scholarly journals Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

2006 ◽  
Vol 398 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Jingzhi Li ◽  
Yunkun Wu ◽  
Xinguo Qian ◽  
Bingdong Sha
Keyword(s):  
Crystal Structure ◽  
Close Contact ◽  
Critical Role ◽  
Peptide Binding ◽  
Structural Basis ◽  
Cellular Processes ◽  
Terminal Form ◽  
Charged Residues ◽  
Domain I

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40–Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a β-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge–charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

scholarly journals Structural basis for UFM1 transfer from UBA5 to UFC1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Manoj Kumar ◽  
Prasanth Padala ◽  
Jamal Fahoum ◽  
Fouad Hassouna ◽  
Tomer Tsaban ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Structural Basis ◽  
Adenylation Domain ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Cellular Processes ◽  
Enzyme Cascade ◽  
Linear Sequence ◽  
C Terminus

AbstractUfmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.

scholarly journals The Structural Basis for Glycerol Permeation by human AQP7

2019 ◽  
Author(s):  
Li Zhang ◽  
Deqiang Yao ◽  
Fu Zhou ◽  
Qing Zhang ◽  
Ying Xia ◽  
...  
Keyword(s):  
Physiological Condition ◽  
Critical Role ◽  
Binding Pocket ◽  
Structural Basis ◽  
Glycerol Metabolism ◽  
Functional Assay ◽  
Substrate Binding Pocket ◽  
Glycerol Molecule ◽  
Cytoplasmic Side

AbstractHuman glycerol channel AQP7 conducts glycerol release from adipocyte and entry into the cells in pancreatic islets, muscles and kidney tubule, and thus regulate glycerol metabolism in those tissues. Compared with other human aquaglyceroporins, AQP7 shows a less conserved “NPA” motif in the center cavity, and a pair of aromatic residues at Ar/R selectivity filter. To understand the structural basis for the glycerol conductance, we crystallized the human AQP7 and determined the structure at 3.7 Å. A substrate binding pocket was found near to the Ar/R filter and the bound glycerol molecule stabilized by R229. In vivo functional assay on human AQP7 as well as AQP3 and AQP10 demonstrated strong glycerol transportation activities at physiological condition. The human AQP7 structure reveals a fully closed conformation with its permeation pathway strictly confined by Ar/R filter at the exoplasmic side and the gate at the cytoplasmic side, and the dislocation of the residues at narrowest parts of glycerol pathway in AQP7 play a critical role in controlling the glycerol flux.

scholarly journals FPD: A comprehensive phosphorylation database in fungi

2016 ◽  
Author(s):  
Youhuang Bai ◽  
Bin Chen ◽  
Yincong Zhou ◽  
Silin Ren ◽  
Qin Xu ◽  
...  
Keyword(s):  
Critical Role ◽  
Fungal Species ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
High Confidence ◽  
Cellular Processes ◽  
Phosphorylation Motif ◽  
Cycle Growth ◽  
Available Information

AbstractProtein phosphorylation, one of the most classic post-translational modification, plays a critical role in the diverse cellular processes including cell cycle, growth and signal transduction pathways. However, the available information of phosphorylation in fungi is limited. Here we provided a Fungi Phosphorylation Database (FPD) that comprises high-confidence in vivo phosphosites identified by MS-based proteomics in various fungal species. This comprehensive phosphorylation database contains 62,272 non-redundant phosphorylation sites in 11,222 proteins across eight organisms, including Aspergillus flavus, Aspergillus nidulans, Fusarium graminearum, Magnaporthe oryzae, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Cryptococcus neoformans. A fungi-specific phosphothreonine motif and several conserved phosphorylation motif were discovered by comparatively analyzing the pattern of phosphorylation sites in fungi, plants and animals.Database URL: http://bis.zju.edu.cn/FPD/index.php

scholarly journals Structural basis of altered potency and efficacy displayed by a major in vivo metabolite of the anti-diabetic PPARγ drug pioglitazone

2018 ◽  
Author(s):  
Sarah A. Mosure ◽  
Jinsai Shang ◽  
Richard Brust ◽  
Jie Zheng ◽  
Patrick R. Griffin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Activation Function ◽  
Structural Basis ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Exchange Mass Spectrometry ◽  
Pparγ Agonist ◽  
A Cell ◽  
And Function

ABSTRACTThe thiazolidinedione (TZD) pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes mellitus that binds and activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). Although TZDs have potent antidiabetic effects, they also display harmful side effects that have necessitated a better understanding of their mechanisms of action. In particular, little is known about the effect of in vivo TZD metabolites on the structure and function of PPARγ. Here, we present a structure-function comparison of Pio and a major in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays compared to Pio. To determine the structural basis of these findings, we solved an X-ray crystal structure of PioOH bound to PPARγ ligand-binding domain (LBD) and compared it to a published Pio-bound crystal structure. PioOH exhibited an altered hydrogen bonding network that could underlie its reduced affinity and potency compared to Pio. Solution-state structural analysis using NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PioOH stabilizes the PPARγ activation function-2 (AF-2) coactivator binding surface better than Pio. In support of AF-2 stabilization, PioOH displayed stabilized coactivator binding in biochemical assays and better transcriptional efficacy (maximal transactivation response) in a cell-based assay that reports on the activity of the PPARγ LBD. These results, which indicate that Pio hydroxylation affects both its potency and efficacy as a PPARγ agonist, contribute to our understanding of PPARγ-binding drug metabolite interactions and may assist in future PPARγ drug design efforts.

