scholarly journals Crystal structure and calcium‐induced conformational changes of diacylglycerol kinase α EF‐hand domains

2019 ◽  
Vol 28 (4) ◽  
pp. 694-706 ◽  
Author(s):  
Daisuke Takahashi ◽  
Kano Suzuki ◽  
Taiichi Sakamoto ◽  
Takeo Iwamoto ◽  
Takeshi Murata ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Diacylglycerol Kinase ◽  
Ef Hand

scholarly journals EF-hand motifs of α, β and γ isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes

1997 ◽  
Vol 321 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Keiko YAMADA ◽  
Fumio SAKANE ◽  
Norio MATSUSHIMA ◽  
Hideo KANOH
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Diacylglycerol Kinase ◽  
Intrinsic Properties ◽  
Apparent Dissociation Constant ◽  
Ef Hand ◽  
Order Of Magnitude ◽  
Hydrophobic Amino Acids ◽  
Ef Hands

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.

scholarly journals Crystal structures of Ca2+–calmodulin bound to NaV C-terminal regions suggest role for EF-hand domain in binding and inactivation

2019 ◽  
Vol 116 (22) ◽  
pp. 10763-10772 ◽  
Author(s):  
Bernd R. Gardill ◽  
Ricardo E. Rivera-Acevedo ◽  
Ching-Chieh Tung ◽  
Filip Van Petegem
Keyword(s):  
Crystal Structure ◽  
Binding Sites ◽  
Binding Mode ◽  
Conformational Switch ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Ef Hand ◽  
Inherited Arrhythmia ◽  
A Site

Voltage-gated sodium (NaV) and calcium channels (CaV) form targets for calmodulin (CaM), which affects channel inactivation properties. A major interaction site for CaM resides in the C-terminal (CT) region, consisting of an IQ domain downstream of an EF-hand domain. We present a crystal structure of fully Ca2+-occupied CaM, bound to the CT of NaV1.5. The structure shows that the C-terminal lobe binds to a site ∼90° rotated relative to a previous site reported for an apoCaM complex with the NaV1.5 CT and for ternary complexes containing fibroblast growth factor homologous factors (FHF). We show that the binding of FHFs forces the EF-hand domain in a conformation that does not allow binding of the Ca2+-occupied C-lobe of CaM. These observations highlight the central role of the EF-hand domain in modulating the binding mode of CaM. The binding sites for Ca2+-free and Ca2+-occupied CaM contain targets for mutations linked to long-QT syndrome, a type of inherited arrhythmia. The related NaV1.4 channel has been shown to undergo Ca2+-dependent inactivation (CDI) akin to CaVs. We present a crystal structure of Ca2+/CaM bound to the NaV1.4 IQ domain, which shows a binding mode that would clash with the EF-hand domain. We postulate the relative reorientation of the EF-hand domain and the IQ domain as a possible conformational switch that underlies CDI.

scholarly journals The EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

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

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.

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

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.

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

1999 ◽  
Vol 293 (3) ◽  
pp. 545-557 ◽  
Author(s):  
Longyin Chen ◽  
Timothy J Rydel ◽  
Fei Gu ◽  
C.Michelle Dunaway ◽  
Stanislaw Pikul ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Catalytic Domain

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

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.

scholarly journals Recognition of an α-helical hairpin in P22 large terminase by a synthetic antibody fragment

2020 ◽  
Vol 76 (9) ◽  
pp. 876-888
Author(s):  
Ravi K. Lokareddy ◽  
Ying-Hui Ko ◽  
Nathaniel Hong ◽  
Steven G. Doll ◽  
Marcin Paduch ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Recombinant Antibody ◽  
Antibody Fragment ◽  
Genome Packaging ◽  
Synthetic Antibody ◽  
High Affinity ◽  
N Terminus ◽  
Helical Hairpin ◽  
Accessible Surface

The genome-packaging motor of tailed bacteriophages and herpesviruses is a multisubunit protein complex formed by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal protein vertex of an empty precursor capsid to power the energy-dependent packaging of viral DNA. Both the ATPase and nuclease activities associated with genome packaging reside in TerL. Structural studies of TerL from bacteriophage P22 have been hindered by the conformational flexibility of this enzyme and its susceptibility to proteolysis. Here, an unbiased, synthetic phage-display Fab library was screened and a panel of high-affinity Fabs against P22 TerL were identified. This led to the discovery of a recombinant antibody fragment, Fab4, that binds a 33-amino-acid α-helical hairpin at the N-terminus of TerL with an equilibrium dissociation constant K d of 71.5 nM. A 1.51 Å resolution crystal structure of Fab4 bound to the TerL epitope (TLE) together with a 1.15 Å resolution crystal structure of the unliganded Fab4, which is the highest resolution ever achieved for a Fab, elucidate the principles governing the recognition of this novel helical epitope. TLE adopts two different conformations in the asymmetric unit and buries as much as 1250 Å2 of solvent-accessible surface in Fab4. TLE recognition is primarily mediated by conformational changes in the third complementarity-determining region of the Fab4 heavy chain (CDR H3) that take place upon epitope binding. It is demonstrated that TLE can be introduced genetically at the N-terminus of a target protein, where it retains high-affinity binding to Fab4.

Sign in / Sign up

Export Citation Format

Share Document