Supertertiary protein structure affects an allosteric network

Laursen, Louise; Kliche, Johanna; Gianni, Stefano; Jemth, Per

doi:10.1073/pnas.2007201117

Cited by 27 publications

(35 citation statements)

References 52 publications

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“…7 ), which is required for inhibition of phosphate transport ( 16 ). Allosteric networks and allosteric communication are essential regulators of PDZ-containing multidomain protein conformational changes and function ( 44 , 45 ).…”

Section: Discussionmentioning

confidence: 99%

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport

Vistrup-Parry

Sneddon

Bach

et al. 2021

Journal of Biological Chemistry

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The type II sodium-dependent phosphate cotransporter (NPT2A) mediates renal phosphate uptake. The NPT2A is regulated by parathyroid hormone (PTH) and fibroblast growth factor 23, which requires Na + /H + exchange regulatory factor-1 (NHERF1), a multidomain PDZ-containing phosphoprotein. Phosphocycling controls the association between NHERF1 and the NPT2A. Here, we characterize the critical involvement of G protein–coupled receptor kinase 6A (GRK6A) in mediating PTH-sensitive phosphate transport by targeted phosphorylation coupled with NHERF1 conformational rearrangement, which in turn allows phosphorylation at a secondary site. GRK6A, through its carboxy-terminal PDZ recognition motif, binds NHERF1 PDZ1 with greater affinity than PDZ2. However, the association between NHERF1 PDZ2 and GRK6A is necessary for PTH action. Ser 162 , a PKCα phosphorylation site in PDZ2, regulates the binding affinity between PDZ2 and GRK6A. Substitution of Ser 162 with alanine (S 162 A) blocks the PTH action but does not disrupt the interaction between NHERF1 and the NPT2A. Replacement of Ser 162 with aspartic acid (S 162 D) abrogates the interaction between NHERF1 and the NPT2A and concurrently PTH action. We used amber codon suppression to generate a phosphorylated Ser 162 (pSer 162 )-PDZ2 variant. K D values determined by fluorescence anisotropy indicate that incorporation of pSer 162 increased the binding affinity to the carboxy terminus of GRK6A 2-fold compared with WT PDZ2. Molecular dynamics simulations predict formation of an electrostatic network between pSer 162 and Asp 183 of PDZ2 and Arg at position −1 of the GRK6A PDZ-binding motif. Our results suggest that PDZ2 plays a regulatory role in PTH-sensitive NPT2A-mediated phosphate transport and phosphorylation of Ser 162 in PDZ2 modulates the interaction with GRK6A.

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Section: Discussionmentioning

confidence: 99%

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport

Vistrup-Parry

Sneddon

Bach

et al. 2021

Journal of Biological Chemistry

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“…The inter-domain energetic communication within the PSG supramodule was also investigated by double mutant cycles [ 58 ]. The so-called ‘double mutant cycles’ methodology is a powerful approach to measuring, directly, the extent of energetic communication between interacting side-chains.…”

Section: Interaction Between Protein Domains—supertertiary Structurementioning

confidence: 99%

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains

et al. 2022

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The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure–function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, influencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts.

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“…In this context, double mutant cycles proved very useful. An extensive double mutant cycle analysis of a PDZ domain, both in isolation and in the context of a supramodule comprising the PDZ domain, an SH3 and a GK domain, showed that allosteric networks are highly sensitive to the supertertiary structure [ 45 ]. In particular, it was found that the presence of the SH3–GK tandem resulted in strong coupling from the bound peptide ligand to the β1β2 loop, β2β3 loop, and α3 helix that was not observed with the single PDZ3 domain.…”

Section: Protein Binding and Allosterymentioning

confidence: 99%

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Pagano

Toto

Malagrinò

et al. 2021

IJMS

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Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted ‘energetic coupling’ describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein–ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

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Supertertiary protein structure affects an allosteric network

Cited by 27 publications

References 52 publications

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport

Multisite NHERF1 phosphorylation controls GRK6A regulation of hormone-sensitive phosphate transport

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

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