scholarly journals Crystal structures of Bax and Bak Reveal Molecular Events Initiating Apoptosis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1490-C1490
Author(s):  
Peter Czabotar ◽  
Jason Brouwer ◽  
Dana Westphal ◽  
Geoff Thompson ◽  
Peter Colman
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Mitochondrial Membrane ◽  
Molecular Mechanisms ◽  
Structural Studies ◽  
Core Domain ◽  
Hydrophobic Groove ◽  
Outer Leaflet ◽  
Molecular Events ◽  
Surface Crystal

A key event in apoptosis is the conversion of Bax or Bak from inert monomers into cytotoxic mitochondrial membrane perforating oligomers. Certain BH3-only relatives can initiate this step through direct interactions, yet the means by which conformational changes are invoked, the nature of the conformational changes themselves, the mechanism by which they insert into membranes and the process by which they perforate these barriers has largely remained a mystery. Our recent structural studies provided the first insights into this process for Bax [1]. We found that BH3 domains activate Bax by binding to a hydrophobic groove on its surface. Crystal structures of these complexes revealed an unexpected conformational change involving dissociation of a previously unrecognized "core" domain from a "latch' domain. A further structure of the freed Bax "core" domains revealed that these form dimers that possess a surface of aromatic residues which we hypothesis engage the outer leaflet of the mitochondrial membrane and induce curvature. We have now extended our studies to include structures of Bax bound to alternative BH3-only proteins providing new insights into key interactions occurring at this interface. Additionally, we have solved structures of activated Bak and of the freed Bak "core" domain dimers. These results further our understanding of the molecular mechanisms by which these highly dynamic proteins engage the mitochondrial membrane and thus control the life/death switch in cells.

scholarly journals Securing SNAREs for assembly

2020 ◽  
Vol 295 (30) ◽  
pp. 10136-10137
Author(s):  
Yongli Zhang ◽  
Jie Yang
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Snare Proteins ◽  
Membrane Tethering ◽  
Target Membrane

SNARE proteins are essential for exocytosis, mediating the fusion of vesicles with their target membrane. Tethering factors play a key role in chaperoning SNARE assembly; however, the underlying molecular mechanisms are not well-understood. Here, Travis et al. report two crystal structures of a yeast tethering factor, the Dsl1 complex, bound with two SNARE proteins, revealing new insights into how tethering factors bridge vesicles to target membranes, recruit multiple SNARE proteins, trigger their conformational changes, and facilitate SNARE assembly.

scholarly journals Molecular mechanisms of substrate-controlled ring dynamics and sub-stepping in a nucleic-acid dependent hexameric motor

2016 ◽  
Author(s):  
Nathan D. Thomsen ◽  
Michael R. Lawson ◽  
Lea B. Witkowsky ◽  
Song Qu ◽  
James M. Berger
Keyword(s):  
Nucleic Acid ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Structural Changes ◽  
Molecular Mechanisms ◽  
Atp Hydrolysis ◽  
Ring Closure ◽  
Structural Studies ◽  
Saxs Data ◽  
Structural Methods

ABSTRACTRing-shaped hexameric helicases and translocases support essential DNA, RNA, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the E. coli Rho transcription termination factor as a model system, we have employed solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. SAXS data show that Rho preferentially adopts an open-ring state in solution, and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish the first structural snapshots of substepping events in a hexameric helicase/translocase.SIGNIFICANCEHexameric, ring-shaped translocases are molecular motors that convert the chemical energy of ATP hydrolysis into the physical movement of protein and nucleic acid substrates. Structural studies of several distinct hexameric translocases have provided insights into how substrates are loaded and translocated; however, the range of structural changes required for coupling ATP turnover to a full cycle of substrate loading and translocation has not been visualized for any one system. Here, we combine low-and high-resolution structural studies of the Rho helicase, defining for the first time the ensemble of conformational transitions required both for substrate loading in solution and for substrate movement by a processive hexameric translocase.

