scholarly journals Structural basis of BAK activation in mitochondrial apoptosis initiation

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Geetika Singh ◽  
Cristina D. Guibao ◽  
Jayaraman Seetharaman ◽  
Anup Aggarwal ◽  
Christy R. Grace ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Basis ◽  
Ligand Affinity ◽  
High Affinity ◽  
Direct Activation ◽  
In Trans ◽  
Hit And Run ◽  
Membrane Poration ◽  
Apoptosis Initiation

AbstractBCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

New insights into the structural basis of integrin activation

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1155-1159 ◽  
Author(s):  
Jian-Ping Xiong ◽  
Thilo Stehle ◽  
Simon L. Goodman ◽  
M. Amin Arnaout
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Basis ◽  
Integrin Activation ◽  
Cellular Functions ◽  
Electron Microscopic ◽  
The Past ◽  
Intracellular Signals ◽  
Bidirectional Signaling ◽  
New Hypothesis

Abstract Integrins are cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane and thus influence most cellular functions. Intracellular signals switch integrins into a ligand-competent state as a result of elicited conformational changes in the integrin ectodomain. Binding of extracellular ligands induces, in turn, structural changes that convey distinct signals to the cell interior. The structural basis of this bidirectional signaling has been the focus of intensive study for the past 3 decades. In this perspective, we develop a new hypothesis for integrin activation based on recent crystallographic, electron microscopic, and biochemical studies.

scholarly journals Structural changes during HCN channel gating defined by high affinity metal bridges

2012 ◽  
Vol 140 (3) ◽  
pp. 279-291 ◽  
Author(s):  
Daniel C.H. Kwan ◽  
David L. Prole ◽  
Gary Yellen
Keyword(s):  
Structural Changes ◽  
Structural Model ◽  
Channel Gating ◽  
Structural Basis ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Membrane Hyperpolarization ◽  
Linker Region ◽  
High Affinity ◽  
Gating Mechanism

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

scholarly journals Two distinct mechanisms of transcriptional regulation by the redox sensor YodB

2016 ◽  
Vol 113 (35) ◽  
pp. E5202-E5211 ◽  
Author(s):  
Sang Jae Lee ◽  
In-Gyun Lee ◽  
Ki-Young Lee ◽  
Dong-Gyun Kim ◽  
Hyun-Jong Eun ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
High Specificity ◽  
Redox Balance ◽  
Structural Basis ◽  
X Ray Crystallography ◽  
Redox Sensor ◽  
Regulatory Systems ◽  
Sense And Respond

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis. This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

scholarly journals ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Mark F. Mabanglo ◽  
Elisa Leung ◽  
Siavash Vahidi ◽  
Thiago V. Seraphim ◽  
Bryan T. Eger ◽  
...  
Keyword(s):  
Small Molecule ◽  
Conformational Changes ◽  
Electrostatic Interaction ◽  
Structural Changes ◽  
Structural Heterogeneity ◽  
Interaction Networks ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Bacterial Cell Death

Abstract Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

scholarly journals Trypsin-Induced Structural Transformation in Aquareovirus

2000 ◽  
Vol 74 (14) ◽  
pp. 6546-6555 ◽  
Author(s):  
Emma L. Nason ◽  
Siba K. Samal ◽  
B. V. Venkataram Prasad
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Three Dimensional ◽  
Structural Basis ◽  
Trypsin Treatment ◽  
A Genome ◽  
The Family ◽  
Large Virus ◽  
Infectious Stage ◽  
Outer Capsid

ABSTRACT Aquareovirus, a member of the familyReoviridae, is a large virus with multiple capsid layers surrounding a genome composed of 11 segments of double-stranded RNA. Biochemical studies have shown that treatment with the proteolytic agent trypsin significantly alters the infectivity of the virus. The most infectious stage of the virus is produced by a 5-min treatment with trypsin. However, prolonged trypsin treatment almost completely abolishes the infectivity. We have used three-dimensional electron cryomicroscopy to gain insight into the structural basis of protease-induced alterations in infectivity by examining the structural changes in the virion at various time intervals of trypsin treatment. Our data show that after 5 min of trypsinization, projection-like spikes made of VP7 (35 kDa), associated with the underlying trimeric subunits, are completely removed. Concurrent with the removal of VP7, conformational changes are observed in the trimeric subunit composed of putative VP5 (71 kDa). The removal of VP7 and the accompanied structural changes may expose regions in the putative VP5 important for cell entry processes. Prolonged trypsinization not only entirely removes the outer capsid layer, producing the poorly infectious core particle, but also causes significant conformational changes in the turret protein. These changes result in shortening of the turret and narrowing of its central channel. The turret, as in orthoreoviruses, is likely to play a major role in the capping and translocation of mRNA during transcription, and the observed conformational flexibility in the turret protein may have implications in rendering the particle transcriptionally active or inactive.