scholarly journals Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

2010 ◽  
Vol 84 (16) ◽  
pp. 8072-8084 ◽  
Author(s):  
Sarah E. Hobdey ◽  
Brian J. Kempf ◽  
Benjamin P. Steil ◽  
David J. Barton ◽  
Olve B. Peersen
Keyword(s):  
Amino Acid ◽  
Rna Binding ◽  
Critical Role ◽  
Polymerase Activity ◽  
Structural Basis ◽  
Wild Type ◽  
Elongation Complex ◽  
Virus Growth ◽  
The Stability

ABSTRACT The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.

scholarly journals Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii

2017 ◽  
Vol 73 (12) ◽  
pp. 706-712
Author(s):  
K. J. Pampa ◽  
S. Madan Kumar ◽  
M. K. Hema ◽  
Karthik Kumara ◽  
S. Naveen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Trafficking ◽  
X Ray Diffraction ◽  
Pyrococcus Horikoshii ◽  
Cellular Processes ◽  
Catalytic Function ◽  
Monomeric Structure ◽  
Domain I ◽  
Substrate Binding Domain ◽  
Metabolism Gene

Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-L-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subunits, presumably near to the active site(s) of the enzyme. The observed dimeric state might be important for the catalytic function of the enzyme.

Ca2+-Operated Transcriptional Networks: Molecular Mechanisms and In Vivo Models

2008 ◽  
Vol 88 (2) ◽  
pp. 421-449 ◽  
Author(s):  
Britt Mellström ◽  
Magali Savignac ◽  
Rosa Gomez-Villafuertes ◽  
Jose R. Naranjo
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Transcriptional Networks ◽  
Calcium Signal ◽  
In Vivo Models ◽  
Cellular Processes ◽  
Calcium Sensors ◽  
Genes Encoding ◽  
Living Organisms

Calcium is the most universal signal used by living organisms to convey information to many different cellular processes. In this review we present well-known and recently identified proteins that sense and decode the calcium signal and are key elements in the nucleus to regulate the activity of various transcriptional networks. When possible, the review also presents in vivo models in which the genes encoding these calcium sensors-transducers have been modified, to emphasize the critical role of these Ca2+-operated mechanisms in many physiological functions.

scholarly journals Structural and histone binding studies of the chromo barrel domain of TIP60

2018 ◽  
Author(s):  
Yuzhe Zhang ◽  
Ming Lei ◽  
Xiao Liang ◽  
Peter Loppnau ◽  
Yanjun Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Peptide Binding ◽  
Side Chain ◽  
Titration Calorimetry ◽  
Binding Studies ◽  
Histone Tails ◽  
Histone Binding ◽  
Cellular Processes ◽  
Peptide Binding Site ◽  
Acetyltransferase Domain

AbstractTIP60 consists of an N-terminal chromo barrel domain (TIP60-CB) and a C-terminal acetyltransferase domain and acetylates histone and non-histone proteins within diverse cellular processes. Whereas the TIP60-CB is thought to recognize histone tails, molecular details of this interaction remain unclear. Here we attempted a quantitative analysis of the interaction between the TIP60-CB and histone peptides, but did not observe any binding through either fluorescence polarization or isothermal titration calorimetry. We solved a crystal structure of the TIP60-CB alone. Analysis of the crystal structure demonstrates a putative peptide binding site that may be occluded by the basic side chain of a residue in a unique β hairpin between the two N-terminal strands of the β barrel.

scholarly journals In vivo experiments do not support the charge zipper model for Tat translocase assembly

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Felicity Alcock ◽  
Merel PM Damen ◽  
Jesper Levring ◽  
Ben C Berks
Keyword(s):  
Structural Model ◽  
Protein Translocation ◽  
Critical Role ◽  
Oligomeric State ◽  
In Vivo Experiments ◽  
Charged Residues ◽  
Twin Arginine Translocase ◽  
Vitro Analysis

The twin-arginine translocase (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat translocation site is formed by substrate-triggered oligomerization of the protein TatA. Walther and co-workers have proposed a structural model for the TatA oligomer in which TatA monomers self-assemble using electrostatic ‘charge zippers’ (Cell (2013) 132: 15945). This model was supported by in vitro analysis of the oligomeric state of TatA variants containing charge-inverting substitutions. Here we have used live cell assays of TatA assembly and function in Escherichia coli to re-assess the roles of the charged residues of TatA. Our results do not support the charge zipper model. Instead, we observe that substitutions of charged residues located in the TatA amphipathic helix lock TatA in an assembled state, suggesting that these charged residues play a critical role in the protein translocation step that follows TatA assembly.

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