scholarly journals Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

2012 ◽  
Vol 198 (3) ◽  
pp. 315-322 ◽  
Author(s):  
Hiroaki Yajima ◽  
Toshihiko Ogura ◽  
Ryo Nitta ◽  
Yasushi Okada ◽  
Chikara Sato ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Regulatory Mechanism ◽  
Reconstruction Algorithm ◽  
Microtubule Dynamics ◽  
Structural Basis ◽  
Cryoelectron Microscopy ◽  
Guanosine Triphosphate ◽  
Gtp Hydrolysis ◽  
Guanosine Diphosphate ◽  
The Stability

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5′-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.

scholarly journals Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

2022 ◽  
Vol 119 (2) ◽  
pp. e2114994119
Author(s):  
Benjamin J. LaFrance ◽  
Johanna Roostalu ◽  
Gil Henkin ◽  
Basil J. Greber ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Total Internal Reflection ◽  
Dynamic Instability ◽  
Wild Type ◽  
Gtp Hydrolysis ◽  
Cryo Electron Microscopy ◽  
Catalytically Active ◽  
The Stability ◽  
Insight Into

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis–deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

scholarly journals Structural changes accompanying GTP hydrolysis in microtubules: information from a slowly hydrolyzable analogue guanylyl-(alpha,beta)-methylene-diphosphonate.

1995 ◽  
Vol 128 (1) ◽  
pp. 117-125 ◽  
Author(s):  
A A Hyman ◽  
D Chrétien ◽  
I Arnal ◽  
R H Wade
Keyword(s):  
Structural Change ◽  
Structural Changes ◽  
Structural Difference ◽  
Structural Basis ◽  
Cryoelectron Microscopy ◽  
Gtp Hydrolysis ◽  
Methylene Diphosphonate ◽  
Moire Patterns ◽  
Alpha Beta

We have used cryoelectron microscopy to try to understand the structural basis for the role of GTP hydrolysis in destabilizing the microtubule lattice. We have measured a structural difference introduced into microtubules by replacing GTP with guanylyl-(alpha,beta)-methylene-diphosphonate (GMPCPP). In a stable GMPCPP microtubule lattice, the moiré patterns change and the tubulin subunits increase in size by 1.5 A. This information provides a clue to the role of hydrolysis in inducing the structural change at the end of a microtubule during the transition from a growing to a shrinking phase.

scholarly journals Rab17 regulates apical delivery of hepatic transcytotic vesicles

2018 ◽  
Vol 29 (23) ◽  
pp. 2887-2897 ◽  
Author(s):  
Anneliese C. Striz ◽  
Anna P. Stephan ◽  
Alfonso López-Coral ◽  
Pamela L. Tuma
Keyword(s):  
Dominant Negative ◽  
Apical Surface ◽  
Wild Type ◽  
Major Focus ◽  
Guanosine Triphosphate ◽  
Gtp Hydrolysis ◽  
Vesicle Docking ◽  
Guanosine Diphosphate ◽  
Selective Interactions ◽  
General Component

A major focus for our laboratory is identifying the molecules and mechanisms that regulate basolateral-to-apical transcytosis in polarized hepatocytes. Our most recent studies have focused on characterizing the biochemical and functional properties of the small rab17 GTPase. We determined that rab17 is a monosumoylated protein and that this modification likely mediates selective interactions with the apically located syntaxin 2. Using polarized hepatic WIF-B cells exogenously expressing wild-type, dominant active/guanosine triphosphate (GTP)-bound, dominant negative/guanosine diphosphate (GDP)-bound, or sumoylation-deficient/K68R rab17 proteins, we confirmed that rab17 regulates basolateral-to-apical transcytotic vesicle docking and fusion with the apical surface. We further confirmed that transcytosis is impaired from the subapical compartment to the apical surface and that GTP-bound and sumoylated rab17 are likely required for apical vesicle docking. Because expression of the GTP-bound rab17 led to impaired transcytosis, whereas wild type had no effect, we further propose that rab17 GTP hydrolysis is required for vesicle delivery. We also determined that transcytosis of three classes of newly synthesized apical residents showed similar responses to rab17 mutant expression, indicating that rab17 is a general component of the transcytotic machinery required for apically destined vesicle docking and fusion.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

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 activation and allosteric modulation of full-length calcium-sensing receptor

2021 ◽  
Vol 7 (23) ◽  
pp. eabg1483
Author(s):  
Tianlei Wen ◽  
Ziyu Wang ◽  
Xiaozhe Chen ◽  
Yue Ren ◽  
Xuhang Lu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Hormone Secretion ◽  
Allosteric Modulation ◽  
Full Length ◽  
Structural Basis ◽  
Endocrine Disorders ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Class C

Calcium-sensing receptor (CaSR) is a class C G protein–coupled receptor (GPCR) that plays an important role in calcium homeostasis and parathyroid hormone secretion. Here, we present multiple cryo–electron microscopy structures of full-length CaSR in distinct ligand-bound states. Ligands (Ca2+ and l-tryptophan) bind to the extracellular domain of CaSR and induce large-scale conformational changes, leading to the closure of two heptahelical transmembrane domains (7TMDs) for activation. The positive modulator (evocalcet) and the negative allosteric modulator (NPS-2143) occupy the similar binding pocket in 7TMD. The binding of NPS-2143 causes a considerable rearrangement of two 7TMDs, forming an inactivated TM6/TM6 interface. Moreover, a total of 305 disease-causing missense mutations of CaSR have been mapped to the structure in the active state, creating hotspot maps of five clinical endocrine disorders. Our results provide a structural framework for understanding the activation, allosteric modulation mechanism, and disease therapy for class C GPCRs.

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.

F-box protein MdAMR1L1 regulates ascorbate biosynthesis in apple by modulating GDP-mannose pyrophosphorylase

2021 ◽  
Author(s):  
Songya Ma ◽  
Huixia Li ◽  
Lan Wang ◽  
Baiyun Li ◽  
Zhengyang Wang ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Transcript Level ◽  
Ethylene Response Factor ◽  
Protein Abundance ◽  
Protein Levels ◽  
Guanosine Diphosphate ◽  
Physiological Processes ◽  
Ethylene Response ◽  
Ubiquitination Pathway ◽  
F Box Protein

Abstract Ascorbate (Asc) is an important antioxidant in plants and humans that plays key roles in various physiological processes. Understanding the regulation of Asc content in fruit plants is important for improving plant resiliency and optimizing Asc in food. Here, we found that both the transcript level and protein abundance of Asc Mannose pathway Regulator 1 Like 1 (MdAMR1L1) was negatively associated with Asc levels during the development of apple (Malus × domestica) fruit. The overexpression or silencing of MdAMR1L1 in apple indicated that MdAMR1L1 negatively regulated Asc levels. However, in the leaves of MdAMR1L1-overexpressing apple lines, the transcript levels of the Asc synthesis gene Guanosine diphosphate-mannose pyrophosphorylase MdGMP1 were increased, while its protein levels and enzyme activity were reduced. This occurred because the MdAMR1L1 protein interacted with MdGMP1 and promoted its degradation via the ubiquitination pathway to inhibit Asc synthesis at the post-translational level. MdERF98, an apple ethylene response factor, whose transcription was modulated by Asc level, is directly bound to the promoter of MdGMP1 to promote the transcription of MdGMP1. These findings provide insights into the regulatory mechanism of Asc biosynthesis in apples and revealed potential opportunities to improve fruit Asc levels.

Sign in / Sign up

Export Citation Format

Share Document