scholarly journals pH-dependent gating mechanism of theHelicobacter pyloriurea channel revealed by cryo-EM

2019 ◽  
Vol 5 (3) ◽  
pp. eaav8423 ◽  
Author(s):  
Yanxiang Cui ◽  
Kang Zhou ◽  
David Strugatsky ◽  
Yi Wen ◽  
George Sachs ◽  
...  
Keyword(s):  
Drug Target ◽  
Carboxyl Terminus ◽  
Site Specific ◽  
Gating Mechanism ◽  
New Strategy ◽  
Periplasmic Domain ◽  
Urea Channel ◽  
Ph Dependent ◽  
H Pylori ◽  
Key Residues

The urea channel ofHelicobacterpylori(HpUreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication ofH. pylori. Here, we present the cryo-EM structures ofHpUreI in closed and open conformations, both at a resolution of 2.7 Å. Our hexameric structures of this small membrane protein (~21 kDa/protomer) resolve its periplasmic loops and carboxyl terminus that close and open the channel, and define a gating mechanism that is pH dependent and requires cooperativity between protomers in the hexamer. Gating is further associated with well-resolved changes in the channel-lining residues that modify the shape and length of the urea pore. Site-specific mutations in the periplasmic domain and urea pore identified key residues important for channel function. Drugs blocking the urea pore based on our structures should lead to a new strategy forH. pylorieradication.

scholarly journals Identification of Key Amino Acid Residues Determining Product Specificity of 2,3-Oxidosqualene Cyclase in Siraitia grosvenorii

Catalysts ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 577 ◽  
Author(s):  
Jing Qiao ◽  
Jiushi Liu ◽  
Jingjing Liao ◽  
Zuliang Luo ◽  
Xiaojun Ma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Bioactive Molecules ◽  
Amino Acid Residues ◽  
Product Specificity ◽  
Oxidosqualene Cyclase ◽  
Siraitia Grosvenorii ◽  
Initial Substrate ◽  
New Strategy ◽  
Key Residues

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during the initial substrate preorganization step, oxidosqualene cyclases (OSCs) are roughly segregated into a dammarenyl cation group that predominantly catalyzes triterpenoid precursor products and a protosteryl cation group which mostly generates sterol precursor products. The mechanism of conversion between two scaffolds is not well understood. Previously, we have characterized a promiscuous OSC from Siraitia grosvenorii (SgCS) that synthesizes a novel cucurbitane-type triterpene cucurbitadienol as its main product. By integration of homology modeling, molecular docking and site-directed mutagenesis, we discover that five key amino acid residues (Asp486, Cys487, Cys565, Tyr535, and His260) may be responsible for interconversions between chair–boat–chair and chair–chair–chair conformations. The discovery of euphol, dihydrolanosterol, dihydroxyeuphol and tirucallenol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into cucurbitane-type and lanostane-type skeletons, and provide a new strategy to identify key residues determining OSC specificity.

A New Strategy for the Site-Specific Modification of Proteinsin Vivo†

Biochemistry ◽  
2003 ◽  
Vol 42 (22) ◽  
pp. 6735-6746 ◽  
Author(s):  
Zhiwen Zhang ◽  
Brian A. C. Smith ◽  
Lei Wang ◽  
Ansgar Brock ◽  
Charles Cho ◽  
...  
Keyword(s):  
Site Specific ◽  
Specific Modification ◽  
New Strategy

scholarly journals Site-Specific Protonation Kinetics of Acidic Side Chains in Proteins Determined by pH-Dependent Carboxyl 13C NMR Relaxation

2015 ◽  
Vol 137 (8) ◽  
pp. 3093-3101 ◽  
Author(s):  
Johan Wallerstein ◽  
Ulrich Weininger ◽  
M. Ashhar I. Khan ◽  
Sara Linse ◽  
Mikael Akke
Keyword(s):  
13C Nmr ◽  
Nmr Relaxation ◽  
Side Chains ◽  
Site Specific ◽  
Ph Dependent ◽  
Kinetics Of

scholarly journals New Strategy for Reversible Modulation of Protein Activity through Site-Specific Conjugation of Small Molecule and Polymer

