Uncovering the Dominant Motion Modes of Allosteric Regulation Improves Allosteric Site Prediction

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named “(p)ppGpp”] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) fromBacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp—but not ppGpp—positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

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Unraveling allosteric landscapes of allosterome with ASD

Nucleic Acids Research ◽

10.1093/nar/gkz958 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xinyi Liu ◽

Shaoyong Lu ◽

Kun Song ◽

Qiancheng Shen ◽

Duan Ni ◽

...

Keyword(s):

Drug Discovery ◽

Protein Function ◽

Target Identification ◽

Allosteric Regulation ◽

Biological Research ◽

Missense Mutations ◽

Allosteric Site ◽

Comprehensive Information ◽

Allosteric Sites ◽

Human Proteins

Abstract Allosteric regulation is one of the most direct and efficient ways to fine-tune protein function; it is induced by the binding of a ligand at an allosteric site that is topographically distinct from an orthosteric site. The Allosteric Database (ASD, available online at http://mdl.shsmu.edu.cn/ASD) was developed ten years ago to provide comprehensive information related to allosteric regulation. In recent years, allosteric regulation has received great attention in biological research, bioengineering, and drug discovery, leading to the emergence of entire allosteric landscapes as allosteromes. To facilitate research from the perspective of the allosterome, in ASD 2019, novel features were curated as follows: (i) >10 000 potential allosteric sites of human proteins were deposited for allosteric drug discovery; (ii) 7 human allosterome maps, including protease and ion channel maps, were built to reveal allosteric evolution within families; (iii) 1312 somatic missense mutations at allosteric sites were collected from patient samples from 33 cancer types and (iv) 1493 pharmacophores extracted from allosteric sites were provided for modulator screening. Over the past ten years, the ASD has become a central resource for studying allosteric regulation and will play more important roles in both target identification and allosteric drug discovery in the future.

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Structural Insights into the Allosteric site of Arabidopsis NADP-Malic Enzyme 2: Role of the Second Sphere Residues in the Regulatory Signal Transmission

10.21203/rs.3.rs-256060/v1 ◽

2021 ◽

Author(s):

Mariel Claudia Gerrard Wheeler ◽

Cintia Lucía Arias ◽

Juliana Juliana da Fonseca Rezende e Mello ◽

Nuria Cirauqui Diaz ◽

Carlos Rangel Rodrigues ◽

...

Keyword(s):

Normal Mode ◽

Conformational Changes ◽

Active Site ◽

Normal Mode Analysis ◽

Signal Transmission ◽

Allosteric Regulation ◽

3D Structure ◽

Mode Analysis ◽

Allosteric Site ◽

Structural Determinants

Abstract Structure-function studies contribute to deciphering how small modifications in the primary structure could introduce desirable characteristics into enzymes without affecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from different species facilitates comparisons between sequence and structure. Specifically, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and effector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also fit into fumarate’s pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identified. The substitution of one of those residues, L62, by a less flexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate affinities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.

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Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

10.1101/346288 ◽

2018 ◽

Author(s):

Fernando Salgado-Polo ◽

Alex Fish ◽

Minos-Timotheos Matsoukas ◽

Tatjana Heidebrecht ◽

Willem-Jan Keune ◽

...

Keyword(s):

Lysophosphatidic Acid ◽

Allosteric Regulation ◽

Catalytic Efficiency ◽

Feedback Mechanism ◽

Allosteric Site ◽

Functional Studies ◽

Physiological Processes ◽

Positive Feedback Mechanism ◽

Experimental Kinetics ◽

Kinetics Of

AbstractAutotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the “pocket” and the “tunnel”. Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

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Probe Confined Dynamic Mapping for G Protein-Coupled Receptor Allosteric Site Prediction

ACS Central Science ◽

10.1021/acscentsci.1c00802 ◽

2021 ◽

Author(s):

Antonella Ciancetta ◽

Amandeep Kaur Gill ◽

Tianyi Ding ◽

Dmitry S. Karlov ◽

George Chalhoub ◽

...

Keyword(s):

G Protein ◽

G Protein Coupled Receptor ◽

Allosteric Site ◽

Site Prediction ◽

Dynamic Mapping ◽

G Protein Coupled

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Structure and allosteric regulation of human IDH3 holoenzyme

10.1101/2020.06.25.170399 ◽

2020 ◽

Author(s):

Pengkai Sun ◽

Yan Liu ◽

Tengfei Ma ◽

Jianping Ding

Keyword(s):

Active Sites ◽

Critical Role ◽

Allosteric Regulation ◽

Kinetic Studies ◽

Tca Cycle ◽

Allosteric Site ◽

Functional Roles ◽

The Tca Cycle ◽

And Function ◽

Key Residues

AbstractHuman NAD-dependent isocitrate dehydrogenase or IDH3 catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the TCA cycle. We here report the structure of the IDH3 holoenzyme, in which the αβ and αγ heterodimers assemble the α2βγ heterotetramer via their clasp domains, and two α2βγ heterotetramers assemble the (α2βγ)2 heterooctamer via the β and γ subunits. The functional roles of the key residues involved in the assembly and allosteric regulation are validated by mutagenesis and kinetic studies. The allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation; the activation signal from the allosteric site is transmitted to the active sites of both heterodimers via the clasp domains; and the N-terminus of the γ subunit plays a critical role in the formation and function of the holoenzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of human IDH3 holoenzyme.

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Structure and allosteric regulation of human NAD-dependent isocitrate dehydrogenase

Cell Discovery ◽

10.1038/s41421-020-00220-7 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Pengkai Sun ◽

Yan Liu ◽

Tengfei Ma ◽

Jianping Ding

Keyword(s):

Isocitrate Dehydrogenase ◽

Active Sites ◽

Critical Role ◽

Allosteric Regulation ◽

Kinetic Studies ◽

Tca Cycle ◽

Allosteric Site ◽

Functional Roles ◽

Γ Subunit ◽

Key Residues

AbstractHuman NAD-dependent isocitrate dehydrogenase or HsIDH3 catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the TCA cycle. HsIDH3 exists and functions as a heterooctamer composed of the αβ and αγ heterodimers, and is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. In this work, we report the crystal structure of HsIDH3 containing a β mutant in apo form. In the HsIDH3 structure, the αβ and αγ heterodimers form the α2βγ heterotetramer via their clasp domains, and two α2βγ heterotetramers form the (α2βγ)2 heterooctamer through insertion of the N-terminus of the γ subunit of one heterotetramer into the back cleft of the β subunit of the other heterotetramer. The functional roles of the key residues at the allosteric site, the pseudo allosteric site, the heterodimer and heterodimer–heterodimer interfaces, and the N-terminal of the γ subunit are validated by mutagenesis and kinetic studies. Our structural and biochemical data together demonstrate that the allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation of the enzyme; the activation signal from the allosteric site is transmitted to the active sites of both αβ and αγ heterodimers via the clasp domains; and the N-terminal of the γ subunit plays a critical role in the formation of the heterooctamer to ensure the optimal activity of the enzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of HsIDH3.

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