scholarly journals Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
María Teresa Bueno-Carrasco ◽  
Jorge Cuéllar ◽  
Marte I. Flydal ◽  
César Santiago ◽  
Trond-André Kråkenes ◽  
...  
Keyword(s):  
Tyrosine Hydroxylase ◽  
Conformational Stability ◽  
Regulatory Mechanisms ◽  
Structural Mechanism ◽  
Regulatory Domains ◽  
Rate Limiting Step ◽  
Tetrameric Structure ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Site Blocking

AbstractTyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.

scholarly journals The Structure of Human Tyrosine Hydroxylase Reveals the Mechanism for Feedback Inhibition by Dopamine

2020 ◽  
Author(s):  
José Valpuesta ◽  
Teresa Bueno-Carrasco ◽  
Jorge Cuellar ◽  
Marte Flydal ◽  
Cesar Santiago ◽  
...  
Keyword(s):  
Tyrosine Hydroxylase ◽  
Active Site ◽  
Structural Changes ◽  
Feedback Inhibition ◽  
Rate Limiting Step ◽  
High Resolution Structure ◽  
Feedback Inhibitor ◽  
Α Helix ◽  
Rate Limiting ◽  
Resolution Structure

Abstract Tyrosine hydroxylase (TH) is a highly regulated enzyme that catalyses the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines. Mutations and dysfunction in this enzyme lead to DA deficiency and parkinsonisms of different severity. An understanding of TH deficiency at the level of structure and stability has been lacking to date, as only structures of truncated TH forms have been available. Here, we used cryoEM to determine the first high-resolution structure of full-length human tetrameric TH in the absence (3.4 Å) and presence (3.8 Å) of the end-product and feedback inhibitor DA bound to the active site. We show that upon DA binding, an α-helix (residues 39-59) included within the flexible N-terminal tail of the regulatory domain, is internalized in the active site. The observed structural changes reveal the molecular basis of the inhibitory and stabilizing DA effect, reversible by TH S40-phosphorylation, which are crucial regulatory mechanisms for catecholamine and TH homeostasis.

scholarly journals Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120

2012 ◽  
Vol 8 ◽  
pp. 1858-1866 ◽  
Author(s):  
Julia Meier ◽  
Kristin Kassler ◽  
Heinrich Sticht ◽  
Jutta Eichler
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Envelope Glycoprotein ◽  
Conformational Stability ◽  
Cellular Receptor ◽  
Binding Assays ◽  
Glycoprotein Gp120 ◽  
Dynamics Simulations ◽  
Key Residues ◽  
Hiv 1

Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids.

scholarly journals Structural insight into toxin secretion by contact-dependent growth inhibition transporters

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jeremy Guerin ◽  
Istvan Botos ◽  
Zijian Zhang ◽  
Karl Lundquist ◽  
James C Gumbart ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Outer Membrane ◽  
Structural Modifications ◽  
Extracellular Loops ◽  
Toxin Secretion ◽  
Dependent Growth ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Outer Membrane Transporters ◽  
Contact Dependent Growth Inhibition

Bacterial contact-dependent growth inhibition (CDI) systems use a type Vb secretion mechanism to export large CdiA toxins across the outer membrane by dedicated outer membrane transporters called CdiB. Here, we report the first crystal structures of two CdiB transporters from Acinetobacter baumannii and Escherichia coli. CdiB transporters adopt a TpsB fold, containing a 16-stranded transmembrane β-barrel connected to two periplasmic domains. The lumen of the CdiB pore is occluded by an N-terminal α-helix and the conserved extracellular loop 6; these two elements adopt different conformations in the structures. We identified a conserved DxxG motif located on strand β1 that connects loop 6 through different networks of interactions. Structural modifications of DxxG induce rearrangement of extracellular loops and alter interactions with the N-terminal α-helix, preparing the system for α-helix ejection. Using structural biology, functional assays, and molecular dynamics simulations, we show how the barrel pore is primed for CdiA toxin secretion.