2014 ◽  
Vol 25 (7) ◽  
pp. 1252-1260 ◽  
Author(s):  
Lei Wang ◽  
Lin Yuan ◽  
Hongwei Wang ◽  
Xiaoli Liu ◽  
Xinming Li ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protein Activity ◽  
Site Specific ◽  
New Strategy

scholarly journals The HP0165-HP0166 Two-Component System (ArsRS) Regulates Acid-Induced Expression of HP1186 α-Carbonic Anhydrase in Helicobacter pylori by Activating the pH-Dependent Promoter

2007 ◽  
Vol 189 (6) ◽  
pp. 2426-2434 ◽  
Author(s):  
Yi Wen ◽  
Jing Feng ◽  
David R. Scott ◽  
Elizabeth A. Marcus ◽  
George Sachs
Keyword(s):  
Carbonic Anhydrase ◽  
Promoter Region ◽  
Low Ph ◽  
Start Codon ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Ph Dependent ◽  
H Pylori

ABSTRACT The periplasmic α-carbonic anhydrase of Helicobacter pylori is essential for buffering the periplasm at acidic pH. This enzyme is an integral component of the acid acclimation response that allows this neutralophile to colonize the stomach. Transcription of the HP1186 α-carbonic anhydrase gene is upregulated in response to low environmental pH. A binding site for the HP0166 response regulator (ArsR) has been identified in the promoter region of the HP1186 gene. To investigate the mechanism that regulates the expression of HP1186 in response to low pH and the role of the HP0165-HP0166 two-component system (ArsRS) in this acid-inducible regulation, Northern blot analysis was performed with RNAs isolated from two different wild-type H. pylori strains (26695 and 43504) and mutants with HP0165 histidine kinase (ArsS) deletions, after exposure to either neutral pH or low pH (pH 4.5). ArsS-dependent upregulation of HP1186 α-carbonic anhydrase in response to low pH was found in both strains. Western blot analysis of H. pylori membrane proteins confirmed the regulatory role of ArsS in HP1186 expression in response to low pH. Analysis of the HP1186 promoter region revealed two possible transcription start points (TSP1 and TSP2) located 43 and 11 bp 5′ of the ATG start codon, respectively, suggesting that there are two promoters transcribing the HP1186 gene. Quantitative primer extension analysis showed that the promoter from TSP1 (43 bp 5′ of the ATG start codon) is a pH-dependent promoter and is regulated by ArsRS in combating environmental acidity, whereas the promoter from TSP2 may be responsible for control of the basal transcription of HP1186 α-carbonic anhydrase.

scholarly journals Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration

2020 ◽  
Author(s):  
Swetha Mohan ◽  
Paul J. Sampognaro ◽  
Andrea R. Argouarch ◽  
Jason C. Maynard ◽  
Anand Patwardhan ◽  
...  
Keyword(s):  
Frontotemporal Lobar Degeneration ◽  
Cathepsin L ◽  
Dependent Manner ◽  
Loss Of Function ◽  
New Strategy ◽  
Lysosomal Proteases ◽  
Ph Dependent ◽  
Increased Activity

Abstract Background: Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases at a range of pH setpoints.Results: In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in variable, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn show increased PGRN processing to granulin F, correlating with increased activity of AEP, in a region-specific manner. Conclusions: This study demonstrates that multiple lysosomal proteases may work in concert to liberate granulins and implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage may represent a new strategy to modulate PGRN and granulin levels in disease.

scholarly journals Discovery of allosteric non-covalent KRAS inhibitors that bind with sub-micromolar affinity and disrupt effector binding

2018 ◽  
Author(s):  
Michael J. McCarthy ◽  
Cynthia V. Pagba ◽  
Priyanka Prakash ◽  
Ali Naji ◽  
Dharini van der Hoeven ◽  
...  
Keyword(s):  
Cell Growth ◽  
Drug Target ◽  
Biochemical Properties ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Cancer Cell Growth ◽  
Switch Regions ◽  
Covalent Inhibitors ◽  
Key Residues ◽  
Effector Binding

AbstractApproximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and a notorious oncogene. Somatic mutations that render KRAS constitutively active lead to uncontrolled cell growth, survival, proliferation, and eventually cancer. KRAS is thus a critical anticancer drug target. However, despite aggressive efforts in recent years, there is no drug on the market that directly targets KRAS. In the current work, we combined molecular simulation and high-throughput virtual screening with a battery of cell-based and biophysical assays to discover a novel, pyrazolopyrimidine-based allosteric KRAS inhibitor that exhibits promising biochemical properties. The compound selectively binds to active KRAS with sub-micromolar affinity, slightly modulates exchange factor activity, disrupts effector Raf binding, significantly reduces signal transduction through mutant KRAS and inhibits cancer cell growth. Moreover, by studying two of its analogues, we identified key chemical features of the compound that are critical for affinity, effect on effector binding and mode of action. We propose a set of specific interactions with key residues at the switch regions of KRAS as critical for abrogating effector binding and reducing the rate of nucleotide exchange. Together, these findings not only demonstrate the viability of direct KRAS inhibition and offer guidance for future optimization efforts, but also show that pyrazolopyrimidine-based compounds may represent a first-in-class lead toward a clinically relevant targeting of KRAS by allosteric non-covalent inhibitors.

scholarly journals Structural and molecular insight into the pH-induced low-permeability of the voltage-gated potassium channel Kv1.2 through dewetting of the water cavity

2019 ◽  
Author(s):  
Juhwan Lee ◽  
Mooseok Kang ◽  
Sangyeol Kim ◽  
Iksoo Chang
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Potassium Ion ◽  
Low Permeability ◽  
Voltage Gated ◽  
Kv Channels ◽  
Proton Concentration ◽  
Electrical Voltage ◽  
Gating Mechanism ◽  
Ph Dependent

AbstractUnderstanding the gating mechanism of ion channel proteins is key to understanding the regulation of cell signaling through these channels. Channel opening and closing are regulated by diverse environmental factors that include temperature, electrical voltage across the channel, and proton concentration. Low permeability in voltage-gated potassium ion channels (Kv) is intimately correlated with the prolonged action potential duration observed in many acidosis diseases. The Kv channels consist of voltage-sensing domains (S1–S4 helices) and central pore domains (S5–S6 helices) that include a selectivity filter and water-filled cavity. The voltage-sensing domain is responsible for the voltage-gating of Kv channels. While the low permeability of Kv channels to potassium ion is highly correlated with the cellular proton concentration, it is unclear how an intracellular acidic condition drives their closure, which may indicate an additional pH-dependent gating mechanism of the Kv family. Here, we show that two residues E327 and H418 in the proximity of the water cavity of Kv1.2 play crucial roles as a pH switch. In addition, we present a structural and molecular concept of the pH-dependent gating of Kv1.2 in atomic detail, showing that the protonation of E327 and H418 disrupts the electrostatic balance around the S6 helices, which leads to a straightening transition in the shape of their axes and causes dewetting of the water-filled cavity and closure of the channel. Our work offers a conceptual advancement to the regulation of the pH-dependent gating of various voltage-gated ion channels and their related biological functions.Author SummaryThe acid sensing ion channels are a biological machinery for maintaining the cell functional under the acidic or basic cellular environment. Understanding the pH-dependent gating mechanism of such channels provides the structural insight to design the molecular strategy in regulating the acidosis. Here, we studied the voltage-gated potassium ion channel Kv1.2 which senses not only the electrical voltage across the channels but also the cellular acidity. We uncovered that two key residues E327 and H418 in the pore domain of Kv1.2 channel play a role as pH-switch in that their protonation control the gating of the pore in Kv1.2 channel. It offered a molecular insight how the acidity reduces the ion permeability in voltage-gated potassium channels.

scholarly journals ML277 specifically enhances the fully activated open state of KCNQ1 by modulating VSD-pore coupling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Panpan Hou ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Yuan Gao ◽  
Jianmin Cui
Keyword(s):  
Long Qt Syndrome ◽  
Xenopus Oocytes ◽  
Heart Rhythm ◽  
Long Qt ◽  
Gating Mechanism ◽  
New Strategy ◽  
Pore Opening ◽  
Qt Syndrome ◽  
Voltage Sensing Domain ◽  
Iks Channel

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form IKs channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in Xenopus oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native IKs currents.

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