Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1’s substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1’s intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD’s role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.

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Crescerin uses a TOG domain array to regulate microtubules in the primary cilium

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0603 ◽

2015 ◽

Vol 26 (23) ◽

pp. 4248-4264 ◽

Cited By ~ 21

Author(s):

Alakananda Das ◽

Daniel J. Dickinson ◽

Cameron C. Wood ◽

Bob Goldstein ◽

Kevin C. Slep

Keyword(s):

Crystal Structure ◽

Binding Activity ◽

Structure And Function ◽

Sensory Disorders ◽

Tubulin Binding ◽

Sensory Cilia ◽

And Function ◽

Binding Determinants ◽

Extracellular Environment

Eukaryotic cilia are cell-surface projections critical for sensing the extracellular environment. Defects in cilia structure and function result in a broad range of developmental and sensory disorders. However, mechanisms that regulate the microtubule (MT)-based scaffold forming the cilia core are poorly understood. TOG domain array–containing proteins ch-TOG and CLASP are key regulators of cytoplasmic MTs. Whether TOG array proteins also regulate ciliary MTs is unknown. Here we identify the conserved Crescerin protein family as a cilia-specific, TOG array-containing MT regulator. We present the crystal structure of mammalian Crescerin1 TOG2, revealing a canonical TOG fold with conserved tubulin-binding determinants. Crescerin1's TOG domains possess inherent MT-binding activity and promote MT polymerization in vitro. Using Cas9-triggered homologous recombination in Caenorhabditis elegans, we demonstrate that the worm Crescerin family member CHE-12 requires TOG domain–dependent tubulin-binding activity for sensory cilia development. Thus, Crescerin expands the TOG domain array–based MT regulatory paradigm beyond ch-TOG and CLASP, representing a distinct regulator of cilia structure.

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Conserved Residues in the C-Terminal Domain Affect the Structure and Function of CYP38 in Arabidopsis

Frontiers in Plant Science ◽

10.3389/fpls.2021.630644 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lujing Shi ◽

Lele Du ◽

Jingru Wen ◽

Xiumei Zong ◽

Wene Zhao ◽

...

Keyword(s):

Thylakoid Membrane ◽

Structure And Function ◽

Wild Type ◽

Yeast Two Hybrid ◽

Conserved Residues ◽

Terminal Domain ◽

Target Binding ◽

And Function ◽

Two Hybrid

Arabidopsis cyclophilin38 (CYP38) is a thylakoid lumen protein critial for PSII assembly and maintenance, and its C-terminal region serves as the target binding domain. We hypothesized that four conserved residues (R290, F294, Q372, and F374) in the C-terminal domain are critical for the structure and function of CYP38. In yeast two-hybrid and protein pull-down assays, CYP38s with single-sited mutations (R290A, F294A, Q372A, or F374A) did not interact with the CP47 E-loop as the wild-type CYP38. In contrast, CYP38 with the R290A/F294A/Q372A/F374A quadruple mutation could bind the CP47 E-loop. Gene transformation analysis showed that the quadruple mutation prevented CYP38 to efficiently complement the mutant phenotype of cyp38. The C-terminal domain half protein with the quadruple mutation, like the wild-type one, could interact with the N-terminal domain or the CP47 E-loop in vitro. The cyp38 plants expressing CYP38 with the quadruple mutation showed a similar BN-PAGE profile as cyp38, but distinct from the wild type. The CYP38 protein with the quadruple mutation associated with the thylakoid membrane less efficiently than the wild-type CYP38. We concluded that these four conserved residues are indispensable as changes of all these residues together resulted in a subtle conformational change of CYP38 and reduced its intramolecular N-C interaction and the ability to associate with the thylakoid membrane, thus impairing its function in chloroplast.

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2072-P: Deletion of the Mitofusins 1 and 2 (Mfn1 and Mfn2) in the Pancreatic Beta Cell Disrupts Mitochondrial Structure and Function In Vitro and Strongly Impairs Glucose-Stimulated Insulin Secretion In Vivo

Diabetes ◽

10.2337/db20-2072-p ◽

2020 ◽

Vol 69 (Supplement 1) ◽

pp. 2072-P

Author(s):

ELENI GEORGIADOU ◽

PAULINE L. CHABOSSEAU ◽

ALEJANDRA TOMAS ◽

ISABELLE LECLERC ◽

GUY A. RUTTER

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Pancreatic Beta Cell ◽

Structure And Function ◽

Insulin Secretion In Vivo ◽

Mitochondrial Structure ◽

Glucose Stimulated Insulin Secretion ◽

And Function

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Structure prediction of SPAK C-terminal domain and analysis of its binding to RFXV/I motifs by homology modelling, docking, and molecular dynamics simulation studies