scholarly journals Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Richard B Cooley ◽  
John P O’Donnell ◽  
Holger Sondermann
Keyword(s):  
Conformational Changes ◽  
Biofilm Formation ◽  
Second Messenger ◽  
Receptor Activation ◽  
Coincidence Detection ◽  
Physiological Adaptation ◽  
Structural Basis ◽  
Metabolizing Enzymes ◽  
High Affinity ◽  
Receptor Dimer

The second messenger c-di-GMP (or cyclic diguanylate) regulates biofilm formation, a physiological adaptation process in bacteria, via a widely conserved signaling node comprising a prototypical transmembrane receptor for c-di-GMP, LapD, and a cognate periplasmic protease, LapG. Previously, we reported a structure-function study of a soluble LapD•LapG complex, which established conformational changes in the receptor that lead to c-di-GMP-dependent protease recruitment (Chatterjee et al., 2014). This work also revealed a basal affinity of c-di-GMP-unbound receptor for LapG, the relevance of which remained enigmatic. Here, we elucidate the structural basis of coincidence detection that relies on both c-di-GMP and LapG binding to LapD for receptor activation. The data indicate that high-affinity for LapG relies on the formation of a receptor dimer-of-dimers, rather than a simple conformational change within dimeric LapD. The proposed mechanism provides a rationale of how external proteins can regulate receptor function and may also apply to c-di-GMP-metabolizing enzymes that are akin to LapD.

scholarly journals E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
James Chen ◽  
Saumya Gopalkrishnan ◽  
Courtney Chiu ◽  
Albert Y Chen ◽  
Elizabeth A Campbell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Structural Basis ◽  
E Coli ◽  
Bacterial Proteins ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Induced Changes

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

scholarly journals Conformational dynamics of androgen receptors bound to agonists and antagonists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hyo Jin Gim ◽  
Jiyong Park ◽  
Michael E. Jung ◽  
K. N. Houk
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Integrity ◽  
Structural Information ◽  
Close Contact ◽  
Conformational Dynamics ◽  
Principal Component ◽  
Binding Pocket ◽  
Structural Basis ◽  
Accelerated Molecular Dynamics

AbstractThe androgen receptor (AR) is critical in the progression of prostate cancer (PCa). Small molecule antagonists that bind to the ligand binding domain (LBD) of the AR have been successful in treating PCa. However, the structural basis by which the AR antagonists manifest their therapeutic efficacy remains unclear, due to the lack of detailed structural information of the AR bound to the antagonists. We have performed accelerated molecular dynamics (aMD) simulations of LBDs bound to a set of ligands including a natural substrate (dihydrotestosterone), an agonist (RU59063) and three antagonists (bicalutamide, enzalutamide and apalutamide) as well as in the absence of ligand (apo). We show that the binding of AR antagonists at the substrate binding pocket alter the dynamic fluctuations of H12, thereby disrupting the structural integrity of the agonistic conformation of AR. Two antagonists, enzalutamide and apalutamide, induce considerable structural changes to the agonist conformation of LBD, when bound close to H12 of AR LBD. When the antagonists bind to the pocket with different orientations having close contact with H11, no significant conformational changes were observed, suggesting the AR remains in the functionally activated (agonistic) state. The simulations on a drug resistance mutant F876L bound to enzalutamide demonstrated that the mutation stabilizes the agonistic conformation of AR LBD, which compromises the efficacy of the antagonists. Principal component analysis (PCA) of the structural fluctuations shows that the binding of enzalutamide and apalutamide induce conformational fluctuations in the AR, which are markedly different from those caused by the agonist as well as another antagonist, bicalutamide. These fluctuations could only be observed with the use of aMD.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Abstract Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

scholarly journals Structural basis for two-way communication between dynein and microtubules

2019 ◽  
Author(s):  
Noritaka Nishida ◽  
Yuta Komori ◽  
Osamu Takarada ◽  
Atsushi Watanabe ◽  
Satoko Tamura ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Conformational Changes ◽  
Structural Changes ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Structural Basis ◽  
Atpase Domain ◽  
Directional Bias ◽  
Α Helix ◽  
Half Turn

AbstractThe movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain of dynein via an a-helical coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulated MTBD either in high-affinity or low-affinity states by introducing a disulfide bond between the coiled-coils and analyzed the resulting structures by NMRand cryo-EM. In the MT-unbound state, the affinity changes of MTBD were achieved by sliding of the N-terminal α-helix by one half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, cryo-EM analysis showed that MT binding induced further sliding of the N-terminal α-helix even without the disulfide bond, which suggests the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.

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