scholarly journals Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase

2019 ◽  
Author(s):  
Eva Absmeier ◽  
Karine F. Santos ◽  
Markus C. Wahl
Keyword(s):  
Water Molecule ◽  
Atpase Activity ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Bound States ◽  
Molecular Mechanisms ◽  
Adenine Nucleotides ◽  
Rna Helicase ◽  
Terminal Region ◽  
List Type

SUMMARYRNA-dependent NTPases can act as RNA/RNA-protein remodeling enzymes and typically exhibit low NTPase activity in the absence of RNA/RNA-protein substrates. How futile intrinsic NTP hydrolysis is prevented is frequently not known. The ATPase/RNA helicase Brr2 belongs to the Ski2-like family of nucleic acid-dependent NTPases and is an integral component of the spliceosome. Comprehensive nucleotide binding and hydrolysis studies are not available for a member of the Ski2-like family. We present crystal structures of Chaetomium thermophilum Brr2 in the apo, ADP-bound and ATPyS-bound states, revealing nucleotide-induced conformational changes and a hitherto unknown ATPyS binding mode. Our results in conjunction with Brr2 structures in other molecular contexts reveal multiple molecular mechanisms that contribute to the inhibition of intrinsic ATPase activity, including an N-terminal region that restrains the RecA-like domains in an open conformation and exclusion of an attacking water molecule, and suggest how RNA substrate binding can lead to ATPase stimulation.HIGHLIGHTSCrystal structures of Brr2 in complex with different adenine nucleotides.The Brr2 N-terminal region counteracts conformational changes induced by ATP binding.Brr2 excludes an attacking water molecule in the absence of substrate RNA.Different helicase families resort to different NTPase mechanisms.

scholarly journals Visualization of Calcium Ion Loss from Rotavirus during Cell Entry

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Eric N. Salgado ◽  
Brian Garcia Rodriguez ◽  
Nagarjun Narayanaswamy ◽  
Yamuna Krishnan ◽  
Stephen C. Harrison
Keyword(s):  
Conformational Changes ◽  
Outer Layer ◽  
Live Cell Imaging ◽  
Molecular Mechanisms ◽  
Cell Imaging ◽  
Tight Binding ◽  
Live Cell ◽  
Membrane Disruption ◽  
Molecular Events

ABSTRACTBound calcium ions stabilize many nonenveloped virions. Loss of Ca2+from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+-stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca2+in vitrostrips away both outer layer proteins; we and others have postulated that the loss of Ca2+triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+sensor, the timing of Ca2+loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca2+sensor, placed on VP5*, monitors the Ca2+concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca2+precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca2+loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.IMPORTANCENonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca2+sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca2+loss from an entering particle and other molecular events in the entry pathway. Specific Ca2+binding stabilizes many other viruses of eukaryotes, and Ca2+loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens.

Crystal structures and catalytic mechanism of theC-methyltransferase Coq5 provide insights into a key step of the yeast coenzyme Q synthesis pathway

2014 ◽  
Vol 70 (8) ◽  
pp. 2085-2092 ◽  
Author(s):  
Ya-Nan Dai ◽  
Kang Zhou ◽  
Dong-Dong Cao ◽  
Yong-Liang Jiang ◽  
Fei Meng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Coenzyme Q ◽  
Computational Simulation ◽  
Binding Pocket ◽  
Multiple Sequence ◽  
Core Domain ◽  
Dimeric Interface ◽  
Adenosyl Methionine

Saccharomyces cerevisiaeCoq5 is anS-adenosyl methionine (SAM)-dependent methyltransferase (SAM-MTase) that catalyzes the onlyC-methylation step in the coenzyme Q (CoQ) biosynthesis pathway, in which 2-methoxy-6-polyprenyl-1,4-benzoquinone (DDMQH2) is converted to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (DMQH2). Crystal structures of Coq5 were determined in the apo form (Coq5-apo) at 2.2 Å resolution and in the SAM-bound form (Coq5-SAM) at 2.4 Å resolution, representing the first pair of structures for the yeast CoQ biosynthetic enzymes. Coq5 displays a typical class I SAM-MTase structure with two minor variations beyond the core domain, both of which are considered to participate in dimerization and/or substrate recognition. Slight conformational changes at the active-site pocket were observed upon binding of SAM. Structure-based computational simulation using an analogue of DDMQH2enabled us to identify the binding pocket and entrance tunnel of the substrate. Multiple-sequence alignment showed that the residues contributing to the dimeric interface and the SAM- and DDMQH2-binding sites are highly conserved in Coq5 and homologues from diverse species. A putative catalytic mechanism of Coq5 was proposed in which Arg201 acts as a general base to initiate catalysis with the help of a water molecule.

