene. The conformational transition of the lid was not correlated to the motions of the β-hairpin structure.
Conclusion
Conformational transitions between the experimentally observed closed and open conformation of the lid were observed by multiple molecular dynamics simulations of B. cepacia lipase. Transitions in both directions occurred without applying restraints or external forces. The opening and closing were driven by the solvent and independent of a bound substrate molecule.
doi:10.1186/1472-6807-9-38
PMCID: PMC2695465  PMID: 19476626
18.  Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium Type III Secretion System 
mBio  2010;1(3):e00158-10.
The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.
IMPORTANCE
Many biological macromolecular complexes are composed of symmetrical homomers, which assemble into larger structures. Some complexes, such as secretion systems, span biological membranes, forming hydrophilic domains to move substrates across lipid bilayers. Type III secretion systems (T3SSs) deliver bacterial effector proteins directly to the host cell cytoplasm and allow for critical interactions between many Gram-negative pathogenic bacterial species and their hosts. Progress has been made towards the goal of determining the three-dimensional structure of the secretion apparatus by determination of high-resolution crystal structures of individual protein subunits and low-resolution models of the assembly, using reconstructions of cryoelectron microscopy images. However, a more refined picture of the localization of periplasmic ring structures within the assembly and their interactions has only recently begun to emerge. This work localizes T3SS transmembrane rings and identifies structural elements that affect substrate switching and are essential to the assembly of components that are inserted into host cell membranes.
doi:10.1128/mBio.00158-10
PMCID: PMC2932509  PMID: 20824104
19.  The Intrinsically Disordered Sem1 Protein Functions as a Molecular Tether During Proteasome Lid Biogenesis 
Molecular cell  2014;53(3):433-443.
Summary
The intrinsically disordered yeast protein Sem1 (DSS1 in mammals) participates in multiple protein complexes, including the proteasome, but its role(s) within these complexes is uncertain. We report that Sem1 enforces the ordered incorporation of subunits Rpn3 and Rpn7 into the assembling proteasome lid. Sem1 uses conserved acidic segments separated by a flexible linker to grasp Rpn3 and Rpn7. The same segments are used for protein binding in other complexes, but in the proteasome lid they are uniquely deployed for recognizing separate polypeptides. We engineered TEV protease-cleavage sites into Sem1 to show that the tethering function of Sem1 is important for the biogenesis and integrity of the Rpn3-Sem1-Rpn7 ternary complex but becomes dispensable once the ternary complex incorporates into larger lid precursors. Thus, although Sem1 is a stoichiometric component of the mature proteasome, it has a distinct, chaperone-like function specific to early stages of proteasome assembly.
doi:10.1016/j.molcel.2013.12.009
PMCID: PMC3947554  PMID: 24412063
20.  Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly 
PLoS Biology  2011;9(3):e1000603.
The authors perform a cryo-EM study of the 1.9 MDa human Mediator-RNA polymerase II-TFIIF assembly, which reveals the structural organization of the human transcription initiation apparatus.
The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly.
Author Summary
Transcription initiation in humans is regulated by a macromolecular complex formed by the RNA polymerase II enzyme (pol II), Mediator, and the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Collectively, these factors are known as the Pre-Initiation Complex (PIC). Although the 1.2 MDa Mediator seems to have a major role in regulating assembly and function of the PIC, a structural understanding of the complex has yet to be established. This study outlines a cryo-EM analysis of the Mediator–pol II assembly in the presence or absence of the dimeric TFIIF complex. We observe that TFIIF is required to stably orient the pol II enzyme within the Mediator–pol II assembly, indicating a novel structural role for TFIIF in transcription initiation. Additionally, we accurately dock the pol II crystal structure within the human Mediator–pol II–TFIIF cryo-EM map. The locations of TFIIH, TBP (a subunit within TFIID), TFIIA, TFIIB, TFIIE, and TFIIF relative to the pol II enzyme itself have been determined by previous studies. These data in combination with the Mediator–pol II–TFIIF structure described here allow us to propose the structural organization of the entire 3.5 MDa human PIC.
doi:10.1371/journal.pbio.1000603
PMCID: PMC3066130  PMID: 21468301
21.  Evidence of a Double-Lid Movement in Pseudomonas aeruginosa Lipase: Insights from Molecular Dynamics Simulations 
PLoS Computational Biology  2005;1(3):e28.
Pseudomonas aeruginosa lipase is a 29-kDa protein that, following the determination of its crystal structure, was postulated to have a lid that stretched between residues 125 and 148. In this paper, using molecular dynamics simulations, we propose that there exists, in addition to the above-mentioned lid, a novel second lid in this lipase. We further show that the second lid, covering residues 210–222, acts as a triggering lid for the movement of the first. We also investigate the role of hydrophobicity in the movement of the lids and show that two residues, Phe214 and Ala217, play important roles in lid movement. To our knowledge, this is the first time that a double-lid movement of the type described in our manuscript has been presented to the scientific community. This work also elucidates the interplay of hydrophobic interactions in the dynamics, and hence the function, of an enzyme.