scholarly journals Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity

2021 ◽  
Author(s):  
Victor Y. Zhao ◽  
João V. Rodrigues ◽  
Elena R. Lozovsky ◽  
Daniel L. Hartl ◽  
Eugene I. Shakhnovich
Keyword(s):  
Catalytic Activity ◽  
Dihydrofolate Reductase ◽  
Strongly Correlated ◽  
Thermal Stabilities ◽  
E Coli ◽  
In Vitro Measurements ◽  
Flux Model ◽  
Α Helix ◽  
Dynamics Simulations

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

scholarly journals Energetic Self-Folding Mechanism in α-Helixes

2019 ◽  
Author(s):  
Adolfo Bastida ◽  
José Zúñiga ◽  
Alberto Requena ◽  
Javier Cerezo
Keyword(s):  
Molecular Dynamics ◽  
Potential Energy Surfaces ◽  
Reasonable Agreement ◽  
Dihedral Angles ◽  
Native Conformation ◽  
Experimental Values ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Energy Surfaces ◽  
Large Peptide

A novel energetic route driving the folding of a polyalanine peptide from an extended conformation to its α-helix native conformation is described, supported by a new method to compute mean potential energy surfaces accurately in terms of the dihedral angles of the peptide chain from extensive Molecular Dynamics simulations. The Energetic Self-Folding (ESF) route arises specifically from the balance between the intrinsic propensity of alanine residues towards the α<sub>R </sub>conformation and two, opposite, effects: the destabilizing interaction with neighbor residues and the stabilizing formation of native hydrogen bonds, with the latter being dominant for large peptide lengths. The ESF mechanism provides simple but robust support to the nucleation-elongation, or zipper models, and offers a quantitative energetic funnel picture of the folding process. The mechanism is validated by the reasonable agreement between the computed folding energies and the experimental values.

scholarly journals Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sean P. Carney ◽  
Wen Ma ◽  
Kevin D. Whitley ◽  
Haifeng Jia ◽  
Timothy M. Lohman ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Variable Number ◽  
Structural Basis ◽  
Variable Step Size ◽  
Dna Unwinding ◽  
Step Size ◽  
Structural Mechanism ◽  
Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability

Biochemistry ◽  
2016 ◽  
Vol 55 (24) ◽  
pp. 3418-3431 ◽  
Author(s):  
José L. Neira ◽  
Felipe Hornos ◽  
Julio Bacarizo ◽  
Ana Cámara-Artigás ◽  
Javier Gómez
Keyword(s):  
Tyrosine Hydroxylase ◽  
Conformational Stability ◽  
Regulatory Domain ◽  
Monomeric Species

scholarly journals Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors

2018 ◽  
Vol 115 (35) ◽  
pp. E8172-E8180 ◽  
Author(s):  
Camille I. Pataki ◽  
João Rodrigues ◽  
Lichao Zhang ◽  
Junyang Qian ◽  
Bradley Efron ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Coarse Grained ◽  
Structural Basis ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Surfaces ◽  
Common Motif ◽  
Cysteine Scanning Mutagenesis ◽  
Α Helix ◽  
Dynamics Simulations ◽  
Integral Proteins

Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent–membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

An α-helix mimetic oligopyridylamide, ADH-31, modulates Aβ42 monomer aggregation and destabilizes protofibril structures: insights from molecular dynamics simulations

2020 ◽  
Vol 22 (48) ◽  
pp. 28055-28073
Author(s):  
Anupamjeet Kaur ◽  
Deepti Goyal ◽  
Bhupesh Goyal
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Helical Conformation ◽  
Aβ Peptide ◽  
Α Helix ◽  
Dynamics Simulations

The molecular dynamics simulations highlighted that ADH-31 inhibited Aβ42 aggregation by constraining Aβ peptide into helical conformation and destabilized Aβ42 trimer as well as protofibril structures.

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