Biology subjects

Grzesiek, S.

Publications and source records attributed to Grzesiek, S..

6 recordsLinked to original sources

bioRxiv2024

A high-resolution analysis of arrestin2 interactions responsible for CCR5 endocytosis

Clathrin-mediated endocytosis (CME) is crucial for regulating G protein-coupled receptors (GPCRs) via phosphorylation-dependent arrestin interactions. Despite detailed structural knowledge on the arrestin interactions with phosphorylated tails of GPCRs, the interplay between receptor phosphorylation and arrestin coupling to the CME machinery is not well understood, in particular due to the weakness and dynamics of the individual molecular interactions. Here we have characterized the interactions of arrestin2, which is activated by the phosphorylated C-terminus of the human chemokine receptor 5 (CCR5), with the main protein constituents of CME, namely clathrin and AP2 by solution NMR spectroscopy, biochemical and cellular assays. The NMR analysis revealed that arrestin2 interacts weakly with clathrin through a single binding site, independent of arrestin2 activation. In contrast, the arrestin2-AP2 interaction is stronger, requires arrestin2 activation by the CCR5 phospho-tail, and depends quantitatively on its degree of phosphorylation. These in vitro results are corroborated by cellular assays, which show that the chemokine-induced formation of a long-lived CCR5-arrestin2 internalization complex depends strongly on the interaction of arrestin2 with AP2, but not with clathrin. Taken together, these findings provide quantitative, atom-scale insights on the first steps of CCR5 endocytosis.

biochemistry↗

bioRxiv2024

Biased agonism of carvedilol in the beta1-adrenergic receptor is governed by conformational exclusion

Only two non-visual arrestins recognize many hundreds of different, intracellularly phosphorylated G protein-coupled receptors (GPCRs). Due to the highly dynamic nature of GPCR*arrestin complexes, the critical determinants of GPCR-arrestin recognition have remained largely unclear. We show here that arrestin2 recruitment to the {beta}1-adrenergic receptor ({beta}1AR) can be induced by an arrestin-activating phosphopeptide that is not covalently linked to the receptor and that the recruitment is independent of the presence and type of the orthosteric receptor ligand. Apparently, the arrestin-receptor interaction is driven by the conformational switch within arrestin induced by the phosphopeptide, whereas the electrostatic attraction towards the receptor phosphosites may only play an auxiliary role. Extensive NMR observations show that in contrast to previous static GPCR*arrestin complex structures, the {beta}1AR complex with the beta-blocker carvedilol and arrestin2 is in a G protein-inactive conformation. The insensitivity to the specific receptor conformation provides a rationale for arrestins promiscuous recognition of GPCRs and explains the arrestin-biased agonism of carvedilol, which largely blocks G protein binding, while still enabling arrestin engagement. Significance statementG protein-coupled receptors regulate cellular signaling through G proteins and arrestins. While G protein interactions are well understood, the molecular basis of arrestin recognition remains unclear due to the dynamic nature of GPCR*arrestin complexes. We show that arrestin recognition of the {beta}1-adrenergic receptor occurs independently of the receptors conformational state or ligand binding. Using NMR, cryo-EM, and biochemical assays, we find that arrestin engagement is driven by a conformational change within arrestin itself, triggered by a non-receptor-attached phosphopeptide, whereas electrostatic attraction towards receptor phosphosites may only play an auxiliary role. These findings provide new insights into arrestin activation, explain its ability to recognize diverse GPCRs, with significant implications for understanding biased signaling mechanisms and designing of selective therapeutic strategies.

biochemistry↗

bioRxiv2023

The molecular basis of Abelson kinase regulation by its αI-helix

Abelson tyrosine kinase (Abl) is regulated by the arrangement of its regulatory core, consisting sequentially of the SH3, SH2 and kinase (KD) domains, where an assembled or disassembled core corresponds to low or high kinase activity, respectively. It was recently established that binding of type II ATP site inhibitors, such as imatinib, generates a force from the KD N-lobe onto the SH3 domain and in consequence disassembles the core. Here, we demonstrate that the C-terminal I-helix exerts an additional force towards the SH2 domain, which correlates both with kinase activity and type II inhibitor-induced disassembly. The I- helix mutation E528K, which is responsible for the ABL1 malformation syndrome, strongly activates Abl by breaking a salt bridge with the KD C-lobe and thereby increasing the force onto the SH2 domain. In contrast, the allosteric inhibitor asciminib strongly reduces Abls activity by fixating the I-helix and reducing the force onto the SH2 domain. These observations are explained by a simple mechanical model of Abl activation involving forces from the KD N-lobe and the I-helix onto the KD/SH2SH3 interface.

biophysics↗

bioRxiv2022

A key GPCR phosphorylation motif discovered in arrestin2-CCR5 phosphopeptide complexes

The two non-visual arrestin isoforms, arrestin2 and arrestin3 recognize and bind hundreds of G protein-coupled receptors (GPCRs) with different phosphorylation patterns leading to distinct functional outcomes. The impact of phosphorylation on arrestin interactions has been well studied only for very few GPCRs. Here we have characterized the interactions between the phosphorylated CC chemokine receptor 5 (CCR5) and arrestin2. We detected several new CCR5 phosphorylation sites, which are necessary for stable complex formation with arrestin2. Crystal structures of arrestin2 in apo form and in complexes with CCR5 C-terminal phosphopeptides together with NMR spectroscopy, biochemical and functional assays revealed three phosphoresidues in a pXpp motif that are essential for the arrestin2 interactions and activation. The same phosphoresidue cluster is present in other receptors, which form stable complexes with arrestin2. We propose that the identified pXpp motif is responsible for robust arrestin2 recruitment in many GPCRs. An analysis of available sequences, structural and functional information on other GPCR*arrestin interactions suggests that a particular arrangement of phosphoresidues within the GPCR intracellular loop 3 and C-terminal tail determines arrestin2 and 3 isoform specificity. Taken together, our findings demonstrate how multi-site phosphorylation controls GPCR*arrestin interactions and provide a framework to probe the intricate details of arrestin activation and signaling. One-sentence summaryA structural and functional analysis of arrestin2 in apo form and complexes with several CCR5 phosphopeptides reveals key phosphorylation sites responsible for stable GPCR*arrestin interactions and their contributions to CCR5-arrestin2 function.

biophysics↗

bioRxiv2021

Filling of a water-free void explains the allosteric regulation of the β1-adrenergic receptor by cholesterol

Proteins often contain cavities, which are usually assumed to be water-filled. Recent high-pressure NMR results indicate that the preactive conformation of the {beta}1-adrenergic receptor ({beta}1AR) contains completely empty cavities (dry voids) of about [~]100 [A]3 volume, which disappear in the active conformation of the receptor. Here we have localized these cavities by X-ray crystallography on xenon-derivatized {beta}1AR crystals. One of the cavities coincides with the binding pocket of cholesterol. Solution NMR data show that addition of the soluble cholesterol analog cholesteryl hemisuccinate (CHS) impedes the formation of the active conformation of the receptor by blocking conserved GPCR microswitches. This wedge-like action explains the function of the cellularly highly abundant cholesterol as a negative allosteric modulator of {beta}1AR. The detailed understanding of GPCR regulation by cholesterol via filling of a dry void and the easy scouting for such voids by xenon may provide new routes for the development of allosteric drugs.

biophysics↗

bioRxiv2020

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism. One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

biophysics↗