A Disordered Loop Mediates Heterogeneous Unfolding of an Ordered Protein by Altering the Native Ensemble

2020 ◽  
Vol 11 (16) ◽  
pp. 6749-6756
Author(s):  
Kabita Bhattacharjee ◽  
Soundhararajan Gopi ◽  
Athi N. Naganathan
Keyword(s):  
Disordered Loop

scholarly journals Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

2019 ◽  
Author(s):  
Sindhu Carmen Sivakumaren ◽  
Hyeseok Shim ◽  
Tinghu Zhang ◽  
Fleur M. Ferguson ◽  
Mark R. Lundquist ◽  
...  
Keyword(s):  
Cancer Metabolism ◽  
Target Genes ◽  
Therapeutic Potential ◽  
Dependent Manner ◽  
Cancer Cell Proliferation ◽  
Lipid Kinase ◽  
Covalent Inhibitors ◽  
Lipid Kinases ◽  
Disordered Loop

SummaryThe PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here we present the discovery and characterization of a novel pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K’s reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders.

scholarly journals Phase separation of Polo-like kinase 4 by autoactivation and clustering drives centriole biogenesis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jung-Eun Park ◽  
Liang Zhang ◽  
Jeong Kyu Bang ◽  
Thorkell Andresson ◽  
Frank DiMaio ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Phase Separation ◽  
Symmetry Breaking ◽  
Genomic Instability ◽  
Chromosome Segregation ◽  
Genomic Stability ◽  
Normal Chromosome ◽  
Tight Control ◽  
Centriole Duplication ◽  
Disordered Loop

Abstract Tight control of centriole duplication is critical for normal chromosome segregation and the maintenance of genomic stability. Polo-like kinase 4 (Plk4) is a key regulator of centriole biogenesis. How Plk4 dynamically promotes its symmetry-breaking relocalization and achieves its procentriole-assembly state remains unknown. Here we show that Plk4 is a unique kinase that utilizes its autophosphorylated noncatalytic cryptic polo-box (CPB) to phase separate and generate a nanoscale spherical condensate. Analyses of the crystal structure of a phospho-mimicking, condensation-proficient CPB mutant reveal that a disordered loop at the CPB PB2-tip region is critically required for Plk4 to generate condensates and induce procentriole assembly. CPB phosphorylation also promotes Plk4’s dissociation from the Cep152 tether while binding to downstream STIL, thus allowing Plk4 condensate to serve as an assembling body for centriole biogenesis. This study uncovers the mechanism underlying Plk4 activation and may offer strategies for anti-Plk4 intervention against genomic instability and cancer.

Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype

Transfusion ◽  
2005 ◽  
Vol 45 (7) ◽  
pp. 1178-1182 ◽  
Author(s):  
Mark H. Yazer ◽  
Greg A. Denomme ◽  
Natisha L. Rose ◽  
Monica M. Palcic
Keyword(s):  
Amino Acid ◽  
Blood Group ◽  
Amino Acid Substitution ◽  
Group B ◽  
Disordered Loop

scholarly journals Zn2+ and Cu2+ Binding to the Extramembrane Loop of Zrt2, a Zinc Transporter of Candida albicans

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 121
Author(s):  
Denise Bellotti ◽  
Adriana Miller ◽  
Magdalena Rowińska-Żyrek ◽  
Maurizio Remelli
Keyword(s):  
Candida Albicans ◽  
Binding Site ◽  
Metal Binding ◽  
Zinc Transporter ◽  
Serine Residue ◽  
Metal Binding Site ◽  
Binding Motifs ◽  
Zip Family ◽  
Acidic Conditions ◽  
Disordered Loop

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126–215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144–153), HxxHxxEHxD (181–193) and the Glu- and Asp- rich sequence DDEEEDxE (161–168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.

scholarly journals Structural basis of SUFU–GLI interaction in human Hedgehog signalling regulation

2013 ◽  
Vol 69 (12) ◽  
pp. 2563-2579 ◽  
Author(s):  
Amy L. Cherry ◽  
Csaba Finta ◽  
Mikael Karlström ◽  
Qianren Jin ◽  
Thomas Schwend ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Negative Regulator ◽  
Structural Basis ◽  
Hedgehog Signalling ◽  
Intrinsically Disordered ◽  
X Ray Scattering ◽  
Cell Proliferation And Differentiation ◽  
Pathway Activation ◽  
Proliferation And Differentiation ◽  
Disordered Loop

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU–GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU–GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

scholarly journals Conformational Switching in Bcl-xL: Enabling Non-Canonic Inhibition of Apoptosis Involves Multiple Intermediates and Lipid Interactions

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 539
Author(s):  
Victor Vasquez-Montes ◽  
Alexander Kyrychenko ◽  
Mauricio Vargas-Uribe ◽  
Mykola V. Rodnin ◽  
Alexey S. Ladokhin
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Active Form ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mitochondrial Outer Membrane ◽  
Spectroscopic Techniques ◽  
Apoptotic Protein ◽  
Disordered Loop

The inhibition of mitochondrial permeabilization by the anti-apoptotic protein Bcl-xL is crucial for cell survival and homeostasis. Its inhibitory role requires the partitioning of Bcl-xL to the mitochondrial outer membrane from an inactive state in the cytosol, leading to its extensive refolding. The molecular mechanisms behind these events and the resulting conformations in the bilayer are unclear, and different models have been proposed to explain them. In the most recently proposed non-canonical model, the active form of Bcl-xL employs its N-terminal BH4 helix to bind and block its pro-apoptotic target. Here, we used a combination of various spectroscopic techniques to study the release of the BH4 helix (α1) during the membrane insertion of Bcl-xL. This refolding was characterized by a gradual increase in helicity due to the lipid-dependent partitioning-coupled folding and formation of new helix αX (presumably in the originally disordered loop between helices α1 and α2). Notably, a comparison of various fluorescence and circular dichroism measurements suggested the presence of multiple Bcl-xL conformations in the bilayer. This conclusion was explicitly confirmed by single-molecule measurements of Förster Resonance Energy Transfer from Alexa-Fluor-488-labeled Bcl-xL D189C to a mCherry fluorescent protein attached at the N-terminus. These measurements clearly indicated that the refolding of Bcl-xL in the bilayer is not a two-state transition and involves multiple membranous intermediates of variable compactness.

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