Current Computer - Aided Drug Design ◽

10.2174/1573409916666200712140941 ◽

2020 ◽

Vol 16 ◽

Author(s):

Mubarak A. Alamri ◽

Ahmed D. Alafnan ◽

Obaid Afzal ◽

Alhumaidi B. Alabbas ◽

Safar M. Alqahtani

Keyword(s):

Molecular Dynamic ◽

Dynamic Stability ◽

Md Simulation ◽

Homology Modelling ◽

Antihypertensive Agents ◽

Structure And Function ◽

Dynamics Simulation ◽

Dimensional Structure ◽

Terminal Domain ◽

And Function

Background: The STE20/SPS1-related proline/alanine-rich kinase (SPAK) is a component of WNKSPAK/OSR1 signaling pathway that plays an essential role in blood pressure regulation. The function of SPAK is mediated by its highly conserved C-terminal domain (CTD) that interacts with RFXV/I motifs of upstream activators, WNK kinases, and downstream substrate, cation-chloride cotransporters. Objective: To determine and validate the three-dimensional structure of the CTD of SPAK and to study and analyze its interaction with the RFXV/I motifs. Methods: A homology model of SPAK CTD was generated and validated through multiple approaches. The model was based on utilizing the OSR1 protein kinase as a template. This model was subjected to 100 ns molecular dynamic (MD) simulation to evaluate its dynamic stability. The final equilibrated model was used to dock the RFQV-peptide derived from WNK4 into the primary pocket that was determined based on the homology sequence between human SPAK and OSR1 CTDs. The mechanism of interaction, conformational rearrangement and dynamic stability of the binding of RFQV-peptide to SPAK CTD were characterized by molecular docking and molecular dynamic simulation. Results: The MD simulation suggested that the binding of RFQV induces a large conformational change due to the distribution of salt bridge within the loop regions. These results may help in understanding the relation between the structure and function of SPAK CTD and to support drug design of potential SPAK kinase inhibitors as antihypertensive agents. Conclusion: This study provides deep insight into SPAK CTD structure and function relationship.

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The enolase in the methionine salvage pathway: crystal structure and function

Acta Crystallographica Section A Foundations of Crystallography ◽

10.1107/s0108767307097395 ◽

2007 ◽

Vol 63 (a1) ◽

pp. s120-s121

Author(s):

A. Fedorov ◽

H. Imker ◽

E. Fedorov ◽

J. Gerlt ◽

S. Almo

Keyword(s):

Crystal Structure ◽

Structure And Function ◽

Salvage Pathway ◽

Methionine Salvage Pathway ◽

And Function ◽

Methionine Salvage

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Interaction of chromium(III) or chromium(VI) with catalase and its effect on the structure and function of catalase: An in vitro study

Food Chemistry ◽

10.1016/j.foodchem.2017.10.062 ◽

2018 ◽

Vol 244 ◽

pp. 378-385 ◽

Cited By ~ 34

Author(s):

Linfeng Chen ◽

Jing Zhang ◽

Yaxian Zhu ◽

Yong Zhang

Keyword(s):

In Vitro Study ◽

Structure And Function ◽

Vitro Study ◽

Chromium Vi ◽

And Function

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Multicomponent differentiation-regulated transcription factors in F9 embryonal carcinoma stem cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1686 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1686-1695 ◽

Cited By ~ 12

Author(s):

M K Shivji ◽

N B La Thangue

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Dna Binding ◽

Embryonal Carcinoma ◽

Regulatory Sequence ◽

Dependent Manner ◽

Sequence Specificity ◽

Dna Sequence Specificity ◽

Cell Extracts

Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p30DR, affinity purified from F9 EC cell extracts produce complexes 1b and 1c. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiation-regulated transcription factors contribute to the cellular E1a-like activity.

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Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon

Journal of General Virology ◽

10.1099/vir.0.81334-0 ◽

2005 ◽

Vol 86 (11) ◽

pp. 3075-3080 ◽

Cited By ~ 63

Author(s):

Paul Targett-Adams ◽

John McLauchlan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Osteosarcoma Cell ◽

Dependent Manner ◽

Entry Site ◽

Subgenomic Replicon ◽

Replication Assay ◽

Cell Extracts ◽

Genotype 1B

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

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