scholarly journals Crystal structure of unliganded TRAP: implications for dynamic allostery

2011 ◽  
Vol 434 (3) ◽  
pp. 427-434 ◽  
Author(s):  
Ali D. Malay ◽  
Masahiro Watanabe ◽  
Jonathan G. Heddle ◽  
Jeremy R. H. Tame
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Structural Changes ◽  
Bacillus Stearothermophilus ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Protein A ◽  
Feedback System ◽  
Vibrational Modes ◽  
Specific Mrna

Allostery is vital to the function of many proteins. In some cases, rather than a direct steric effect, mutual modulation of ligand binding at spatially separated sites may be achieved through a change in protein dynamics. Thus changes in vibrational modes of the protein, rather than conformational changes, allow different ligand sites to communicate. Evidence for such an effect has been found in TRAP (trp RNA-binding attenuation protein), a regulatory protein found in species of Bacillus. TRAP is part of a feedback system to modulate expression of the trp operon, which carries genes involved in tryptophan synthesis. Negative feedback is thought to depend on binding of tryptophan-bound, but not unbound, TRAP to a specific mRNA leader sequence. We find that, contrary to expectations, at low temperatures TRAP is able to bind RNA in the absence of tryptophan, and that this effect is particularly strong in the case of Bacillus stearothermophilus TRAP. We have solved the crystal structure of this protein with no tryptophan bound, and find that much of the structure shows little deviation from the tryptophan-bound form. These data support the idea that tryptophan may exert its effect on RNA binding by TRAP through dynamic and not structural changes, and that tryptophan binding may be mimicked by low temperature.

scholarly journals MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase

2019 ◽  
Vol 5 (10) ◽  
pp. eaax0059
Author(s):  
Hanseong Kim ◽  
Sojin An ◽  
Yeo Reum Park ◽  
Hara Jang ◽  
Heeseon Yoo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Methane Monooxygenase ◽  
Regulatory Protein ◽  
Access Route ◽  
Inhibitory Mechanism ◽  
Ambient Conditions ◽  
Soluble Methane Monooxygenase ◽  
Auxiliary Protein ◽  
Binding Component

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions. The maximum catalytic activity of hydroxylase (MMOH) is achieved through the interplay of its regulatory protein (MMOB) and reductase. An additional auxiliary protein, MMOD, functions as an inhibitor of MMOH; however, its inhibitory mechanism remains unknown. Here, we report the crystal structure of the MMOH-MMOD complex from Methylosinus sporium strain 5 (2.6 Å). Its structure illustrates that MMOD associates with the canyon region of MMOH where MMOB binds. Although MMOD and MMOB recognize the same binding site, each binding component triggers different conformational changes toward MMOH, which then respectively lead to the inhibition and activation of MMOH. Particularly, MMOD binding perturbs the di-iron geometry by inducing two major MMOH conformational changes, i.e., MMOH β subunit disorganization and subsequent His147 dissociation with Fe1 coordination. Furthermore, 1,6-hexanediol, a mimic of the products of sMMO, reveals the substrate access route.

scholarly journals The role of ATP in the function of human ORC4 protein

2010 ◽  
Vol 75 (3) ◽  
pp. 317-322
Author(s):  
Aleksandra Divac ◽  
Branko Tomic ◽  
Jelena Kusic
Keyword(s):  
Conformational Changes ◽  
Origin Recognition Complex ◽  
Structural Changes ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Structural Role ◽  
A Subunit ◽  
Dna Substrates ◽  
Origin Recognition

Human ORC4 protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of ATPases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.

Regulatory Features of the trp Operon and the Crystal Structure of the trp RNA-binding Attenuation Protein from Bacillus stearothermophilus

1999 ◽  
Vol 289 (4) ◽  
pp. 1003-1016 ◽  
Author(s):  
Xiao-ping Chen ◽  
Alfred A. Antson ◽  
Min Yang ◽  
Pan Li ◽  
Chris Baumann ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bacillus Stearothermophilus ◽  
Rna Binding ◽  
Trp Operon

scholarly journals diffBUM-HMM: a robust statistical modeling approach for detecting RNA flexibility changes in high-throughput structure probing data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Paolo Marangio ◽  
Ka Ying Toby Law ◽  
Guido Sanguinetti ◽  
Sander Granneman
Keyword(s):  
High Throughput ◽  
Conformational Changes ◽  
Rna Structure ◽  
Structural Changes ◽  
Rna Binding ◽  
Structural Information ◽  
Sequence Coverage ◽  
Structure Probing ◽  
Generation Sequencing ◽  
Higher Sensitivity

AbstractAdvancing RNA structural probing techniques with next-generation sequencing has generated demands for complementary computational tools to robustly extract RNA structural information amidst sampling noise and variability. We present diffBUM-HMM, a noise-aware model that enables accurate detection of RNA flexibility and conformational changes from high-throughput RNA structure-probing data. diffBUM-HMM is widely compatible, accounting for sampling variation and sequence coverage biases, and displays higher sensitivity than existing methods while robust against false positives. Our analyses of datasets generated with a variety of RNA probing chemistries demonstrate the value of diffBUM-HMM for quantitatively detecting RNA structural changes and RNA-binding protein binding sites.