Synopsis
Lipases hydrolyse long-chain fatty acid esters at water-oil interfaces through the mechanism of interfacial activation mediated by the movement of a lid subdomain that covers the active site. Studying lid movement is an area of active research in the field of protein dynamics. The lipase from Pseudomonas aeruginosa is a 29-kDa protein that was previously crystallized in the open conformation, and as expected, an approximately 20-residue lid subdomain was identified. In the present study, the authors report extensive molecular dynamics simulations of the P. aeruginosa lipase. They show that this protein has two lids covering the substrate-binding pocket. The first lid is the one proposed from the known crystal structure. The second lid, a much shorter one, lies over the binding pocket facing the first lid. Furthermore, using position-restrained simulations, these authors show that movement of the second lid may actually be a trigger for the movement of the first, and that this triggering action is driven by hydrophobic contacts between the two lids. This computational study paves a way for experimentalists to study the structure and dynamics of this protein in greater detail in order to understand coupled subdomain movements in a comprehensive fashion.
doi:10.1371/journal.pcbi.0010028
PMCID: PMC1187864  PMID: 16110344
22.  Robotically-Assisted Titration Coupled to Ion Mobility-Mass Spectrometry Reveals the Interface Structures and Analysis Parameters Critical for Multiprotein Topology Mapping 
Analytical chemistry  2013;85(23):11360-11368.
Multiprotein complexes have three-dimensional shapes and dynamic functions that impact almost every aspect of biochemistry. Despite this, our ability to rapidly assess the structures of such macromolecules lags significantly behind high-throughput efforts to identify their function, especially in the context of human disease. Here, we describe results obtained by coupling ion mobility-mass spectrometry with automated robotic sampling of different solvent compositions. This combination of technologies has allowed us to explore an extensive set of solution conditions for a group of eight protein homo-tetramers, representing a broad sample of protein structure and stability. We find that altering solution ionic strength in concert with dimethylsulfoxide content is sufficient to disrupt the protein-protein interfaces of all of the complexes studied here. Ion mobility measurements captured for both intact assemblies and subcomplexes matched expected values from available X-ray structures in all cases save two. For these exceptions, we find that distorted subcomplexes result from extreme disruption conditions, and are accompanied by small shifts in intact tetramers size, thus enabling the removal of distorted subcomplex data in downstream models. Furthermore, we find strong correlations between the relative intensities of disrupted protein tetramers and the relative number and type of interactions present at interfaces as a function of disrupting agent added. In most cases, this correlation appears strong enough to quantify various types of protein interfacial interactions within unknown proteins following appropriate calibration.
doi:10.1021/ac402276k
PMCID: PMC3895495  PMID: 24164205
23.  The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants 
Molecular Biology of the Cell  2001;12(2):383-392.
In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.
PMCID: PMC30950  PMID: 11179422
24.  Prediction of a common structural scaffold for proteasome lid, COP9-signalosome and eIF3 complexes 
BMC Bioinformatics  2005;6:71.
Background
The 'lid' subcomplex of the 26S proteasome and the COP9 signalosome (CSN complex) share a common architecture consisting of six subunits harbouring a so-called PCI domain (proteasome, CSN, eIF3) at their C-terminus, plus two subunits containing MPN domains (Mpr1/Pad1 N-terminal). The translation initiation complex eIF3 also contains PCI- and MPN-domain proteins, but seems to deviate from the 6+2 stoichiometry. Initially, the PCI domain was defined as the region of detectable sequence similarity between the components mentioned above.
Results
During an exhaustive bioinformatical analysis of proteasome components, we detected multiple instances of tetratrico-peptide repeats (TPR) in the N-terminal region of most PCI proteins, suggesting that their homology is not restricted to the PCI domain. We also detected a previously unrecognized PCI domain in the eIF3 component eIF3k, a protein whose 3D-structure has been determined recently. By using profile-guided alignment techniques, we show that the structural elements found in eIF3k are most likely conserved in all PCI proteins, resulting in a structural model for the canonical PCI domain.
Conclusion
Our model predicts that the homology domain PCI is not a true domain in the structural sense but rather consists of two subdomains: a C-terminal 'winged helix' domain with a key role in PCI:PCI interaction, preceded by a helical repeat region. The TPR-like repeats detected in the N-terminal region of PCI proteins most likely form an uninterrupted extension of the repeats found within the PCI domain boundaries. This model allows an interpretation of several puzzling experimental results.
doi:10.1186/1471-2105-6-71
PMCID: PMC1274264  PMID: 15790418
25.  Reconfiguration of the proteasome during chaperone-mediated assembly 
Nature  2013;497(7450):10.1038/nature12123.
The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.
doi:10.1038/nature12123
PMCID: PMC3687086  PMID: 23644457
proteasome; chaperone; single particle cryoEM; ATPase

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