Biology subjects

Stathopulos, P.

Publications and source records attributed to Stathopulos, P..

3 recordsLinked to original sources

bioRxiv2024

An AI-informed NMR structure reveals a LETM1 F-EF-hand for two-way mitochondrial calcium regulation.

AlphaFold2 can accurately predict high-resolution protein structure from sequence but does not account for solvent conditions, ligands, post-translational modifications, lowly populated states or rare folds. Human leucine zipper EF-hand transmembrane protein-1 (LETM1) has one sequence-identifiable EF-hand but whether and how calcium (Ca2+) binding affects structure and function remains enigmatic. Here, we developed an approach that used highly confident AlphaFold2 C positions to guide nuclear Overhauser effect (NOE) assignments and structure calculation of the LETM1 EF-hand in the presence of Ca2+. The resultant NMR structure exposes pairing between a partial loop-helix and full helix-loop-helix, forming an unprecedented F-EF-hand domain with non-canonical Ca2+ coordination but enhanced hydrophobicity for protein interactions compared to calmodulin. The structure also reveals the basis for pH sensing by His662, linking the canonical and partial EF-hands. Functionally, mutations that augmented or weakened Ca2+ binding led to increased and decreased matrix Ca2+ levels, respectively, establishing F-EF as a two-way mitochondrial Ca2+ potentiator. Collectively, we show how AlphaFold2 can be synergized with NMR data to produce solution-specific structures, revealing here an extraordinary LETM1 F-EF-hand sensor.

cell biology↗

bioRxiv2022

The human MRS2 magnesium binding domain is a regulatory feedback switch for channel activity.

Mitochondrial RNA splicing protein 2 (MRS2) forms a major magnesium (Mg2+) entry channel into the matrix. While MRS2 contains two transmembrane domains that constitute a pore, most of the protein resides within the matrix. Yet, the precise structural and functional role of this obtrusive amino terminal domain (NTD) in human MRS2 function is unknown. Here, we show that the MRS2 NTD self-associates into a homodimer, contrasting the pentameric assembly of CorA, an orthologous bacterial channel. Mg2+ and calcium suppress lower and higher order oligomerization of MRS2 NTD, while cobalt has no effect on the NTD but disassembles full-length MRS2. Mutating pinpointed residues mediating Mg2+ binding to the NTD, not only selectively decreases Mg2+ binding affinity [~]7-fold but also abrogates Mg2+ binding-induced changes in secondary, tertiary and quaternary structures. Disruption of NTD Mg2+ binding strikingly potentiates mitochondrial Mg2+ uptake in wild-type and Mrs2 knockout cells. Our work exposes a mechanism for human MRS2 autoregulation by negative feedback from the NTD and identifies a novel gain of function mutant with broad applicability to future Mg2+ signaling research.

biophysics↗

bioRxiv2019

Molecular Basis for G-protein-Coupled Receptor (GPCR) Activation and Biased Signalling at the Platelet Thrombin Receptor Proteinase Activated Receptor-4 (PAR4)

Proteinase Activated Receptor-4 (PAR4) is a member of the proteolytically-activated PAR family of G-Protein-coupled Receptors (GPCRs). PARs are activated following proteolytic cleavage of the receptor N-terminus by enzymes such as thrombin, trypsin, and cathepsin-G to reveal the receptor-activating motif termed the tethered ligand. The tethered ligand binds intramolecularly to the receptor and triggers receptor signalling and cellular responses. In spite of this unusual mechanism of activation, PARs are fundamentally peptide receptors and can also be activated by exogenous application of short synthetic peptides derived from the tethered ligand sequence. In order to gain a better understanding of the molecular basis for PAR4-dependent signalling, we examined signalling responses to a library of peptides derived from the canonical PAR4 activating peptide (PAR4-AP), AYPGKF-NH2. We examined peptide residues involved in activation of the Gq/11-coupled calcium signalling pathway, {beta}-arrestin recruitment, and mitogen-activated protein kinase pathway activation. The peptide N-methyl-alanine-YPGKF-NH2 was identified as a compound that is a poor activator of PAR4-dependent calcium signalling but was fully competent in recruiting {beta}-arrestin-1 and -2. In order to gain a better understanding of the ligand-binding pocket, we used in silico docking to identify key residues involved in PAR4 interaction with AYPGKF-NH2. The predicted interactions were verified by site-directed mutagenesis and analysis of calcium signalling and {beta}-arrestin-1/-2 recruitment following proteolytic activation (with thrombin) or activation with the synthetic agonist peptide (AYPGKF-NH2). We determined that a key extracellular loop-2 aspartic acid residue (Asp230) is critical for signalling following both proteolytic and peptide activation of PAR4. Finally, we investigated platelet aggregation in response to AyPGKF-NH2 (a peptide with D-tyrosine in position two) which is unable to activate calcium signalling, and AYPGRF-NH2 a peptide that is equipotent to the parental peptide AYPGKF-NH2 for calcium signalling but is more potent at recruiting {beta}-arrestins. We found that AyPGKF-NH2 fails to activate platelets while AYPGRF-NH2 causes a platelet aggregation response that is greater than that seen with the parental peptide and is comparable to that seen with thrombin stimulation. Overall, these studies uncover molecular determinants for agonist binding and signalling through a non-canonically activated GPCR and provide a template for development of small molecule modulators of PAR4.

pharmacology and toxicology↗