Uracil/H+ Symport by FurE Refines Aspects of the Rocking-bundle Mechanism of APC-type Transporters

Zantza, Iliana; Pyrris, Yiannis; Raniolo, Stefano; Papadaki, Georgia; Lambrinidis, George; Limongelli, Vittorio; Diallinas, George; Mikros, Emmanuel

doi:10.1016/j.jmb.2023.168226

Cited by 4 publications

(2 citation statements)

References 77 publications

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“…PCV-MetaD calculations have been successfully employed by us and other groups to study large scale and long timescale conformational changes in different protein systems. 83–89 In the case of A2A, the path is defined considering pAS as intermediate state between the terminal active and inactive state, and including the residues involved in the conformational transitions observed during the multi-microseconds MD calculations (see Experimental Section for details).…”

Section: Resultsmentioning

confidence: 99%

Minute-timescale simulations of G Protein Coupled Receptor A2A activation mechanism reveal a receptor pseudo-active state

D’Amore,

Conflitti,

Marinelli

et al. 2023

Preprint

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G protein-coupled receptors (GPCRs) are a most prominent pharmacological target, regulating various biological functions and targeted by over one third of marketed drugs. These receptors are activated by orthosteric ligands and undergo large conformational changes that lead to coupling effector proteins. Despite their relevance, many aspects of their activation mechanism remain unclear, hampering rational drug design studies. In this work, we elucidate the activation mechanism of the adenosine A2A receptor (A2AR), a class A GPCR, using molecular dynamics simulations and free energy calculations. We have explored the entire conformational landscape of A2AR in its basal apo form and in differently ligated conditions, identifying the receptor's lowest energy functional states. Among these is a novel pseudo-active state (pAS) stabilised by specific "microswitch" residues interactions like the R5.66/E6.30salt bridge. In this form, A2A is able to couple β-arrestin over G proteins, enlightening possible routes for biased signalling. Our findings shed light on GPCR activation dynamics, paving the way to the rational design of drugs for various diseases involving A2AR, including cancer, inflammation, cardiovascular, Parkinson's, and Alzheimer's diseases.

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Section: Resultsmentioning

confidence: 99%

Minute-timescale simulations of G Protein Coupled Receptor A2A activation mechanism reveal a receptor pseudo-active state

D’Amore,

Conflitti,

Marinelli

et al. 2023

Preprint

Self Cite

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“…Protein model construction and MD simulations are described in detail elsewhere [ 52 ]. In brief, homology models of FurE were constructed based on Mhp1 crystal structures 2JLN, 4D1B, 2X79.…”

Section: Methodsmentioning

confidence: 99%

The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit

Pyrris,

Papadaki,

Mikros

et al. 2024

Microb Cell

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FurE is a H+ symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus Aspergillus nidulans. Being member of the NCS1 protein family, FurE is structurally related to the APC-superfamily of transporters. APC-type transporters are characterised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism. Most APC-type transporters possess two extra C-terminal TMS segments (TMS11-12), the function of which remains elusive. Here we present a systematic mutational analysis of TMS11-12 of FurE and show that two specific aromatic residues in TMS12, Trp473 and Tyr484, are essential for ER-exit and trafficking to the plasma membrane (PM). Molecular modeling shows that Trp473 and Tyr484 might be essential through dynamic interactions with residues in TMS2 (Leu91), TMS3 (Phe111), TMS10 (Val404, Asp406) and other aromatic residues in TMS12. Genetic analysis confirms the essential role of Phe111, Asp406 and TMS12 aromatic residues in FurE ER-exit. We further show that co-expression of FurE-Y484F or FurE-W473A with wild-type FurE leads to a dominant negative phenotype, compatible with the concept that FurE molecules oligomerize or partition in specific microdomains to achieve concentrative ER-exit and traffic to the PM. Importantly, truncated FurE versions lacking TMS11-12 are unable to reproduce a negative effect on the trafficking of co-expressed wild-type FurE. Overall, we show that TMS11-12 acts as an intramolecular chaperone for proper FurE folding, which seems to provide a structural code for FurE partitioning in ER-exit sites.

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Minute-timescale free-energy calculations reveal a pseudo-active state in the adenosine A2A receptor activation mechanism

D’Amore,

Conflitti,

Marinelli

et al. 2024

Chem

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Uracil/H+ Symport by FurE Refines Aspects of the Rocking-bundle Mechanism of APC-type Transporters

Cited by 4 publications

References 77 publications

Minute-timescale simulations of G Protein Coupled Receptor A2A activation mechanism reveal a receptor pseudo-active state

Minute-timescale simulations of G Protein Coupled Receptor A2A activation mechanism reveal a receptor pseudo-active state

The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit

Minute-timescale free-energy calculations reveal a pseudo-active state in the adenosine A2A receptor activation mechanism

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