Inhibition of RNA Helicase Brr2 by the C-Terminal Tail of the Spliceosomal Protein Prp8

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 80-84 ◽  
Author(s):  
Sina Mozaffari-Jovin ◽  
Traudy Wandersleben ◽  
Karine F. Santos ◽  
Cindy L. Will ◽  
Reinhard Lührmann ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Retinitis Pigmentosa ◽  
Adenosine Triphosphatase ◽  
Rna Binding ◽  
Protein A ◽  
Rna Helicase ◽  
Core Component ◽  
Spliceosomal Protein ◽  
Spliceosome Activation ◽  
Biochemical Analyses

The Ski2-like RNA helicase Brr2 is a core component of the spliceosome that must be tightly regulated to ensure correct timing of spliceosome activation. Little is known about mechanisms of regulation of Ski2-like helicases by protein cofactors. Here we show by crystal structure and biochemical analyses that the Prp8 protein, a major regulator of the spliceosome, can insert its C-terminal tail into Brr2’s RNA-binding tunnel, thereby intermittently blocking Brr2’s RNA-binding, adenosine triphosphatase, and U4/U6 unwinding activities. Inefficient Brr2 repression is the only recognizable phenotype associated with certain retinitis pigmentosa–linked Prp8 mutations that map to its C-terminal tail. Our data show how a Ski2-like RNA helicase can be reversibly inhibited by a protein cofactor that directly competes with RNA substrate binding.

scholarly journals Functional divergence caused by mutations in an energetic hotspot in ERK2

2019 ◽  
Vol 116 (31) ◽  
pp. 15514-15523 ◽  
Author(s):  
Clinton A. Taylor ◽  
Kevin W. Cormier ◽  
Shannon E. Keenan ◽  
Svetlana Earnest ◽  
Steve Stippec ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Thermal Stability ◽  
Conformational Changes ◽  
Structural Changes ◽  
Functional Divergence ◽  
Tumor Resistance ◽  
Dual Specificity ◽  
Extracellular Signal ◽  
Dual Specificity Phosphatases ◽  
Increased Activity

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

scholarly journals Structural and Immunogenic Effects of Multiple Hapten Loading on Carrier Protein

2010 ◽  
Vol 93 (1) ◽  
pp. 59-65 ◽  
Author(s):  
K Vikas Singh ◽  
Pooja Khanna ◽  
C Raman Suri ◽  
Mohan L Garg
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Protein A ◽  
Blue Shift ◽  
Molar Ratio ◽  
Carrier Protein ◽  
Sequential Approach ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Immunogenic Properties

Abstract Haptens are low-molecular-weight compounds that are usually nonimmunogenic in nature. These compounds are, in general, conjugated with carrier proteins to elicit an immune response for antibody production. In this work, we report the effect of multiple hapten loading on carrier protein after conjugation by monitoring the structural and immunogenic properties of the protein. Biochemical conjugation of carboxylated hapten (atrazine derivative) to bovine serum albumin via ε-amino groups of lysine residues was monitored by the intrinsic fluorescence intensity of tryptophan residues of protein. A significant blue shift of emission maxima confirmed the conformational changes with increasing molar ratio of hapten:protein. Circular dichroism spectroscopy suggested a decreasing trend for α-helical and increased formation of β-sheet structures in hapten-loaded protein. A further insight was sought by using molecular modeling methods for understanding of structural changes in the native protein post-hapten conjugation. A sequential approach for hapten loading on the carrier confirmed that initial binding could affect the possible binding sites for subsequent incorporation of hapten molecules. These changes play a major role in the immunogenic response of hapten-carrier conjugate. The approach taken to develop this model is promising, and can be generalized for studies with other protein-hapten combinations.

Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus

1999 ◽  
Vol 291 (3) ◽  
pp. 727 ◽  
Author(s):  
Xiao-ping Chen ◽  
Alfred A. Antson ◽  
Min Yang ◽  
Pan Li ◽  
Chris Baumann ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Bacillus Stearothermophilus ◽  
Rna Binding ◽  
Trp Operon

scholarly journals Crystal Structure of Human AUH Protein, a Single-Stranded RNA Binding Homolog of Enoyl-CoA Hydratase

Structure ◽  
2001 ◽  
Vol 9 (12) ◽  
pp. 1253-1263 ◽  
Author(s):  
Kazuki Kurimoto ◽  
Shuya Fukai ◽  
Osamu Nureki ◽  
Yutaka Muto ◽  
Shigeyuki Yokoyama
Keyword(s):  
Crystal Structure ◽  
Rna Binding ◽  
Protein A ◽  
Enoyl Coa Hydratase

scholarly journals MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase

2018 ◽  
Author(s):  
Hanseong Kim ◽  
Sojin An ◽  
Yeo Reum Park ◽  
Hara Jang ◽  
Sang Ho Park ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Activity ◽  
Structural Changes ◽  
Methane Monooxygenase ◽  
Regulatory Protein ◽  
Inhibitory Mechanism ◽  
Ambient Conditions ◽  
Soluble Methane Monooxygenase ◽  
Access Channel ◽  
Hydrocarbon Substrates

SummarySoluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions1-3. The maximum catalytic activity of hydroxylase (MMOH) is achieved via interplay of its regulatory protein (MMOB) and reductase4-6. An additional auxiliary protein, MMOD, is believed to function as an inhibitor of the catalytic activity of MMOH; however, the mechanism of its action remains unknown7,8. Herein, we report the crystal structure of MMOH–MMOD complex fromMethylosinus sporiumstrain 5 (2.6 Å), which illustrates that two molecules of MMOD associate symmetrically with the canyon region of MMOH in a manner similar to MMOB, indicating that MMOD competes with MMOB for MMOH recognition. Further, MMOD binding disrupts the geometry of the di-iron centre and opens the substrate access channel. Notably, the electron density of 1,6-hexanediol at the substrate access channel mimics products of sMMO in hydrocarbon oxidation. The crystal structure of MMOH–MMOD unravels the inhibitory mechanism by which MMOD suppresses the MMOH catalytic activity, and reveals how hydrocarbon substrates/products access to the di-iron centre.

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