scholarly journals Unloxing the assembly and activation mechanism of Cre recombinase using Cryo-EM

2021 ◽  
Author(s):  
Kye Stachowski ◽  
Andrew Norris ◽  
Devante Potter ◽  
Vicki Wysocki ◽  
Mark Foster
Keyword(s):  
Conformational Changes ◽  
Site Selection ◽  
Dna Bending ◽  
Dna Recombination ◽  
Cre Recombinase ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Conformational Sampling ◽  
Protein Motifs ◽  
Activation Mechanisms

Mechanistic understanding of the structural basis for DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes (intasomes). These structural and biochemical studies have suggested that conformational changes and DNA bending in presynaptic complexes underlie site-selection and activation mechanisms of Cre recombinase. Here we used protein engineering and various DNA substrates to isolate the Cre-loxP (54 kDa), Cre2-loxP (110 kDa), and Cre4-loxP2 assembly intermediates, and determined their structures using cryo-EM to resolutions of 3.9 Å, 4.5 Å, and 3.2 Å, respectively. Progressive DNA bending along the assembly pathway enables formation of increasingly intimate protein-protein interfaces. Insufficient stabilization of important protein motifs observed during the assembly process provides a compelling explanation for the observed half-the-sites activity, and preferential bottom strand cleavage of loxP sequences. We found that selection of loxP sites is largely dependent on the ability for Cre to bend and stabilize the spacer region between two recombinase binding elements. Application of 3D variability analysis to the tetramer data reveals a propensity for motion along the pathway between protomer activation and Holliday junction isomerization. These findings help us to better understand loxP site specificity, controlled activation of alternating protomers, the basis for the observed bias of strand cleavage order, and the importance of conformational sampling, especially with regards to site-selection and activity among Cre variants. Furthermore, our findings provide invaluable information for the rational development of designer, site-specific recombinases for use as gene editing technologies.

scholarly journals Structural insights into the activation of human calcium-sensing receptor

2021 ◽  
Author(s):  
Xiaochen Chen ◽  
Lu Wang ◽  
Zhanyu Ding ◽  
Qianqian Cui ◽  
Li Han ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Transmembrane Domains ◽  
Structural Basis ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Cryo Electron Microscopy ◽  
Activation Mechanisms ◽  
G Protein Coupled ◽  
Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease

2020 ◽  
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

ABSTRACTCas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 Å and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

scholarly journals Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease

2021 ◽  
Vol 49 (7) ◽  
pp. 4120-4128
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

Abstract Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

scholarly journals Structural insights into the activation of human calcium-sensing receptor

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Xiaochen Chen ◽  
Lu Wang ◽  
Qianqian Cui ◽  
Zhanyu Ding ◽  
Li Han ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Bound States ◽  
Large Scale ◽  
Transmembrane Domains ◽  
Structural Basis ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Activation Mechanisms ◽  
G Protein Coupled

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of 7-transmembrane domains, and inter- and intrasubunit conformational changes of 7-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca2+ ions and L-amino acid leading to the activation of the receptor.

scholarly journals Expression and purification of Protease Activated Receptor 4 (PAR4) and analysis with histidine hydrogen deuterium exchange

2019 ◽  
Author(s):  
Maria de la Fuente ◽  
Xu Han ◽  
Masaru Miyagi ◽  
Marvin T. Nieman
Keyword(s):  
Family Member ◽  
Conformational Changes ◽  
Deuterium Exchange ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Hydrogen Deuterium Exchange ◽  
Expression And Purification ◽  
Agonist Peptide ◽  
Hydrogen Deuterium ◽  
Tethered Ligand

ABSTRACTProtease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by proteolyis of the N-terminus, which exposes a tethered ligand that interacts with the receptor. Numerous studies have focused on the signaling pathways mediated by PARs. However, the structural basis for initiation of these pathways is unknown. Here, we describe a strategy for the expression and purification of PAR4. This is the first PAR family member to be isolated without stabilizing modifications for biophysical studies. We monitored PAR4 activation with histidine-hydrogen deuterium exchange (His-HDX). PAR4 has 9 histidines that are spaced throughout the protein allowing a global view of solvent accessible and non-accessible regions. Peptides containing each of the 9 His residues were used to determine the t1/2 for each His residue in apo or thrombin activated PAR4. The thrombin cleaved PAR4 had a 2-fold increase (p > 0.01) in t1/2 values observed for four histidine residues (His180, His229, His240, and His380) demonstrating that these regions have decreased solvent accessibility upon thrombin treatment. In agreement, thrombin cleaved PAR4 also was resistant to thermolysin digestion. In contrast, activation with the PAR4 agonist peptide was digested at the same rate as apo PAR4. Further analysis showed the C-terminus is protected in thrombin activated PAR4 compared to uncleaved or agonist peptide treated PAR4. The studies described here are the first to examine the tethered ligand activation mechanism for a PAR family member using biophysically and shed light on the overall conformational changes that follow activation of PARs by a protease.

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.

scholarly journals Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

2020 ◽  
Author(s):  
Polina Isaikina ◽  
Ching-Ju Tsai ◽  
Nikolaus Dietz ◽  
Filip Pamula ◽  
Anne Grahl ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Binding Mode ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Cc Chemokine ◽  
Gi Protein ◽  
Agonist And Antagonist ◽  
Cc Chemokine Receptor 5 ◽  
Chemokine Receptor 5

AbstractThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism.One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

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