scholarly journals Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Zhixun Shen ◽  
Sichen Liu ◽  
Xinxin Li ◽  
Zhengpeng Wan ◽  
Youxiang Mao ◽  
...  
Keyword(s):  
B Cell ◽  
Conformational Changes ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Spatial Relationship ◽  
Extracellular Domain ◽  
Cell Receptor ◽  
B Cell Activation ◽  
Downstream Signaling ◽  
Molecular Events

B lymphocytes use B cell receptors (BCRs) to recognize antigens. It is still not clear how BCR transduces antigen-specific physical signals upon binding across cell membrane for the conversion to chemical signals, triggering downstream signaling cascades. It is hypothesized that through a series of conformational changes within BCR, antigen engagement in the extracellular domain of BCR is transduced to its intracellular domain. By combining site-specific labeling methodology and FRET-based assay, we monitored conformational changes in the extracellular domains within BCR upon antigen engagement. Conformational changes within heavy chain of membrane-bound immunoglobulin (mIg), as well as conformational changes in the spatial relationship between mIg and Igβ were observed. These conformational changes were correlated with the strength of BCR activation and were distinct in IgM- and IgG-BCR. These findings provide molecular mechanisms to explain the fundamental aspects of BCR activation and a framework to investigate ligand-induced molecular events in immune receptors.

scholarly journals A Time-Resolved Diffusion Technique for Detection of the Conformational Changes and Molecular Assembly/Disassembly Processes of Biomolecules

2021 ◽  
Vol 12 ◽  
Author(s):  
Yusuke Nakasone ◽  
Masahide Terazima
Keyword(s):  
Intermolecular Interaction ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Dna Interaction ◽  
Molecular Assembly ◽  
Kinetic Measurements ◽  
Time Resolved ◽  
Molecular Events ◽  
Diffusion Technique ◽  
Sensor Proteins

Biological liquid–liquid phase separation (LLPS) is driven by dynamic and multivalent interactions, which involves conformational changes and intermolecular assembly/disassembly processes of various biomolecules. To understand the molecular mechanisms of LLPS, kinetic measurements of the intra- and intermolecular reactions are essential. In this review, a time-resolved diffusion technique which has a potential to detect molecular events associated with LLPS is presented. This technique can detect changes in protein conformation and intermolecular interaction (oligomer formation, protein-DNA interaction, and protein-lipid interaction) in time domain, which are difficult to obtain by other methods. After the principle and methods for signal analyses are described in detail, studies on photoreactive molecules (intermolecular interaction between light sensor proteins and its target DNA) and a non-photoreactive molecule (binding and folding reaction of α-synuclein upon mixing with SDS micelle) are presented as typical examples of applications of this unique technique.

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 studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qing-Tao He ◽  
Peng Xiao ◽  
Shen-Ming Huang ◽  
Ying-Li Jia ◽  
Zhong-Liang Zhu ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Nmr Spectrum ◽  
H Nmr ◽  
Interaction Sites ◽  
Receptor Interactions ◽  
Remote Sites ◽  
Genetic Code Expansion ◽  
G Protein Coupled ◽  
Interaction Change

AbstractArrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.

scholarly journals Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingmin Yuan ◽  
Zongyang Lv ◽  
Melanie J. Adams ◽  
Shaun K. Olsen
Keyword(s):  
Complex Formation ◽  
Crystal Structures ◽  
Molecular Mechanisms ◽  
Conformational State ◽  
Biochemical Data ◽  
Conformational States ◽  
Product Release ◽  
Closed Conformation ◽  
Open Conformation ◽  
Conjugating Enzymes

AbstractE1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

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