scholarly journals Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

2019 ◽  
Author(s):  
Zhu Liu ◽  
Xu Dong ◽  
Hua-Wei Yi ◽  
Ju Yang ◽  
Zhou Gong ◽  
...  
Keyword(s):  
Single Molecule ◽  
Complex Structure ◽  
Binding Mode ◽  
Structural Basis ◽  
Ubiquitinated Proteins ◽  
Single Molecule Fret ◽  
Compact State ◽  
A Charge ◽  
Substrate Proteins ◽  
Ubiquitinated Substrate

ABSTRACTThe interaction between K48-linked ubiquitin (Ub) chain and Rpn13 is important for proteasomal degradation of ubiquitinated substrate proteins. Only the complex structure between the N-terminal domain of Rpn13 (Rpn13NTD) and Ub monomer has been characterized, and it remains unclear how Rpn13 specifically recognizes K48-linked Ub chain. Using single-molecule FRET, here we show that K48-linked diubiquitin (K48-diUb) fluctuates among three distinct conformational states, and a preexisting compact state is selectively enriched by Rpn13NTD. The same binding mode is observed for full-length Rpn13 and longer K48-linked Ub chain. Using solution NMR spectroscopy, we have solved the complex structure between Rpn13NTD and K48-diUb. In the structure, Rpn13NTD simultaneously interacts with proximal and distal Ub subunits of K48-diUb that remain associated in the complex, thus corroborating smFRET findings. The proximal Ub interacts with Rpn13NTD similarly as the Ub monomer in the known Rpn13NTD:Ub structure, while the distal Ub binds to a largely electrostatic surface of Rpn13NTD. Thus, a charge reversal mutation in Rpn13NTD can weaken the interaction between Rpn13 and K48-linked Ub chain, causing accumulation of ubiquitinated proteins. Moreover, blockage of the access of the distal Ub to Rpn13NTD with a proximity attached Ub monomer can also disrupt the interaction between Rpn13 and K48-diUb. Together, the bivalent interaction of K48-linked Ub chain with Rpn13 provides the structural basis for Rpn13 linkage selectivity, which opens a new window for modulating proteasomal function.

scholarly journals Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription

2015 ◽  
Vol 112 (44) ◽  
pp. 13467-13472 ◽  
Author(s):  
Danya J. Martell ◽  
Chandra P. Joshi ◽  
Ahmed Gaballa ◽  
Ace George Santiago ◽  
Tai-Yen Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Dynamic Equilibrium ◽  
Specific Binding ◽  
Binding Mode ◽  
Biased Sampling ◽  
Single Molecule Fret ◽  
Dead End ◽  
The Dead

Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ70-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator−DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)—a Cu+-responsive MerR-family metalloregulator—modulates RNAP interactions with CueR’s cognate suboptimal promoter PcopA, and how RNAP affects CueR−PcopAinteractions. We find that RNAP can form two noninterconverting complexes at PcopAin the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a “biased sampling” instead of “dynamic equilibrium shifting” mechanism in regulating transcription initiation; it modulates RNAP’s binding–unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopAinto its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.

scholarly journals Ultrafast Brownian-ratchet mechanism for protein translocation by a AAA+ machine

2020 ◽  
Author(s):  
Hisham Mazal ◽  
Marija Iljina ◽  
Inbal Riven ◽  
Gilad Haran
Keyword(s):  
Single Molecule ◽  
Protein Translocation ◽  
Conformational Dynamics ◽  
Brownian Ratchet ◽  
Time Dynamics ◽  
Single Molecule Fret ◽  
Differential Behavior ◽  
Amplitude Fluctuations ◽  
Translocation Mechanism ◽  
Substrate Proteins

AbstractAAA+ ring-shaped machines, such as ClpB and Hsp104, mediate substrate translocation through their central channel by a set of pore loops. Recent structural studies suggested a universal hand-over-hand translocation mechanism, in which pore loops are moving rigidly in tandem with their corresponding subunits. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB and their response to substrate binding, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond timescale, and change their conformation upon interaction with substrate proteins. Pore-loop conformational dynamics are modulated by nucleotides and strongly correlate with disaggregation activity. The differential behavior of the pore loops along the axial channel points to a fast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process.

scholarly journals Structural basis of recognition and destabilization of histone H2B ubiquitinated nucleosome by DOT1L histone H3 Lys79 methyltransferase

2018 ◽  
Author(s):  
Seongmin Jang ◽  
Chanshin Kang ◽  
Han-Sol Yang ◽  
Taeyang Jung ◽  
Hans Hebert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cross Talk ◽  
Molecular Basis ◽  
Histone H3 ◽  
Structural Basis ◽  
Histone H2b ◽  
Single Molecule Fret ◽  
Catalytic Function ◽  
Histone H3 Methylation ◽  
Hydrophobic Helix

AbstractDOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B-ubiquitination. Here, we present cryo-EM structures of DOT1L complex with unmodified and H2B-ubiquitinated nucleosomes, showing that DOT1L recognizes H2B-ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L respectively. Furthermore, the structures combined with single-molecule FRET experiment show that H2B-ubiquitination enhances a non-catalytic function of DOT1L destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

scholarly journals DNA-sequence dependent positioning of the linker histone in a chromatosome: a single-pair FRET study

2020 ◽  
Author(s):  
Madhura De ◽  
Mehmet Ali Oeztuerk ◽  
Katalin Toth ◽  
Rebecca C. Wade
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Binding Mode ◽  
Linker Histone ◽  
Single Pair ◽  
Globular Domain ◽  
Binding Modes ◽  
Single Molecule Fret ◽  
Fret Efficiency ◽  
Order Structures

The linker histone (LH) associates with the nucleosome with its globular domain (gH) binding in an on or off-dyad binding mode. The positioning of the LH may play a role in the compaction of higher-order structures of chromatin. Preference for different binding modes has been attributed to the LHs amino acid sequence. We here study the effect of the linker DNA (L-DNA) sequence on the positioning of a full-length LH, Xenopus laevis H1.0b, by employing single-molecule FRET spectroscopy. Chromatosomes were fluorescently labelled on one of the two 40bp long L-DNA arms, and on the gH. We varied 11bp of DNA flanking the core (non-palindromic Widom 601) of each chromatosome construct, making them either A-tract, purely GC, or mixed, with 64% AT. The gH consistently exhibited higher FRET efficiency with the L-DNA containing the A-tract, than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA, compared to the pure-GC stretch. We explain our observations with a FRET-distance restrained model that shows that the gH binds on-dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove.

scholarly journals F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

2016 ◽  
Vol 113 (21) ◽  
pp. E2916-E2924 ◽  
Author(s):  
Mitsuhiro Sugawa ◽  
Kei-ichi Okazaki ◽  
Masaru Kobayashi ◽  
Takashi Matsui ◽  
Gerhard Hummer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motor ◽  
Principal Component ◽  
Structural Basis ◽  
F1 Atpase ◽  
Single Molecule Fret ◽  
Mechanochemical Coupling ◽  
Central Shaft

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3β3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

2021 ◽  
Vol 7 (5) ◽  
pp. eabc6479
Author(s):  
Sarah A. Mosure ◽  
Timothy S. Strutzenberg ◽  
Jinsai Shang ◽  
Paola Munoz-Tello ◽  
Laura A. Solt ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Binding Mode ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Heme Binding ◽  
Endogenous Ligand ◽  
Interaction Domain ◽  
Response Elements ◽  
Biochemical Assays ◽  
Chemical Cross Linking

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

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