Crystal Structure of Streptococcus pyogenes Csn2 Reveals Calcium-Dependent Conformational Changes in Its Tertiary and Quaternary Structure

AbstractRET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFRα1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.HighlightsCrystal structure of zebrafish RET cadherin-like module reveals conformational flexibility at the calcium-dependent CLD2-CLD3 interfaceComparison of X-ray and cryo-EM structures indicate conformational differences between unliganded and liganded RET involving a clade-specific CLD3 loopStrict spatial separation of RETECD C-termini is imposed by each cysteine-rich domain interaction with GFL dimerDifferences in co-receptor engagement and higher-order ligand-bound RET complexes indicate potentially divergent signalling mechanisms

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122952 ◽

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).

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Crystal structure of the conserved hypothetical cytosolic protein Xcc0516 fromXanthomonas campestrisreveals a novel quaternary structure assembled by five four-helix bundles

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.21105 ◽

2006 ◽

Vol 65 (3) ◽

pp. 783-786 ◽

Cited By ~ 6

Author(s):

Li-Ying Lin ◽

Chung-Lun Ching ◽

Ko-Hsin Chin ◽

Shan-Ho Chou ◽

Nei-Li Chan

Keyword(s):

Crystal Structure ◽

Quaternary Structure ◽

Cytosolic Protein

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The first crystal structure of the peptidase domain of the U32 peptidase family

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715019549 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2505-2512 ◽

Cited By ~ 4

Author(s):

Magdalena Schacherl ◽

Angelika A. M. Montada ◽

Elena Brunstein ◽

Ulrich Baumann

Keyword(s):

Crystal Structure ◽

Catalytic Mechanism ◽

Quaternary Structure ◽

Catalytic Domain ◽

Three Dimensional ◽

Zinc Ion ◽

Crystal Structure Analysis ◽

Dimensional Structure ◽

Sequence Motifs ◽

Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

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Improved resolution crystal structure of Acanthamoeba actophorin reveals structural plasticity not induced by microgravity

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21011419 ◽

2021 ◽

Vol 77 (12) ◽

Author(s):

Stephen Quirk ◽

Raquel L. Lieberman

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Actin Filaments ◽

Space Station ◽

Acanthamoeba Castellanii ◽

Test Case ◽

Molecular Replacement ◽

International Space ◽

Microgravity Conditions ◽

Turn Region

Actophorin, a protein that severs actin filaments isolated from the amoeba Acanthamoeba castellanii, was employed as a test case for crystallization under microgravity. Crystals of purified actophorin were grown under microgravity conditions aboard the International Space Station (ISS) utilizing an interactive crystallization setup between the ISS crew and ground-based experimenters. Crystals grew in conditions similar to those grown on earth. The structure was solved by molecular replacement at a resolution of 1.65 Å. Surprisingly, the structure reveals conformational changes in a remote β-turn region that were previously associated with actophorin phosphorylated at the terminal residue Ser1. Although crystallization under microgravity did not yield a higher resolution than crystals grown under typical laboratory conditions, the conformation of actophorin obtained from solving the structure suggests greater flexibility in the actophorin β-turn than previously appreciated and may be beneficial for the binding of actophorin to actin filaments.

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The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714023827 ◽

2014 ◽

Vol 70 (12) ◽

pp. 3212-3225 ◽

Cited By ~ 16

Author(s):

Tiila-Riikka Kiema ◽

Rajesh K. Harijan ◽

Malgorzata Strozyk ◽

Toshiyuki Fukao ◽

Stefan E. H. Alexson ◽

...

Keyword(s):

Crystal Structure ◽

Reaction Mechanism ◽

Active Site ◽

Quaternary Structure ◽

Fatty Acyl ◽

Physiological Importance ◽

Loop Domain ◽

Solution Studies ◽

Hydrolysis Of ◽

Insight Into

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of theZoogloea ramigerabiosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

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Crystal structure of SARS-CoV-2 main protease in complex with a Chinese herb inhibitor shikonin

10.1101/2020.06.16.155812 ◽

2020 ◽

Author(s):

Jian Li ◽

Xuelan Zhou ◽

Yan Zhang ◽

Fanglin Zhong ◽

Cheng Lin ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Drug Targets ◽

Chinese Herb ◽

Binding Modes ◽

High Potency ◽

Main Protease ◽

Covalent Inhibitors ◽

Catalytic Dyad ◽

Important Drug

AbstractMain protease (Mpro, also known as 3CLpro) has a major role in the replication of coronavirus life cycle and is one of the most important drug targets for anticoronavirus agents. Here we report the crystal structure of main protease of SARS-CoV-2 bound to a previously identified Chinese herb inhibitor shikonin at 2.45 angstrom resolution. Although the structure revealed here shares similar overall structure with other published structures, there are several key differences which highlight potential features that could be exploited. The catalytic dyad His41-Cys145 undergoes dramatic conformational changes, and the structure reveals an unusual arrangement of oxyanion loop stabilized by the substrate. Binding to shikonin and binding of covalent inhibitors show different binding modes, suggesting a diversity in inhibitor binding. As we learn more about different binding modes and their structure-function relationships, it is probable that we can design more effective and specific drugs with high potency that can serve as effect SARS-CoV-2 anti-viral agents.

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Crystal structure of the stromelysin catalytic domain at 2.0 Å resolution: inhibitor-induced conformational changes 1 1Edited by R. Huber

Journal of Molecular Biology ◽

10.1006/jmbi.1999.3147 ◽

1999 ◽

Vol 293 (3) ◽

pp. 545-557 ◽

Cited By ~ 45

Author(s):

Longyin Chen ◽

Timothy J Rydel ◽

Fei Gu ◽

C.Michelle Dunaway ◽

Stanislaw Pikul ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Catalytic Domain

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Controlling Quaternary Structure Assembly: Subunit Interface Engineering and Crystal Structure of Dual Chain Avidin

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.04.044 ◽

2006 ◽

Vol 359 (5) ◽

pp. 1352-1363 ◽

Cited By ~ 10

Author(s):

Vesa P. Hytönen ◽

Jarno Hörhä ◽

Tomi T. Airenne ◽

Einari A. Niskanen ◽

Kaisa J. Helttunen ◽

...

Keyword(s):

Crystal Structure ◽

Quaternary Structure ◽

Interface Engineering ◽

Subunit Interface ◽

Structure Assembly

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Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2020438118 ◽

2020 ◽

Vol 118 (1) ◽

pp. e2020438118

Author(s):

Ryuta Kanai ◽

Flemming Cornelius ◽

Haruo Ogawa ◽

Kanna Motoyama ◽

Bente Vilsen ◽

...

Keyword(s):

Crystal Structure ◽

Crystal Structures ◽

Conformational Changes ◽

Ground State ◽

Sodium Pump ◽

Structural Features ◽

Animal Cells ◽

Inhibitory Potency ◽

New Members ◽

Wide Range

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3− complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.

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