Controllable Activation of Nanoscale Dynamics in a Disordered Protein Alters Binding Kinetics

Callaway, David J.E.; Matsui, Tsutomu; Weiß, Thomas; Stingaciu, Laura R.; Stanley, Christopher B.; Heller, William T.; Bu, Zimei

doi:10.1016/j.jmb.2017.03.003

Cited by 14 publications

(13 citation statements)

References 70 publications

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“…The overall molecular size distribution profile of the WT protein that fits the SANS data are consistent with the SEC-SAXS data (Table 1) of monomeric NHERF1 reported in our previous studies (40). The close agreement between the R g values confirms deuteration does not affect the conformation of NHERF1.…”

Section: Global Conformational Changes In Wt and Mutant Nherf1 From Sanssupporting

confidence: 89%

“…7C). We attribute the slower kinetic process to conformational changes in the C-terminal tail of NHERF1 upon docking with ezFERM accompanied by a weak-binding interaction (16,40,43). This two-step kinetic model is well-supported by the published X-ray structure of the 38-residue EB domain (amino acids 321-358) from NHERF1 bound to the moesin FERM domain (43).…”

Section: Binding Of Ezferm To Nherf1 Mutantssupporting

confidence: 59%

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Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly

Bhattacharya

Stanley

Heller

et al. 2019

Journal of Biological Chemistry

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The Na ؉ /H ؉ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes. Signal transduction in the biological milieu is a dynamic exchange of protein-protein interactions (1, 2), coordinated by a diverse family of scaffolding proteins (2-4). Structural mod-ularity of the scaffolds is fine-tuned for linking and transporting the different binding partners to the supramolecular signaling complexes. Well-known scaffold modules include Src homology 3, Src homology 2, PTB, WW, and the more abundant PDZ domains, which are ubiquitous for recruiting diverse protein targets (3, 4). As mediators of protein-protein interactions, PDZ domains have been shown to possess remarkable specificity for C-terminal binding motifs as well as internal peptide sequences of proteins (3). PDZ domains have emerged as key organizers of protein complexes at the plasma membrane, cytoplasmic tails of membrane proteins, and ion channels to promote their transport and localization to the cell surface for signaling (5). Full-length NHERF1 includes tandem PDZ domains, PDZ1 and PDZ2, and a C-terminal ezrin-binding domain (EBD) 4 juxtaposed with a PDZ-binding type 1 motif (Fig. 1). The EBD is a specific target of the FERM domain from ezrin (ezFERM), which disrupts the autoinhibition between the PDZ2 domain and the C-terminal tail in NHERF1 (6). Binding of ezrin with NHERF1 allosterically increases the PDZ domain ...

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Section: Global Conformational Changes In Wt and Mutant Nherf1 From Sanssupporting

confidence: 89%

Section: Binding Of Ezferm To Nherf1 Mutantssupporting

confidence: 59%

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly

Bhattacharya

Stanley

Heller

et al. 2019

Journal of Biological Chemistry

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“…SEC-SAXS studies were performed at SSRL Beamline 4–2 to mitigate aggregation, as described previously 59 , 60 . The details were summarized in Table 2 .…”

Section: Methodsmentioning

confidence: 99%

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus

Panigrahi

Matsui

Song

et al. 2018

Sci Rep

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Proteins with multifunctional regulatory domains often demonstrate structural plasticity or protein disorder, allowing the binding of multiple regulatory factors and post-translational modifications. While the importance of protein disorder is clear, it also poses a challenge for in vitro characterization. Here, we report protein intrinsic disorder in a plant molecular system, which despite its prevalence is less studied. We present a detailed biophysical characterization of the entire cytoplasmic N-terminal domain of Brassica napus diacylglycerol acyltransferase, (DGAT1), which includes an inhibitory module and allosteric binding sites. Our results demonstrate that the monomeric N-terminal domain can be stabilized for biophysical characterization and is largely intrinsically disordered in solution. This domain interacts with allosteric modulators of DGAT1, CoA and oleoyl-CoA, at micromolar concentrations. While solution scattering studies indicate conformational heterogeneity in the N-terminal domain of DGAT1, there is a small gain of secondary structure induced by ligand binding.

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“…University of Arizona, Tucson, AZ, USA, 2 Dept Chem/Biochem, Univ Arizona, Tucson, AZ, USA. There has been progress in designing artificial enzymes, however their proficiency is still below naturally occurring enzymes and there have been almost no studies on the mechanistic details and dynamical nature of these enzymes.…”

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confidence: 99%

Exploring the Effects of Directed Evolution on the Dynamics of Artificial Retro Aldolases

Schafer

Zoi

Schwartz

2019

Biophysical Journal

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situation at a pulsating neutron source and several unique features specific only for the SNS-NSE, high data collection efficiency is achieved allowing nearly gapless coverage of a broad wave-vector-time-range with only a few scattering angle settings. The SNS-NSE instrument is particularly suitable to investigate slow dynamical processes and unravel molecular motions at nanoscopic and mesoscopic scale in biophysics and biochemistry. Investigation of macromolecular assemblies of great importance to human health is one of primary applications of the SNS-NSE, attracting users with interest in biophysics and medical science all over the world. Neutron Spin Echo technique can be successfully applied to access the domain dynamics of proteins and enzyme's, domain dynamics that is strongly related with folding-unfolding processes within proteins 1-2 and enzymatic/catalytic reactions in enzymes. Studies of lipid systems and biological membranes are also carried out at SNS-NSE to investigate how cell membranes organize proteins and lipids to accomplish vital physiological processes 3-4 , and to observe the disruptive effects of antiinflammatory medication on membrane cell organization and the transport process thru cell membranes 5 .

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Controllable Activation of Nanoscale Dynamics in a Disordered Protein Alters Binding Kinetics

Cited by 14 publications

References 70 publications

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly

Intrinsic disorder in the regulatory N-terminal domain of diacylglycerol acyltransferase 1 from Brassica napus

Exploring the Effects of Directed Evolution on the Dynamics of Artificial Retro Aldolases

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