The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

doi:10.1016/0092-8674(84)90278-2

Cell

Volume 38, Issue 3, October 1984, Pages 835-840

https://doi.org/10.1016/0092-8674(84)90278-2 Get rights and content

Abstract

In a previous study, a mutant of tyrosyl-tRNA synthetase in which a threonine residue (Thr51) was converted to proline dramatically improved the affinity of the enzyme for its ATP substrate. How does Pro51 improve the enzyme's affinity for ATP? A priori, Pro51 might interact directly with the ATP, or it might distort the polypeptide backbone and thereby force new or improved contacts elsewhere from the enzyme to ATP. By making mutants of the Pro51 enzyme at two residues that make hydrogen bonds to the ATP substrate, we show that Pro51 greatly improves the strength of one of these contacts. Thus the propagation of a structural change in an enzyme induced by mutation may be detected by the introduction of further mutations.

References (28)

D.G. Barker et al.
Conserved cysteine and histidine residues in the structures of the tyrosyl and methionyl-tRNA synthetases
FEBS Lett.
(1982)
T.N. Bhat et al.
Tyrosyl-tRNA synthetase forms a mononucleotide-binding fold
J. Mol. Biol.
(1982)
D.M. Blow et al.
Structural homology in the amino-terminal domains of two aminoacyl-tRNA synthetases
J. Mol. Biol.
(1983)
D.T. Denhardt
A membrane-filter technique for the detection of complementary DNA
Biochem. Biophys. Res. Commun.
(1966)
C.A. Hutchison et al.
Mutagenesis at a specific position in a DNA sequence
J. Biol. Chem.
(1978)
J. Rubin et al.
Amino acid activation in crystalline tyrosyl-tRNA synthetase from Bacillus stearothermophilus
J. Mol. Biol.
(1981)
C. Zelwer et al.
Crystal structure of Escherichia coli methionyl-tRNA synthetase at 2.5 A resolution
J. Mol. Biol.
(1982)
G. Biesecker et al.
Sequence and structure of D-glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus
Nature
(1977)
M.D. Biggin et al.
Buffer gradient gels and ³⁵S label as an aid to rapid DNA sequence determination
R. Calendar et al.
Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis
Biochemistry
(1966)

S.N. Cohen et al.

Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA

G. Dalbadie-McFarland et al.

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function

A.R. Fersht et al.

Demonstration of two reaction pathways for the aminoacylation of tRNA

A.R. Fersht et al.

Ligand binding and enzymic catalysis coupled through subunits: tyrosyl tRNA synthetase

Biochemistry

(1975)

Cited by (538)

Emerging maps of allosteric regulation in cellular networks
2023, Current Opinion in Structural Biology
Allosteric regulation is classically defined as action at a distance, where a perturbation outside of a protein active site affects function. While this definition has motivated many studies of allosteric mechanisms at the level of protein structure, translating these insights to the allosteric regulation of entire cellular processes – and their crosstalk – has received less attention, despite the broad importance of allostery for cellular regulation foreseen by Jacob and Monod. Here, we revisit an evolutionary model for the widespread emergence of allosteric regulation in colocalized proteins, describe supporting evidence, and discuss emerging advances in mapping allostery in cellular networks that link precise and often allosteric perturbations at the molecular level to functional changes at the pathway and systems levels.
Potent Ca<inf>V</inf>3.2 channel inhibitors exert analgesic effects in acute and chronic pain models
2022, Biomedicine and Pharmacotherapy
Pain is the most common presenting physical symptom and a primary reason for seeking medical care, which chronically affects people's mental health and social life. Ca_V3.2 channel plays an essential role in the peripheral processing maintenance of pain states. This study was designed to identify novel drug candidates targeting the Ca_V3.2 channel. Whole-cell patch-clamp, cellular thermal shift assay, FlexStation, in vivo and in vitro Ca_V3.2 knock-down, site-directed mutagenesis, and double-mutant cycle analysis were employed to explore the pain-related receptors and ligand-receptor direct interaction. We found that toddaculin efficiently inhibits the Ca_V3.2 channel and significantly reduced the excitability of dorsal root ganglion neurons and pain behaviors. The Carbonyl group of coumarins directly interacts with the pore domain of Ca_V3.2 via van der Waals (VDW) force. Docking with binding pockets further led us to identify glycycoumarin, which exhibited more potent inhibition on the Ca_V3.2 channel and better analgesic activity than the parent compound. Toddaculin and its analog showed beneficial therapeutic effects in pain models. Toddaculin binding pocket on Ca_V3.2 might be a promising docking site for the design of drugs.
Noncovalent interactions: A brief account of a long history
2022, Journal of Physical Organic Chemistry
The history of noncovalent interactions is outlined, starting with early interpretations of the symmetry of crystals, then with the fast development of coordination chemistry, and of fundamental analyses in the last century. An attempt is made to provide for the practicing scientists who use noncovalent interactions a historical background of the field, paying credit to often forgotten predecessors. A separate section shows that only in the 20th century instrumentation provided a firm basis for the exploration of noncovalent interactions. Selected examples illustrate the early and rather accurate calculations of some noncovalent energies. With the advent of supramolecular chemistry, noncovalent interactions became a mainstream field of science; this allowed further insight into the nature and strength of these seemingly weak forces. Intermolecular interactions in biological systems have also received early attention. As far as possible, exact references are given for the original publications.
Unraveling the mechanism of enantio-controlling switches of an alcohol dehydrogenase toward sterically small ketone†
2022, Catalysis Science and Technology
Efficient synthesis of chiral compounds under mild conditions is highly desirable in the chemical and pharmaceutical communities, but it often faces difficulties. Although various enzymes have been harnessed as biocatalysts in asymmetric reduction, identifying key residues that function as stereoselectivity-switches at which amino acid exchange events can be designed by rational design or directed evolution, constitutes a major challenge. Here, we address this question by using the Zn-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme in the asymmetric reduction of a difficult-to-reduce ketone (tetrahydrofuran-3-one). Following the theoretical analysis of epistatic mutational effects and investigating the results of deconvoluting multi-mutational variants, two crucial amino acid positions 294 and 295 were identified as the “switches” that specifically control the inversion of product chirality. In order to shed light on the concealed mechanistic intricacies, molecular dynamics (MD) simulations along with quantum mechanical (QM) calculations were performed on the variants, which unveiled the distinct H-bonds formed with the furan ring O-atom in pro-(S) and pro-(R) orientations of positions 294 and 295 leading to stereospecific formation of the product.
The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach
2021, Journal of Molecular Biology
The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (β/⍺)₈-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.
Endocannabinoid activation of the TRPV1 ion channel is distinct from activation by capsaicin
2021, Journal of Biological Chemistry
Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the “tail-up, head-down” configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.

View all citing articles on Scopus

View full text

Cell

ArticleThe use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

Abstract

FEBS Lett.

J. Mol. Biol.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Sequence and structure of D-glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

Nature

Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination

Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis

Biochemistry

Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function

Demonstration of two reaction pathways for the aminoacylation of tRNA

Ligand binding and enzymic catalysis coupled through subunits: tyrosyl tRNA synthetase

Biochemistry

Article
The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

Buffer gradient gels and ³⁵S label as an aid to rapid DNA sequence determination