An Ensemble of Rapidly Interconverting Orientations in Electrostatic Protein–Peptide Complexes Characterized by NMR Spectroscopy

Guan, Jia-Ying; Foerster, Johannes M.; Drijfhout, Jan W.; Timmer, Monika; Blok, Anneloes; Ullmann, G. Matthias; Ubbink, Marcellus

doi:10.1002/cbic.201300623

Cited by 10 publications

(12 citation statements)

References 63 publications

(121 reference statements)

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“…It is possible that capsaicin could form an initial encounter complex with the TRPV1 molecule before binding more tightly in a manner mediated by the Y511 side chain conformational flip ( 62 ). Alternatively, it is possible that the absence of long-lived encounters between capsaicin and TRPV1 in the simulations imply that this ligand, in contrast to RTX, interacts only transiently with the channel.…”

Section: Discussionmentioning

confidence: 99%

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Hanson

Newstead

Swartz

et al. 2015

Biophysical Journal

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Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1-S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1-S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1-S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular leaflet and mechanisms of binding site access by capsaicin.

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Section: Discussionmentioning

confidence: 99%

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Hanson

Newstead

Swartz

et al. 2015

Biophysical Journal

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“…(Martin et al, 2001) Because of the bias of the 40's loop for the 6-O-sulfated GalNAc in CS644, the non-reducing end (where the 6-O-sulfation is located) becomes associated with the 40s loop, thus the reducing end is more likely to be located at the N-loop. Because heterogeneous protein-ligand interactions are known to produce small chemical shift perturbations (Guan et al, 2014), the small chemical shift changes of the N-loop and N-terminal residues observed in the titration of CS644 may also be a manifestation of the heterogeneity in binding orientations of CS644. The change in chemical shift migration direction as the ratio of CS644-to-CCL5 goes beyond one maybe a sign of multiple ligand binding involving the N-loop.…”

Section: Discussionmentioning

confidence: 99%

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands

et al. 2015

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Summary Interactions of the chemokine CCL5 (RANTES) with glycosaminoglycans (GAGs) are crucial to the CCL5-mediated inflammation process. However, structural information on interactions between CCL5 and longer GAG fragments is lacking. In this study, the interactions between oligosaccharides derived from chondroitin sulfate and a dimeric variant of CCL5 were investigated using solution NMR. The data indicate that, in addition to the BBXB motif in the 40s loop, GAGs also contact residues in the N-loop in a manner similar to interactions between chemokine and the receptor N-terminus, and leading to possible stabilization of the dimer. Using TEMPO-tagged hexasaccharides, the binding orientation of the hexasaccharides was shown to be highly dependent on the sulfation pattern of the GalNAc groups. Finally, a model of the CCL5 dimer complexed to CS hexasaccharides was constructed using paramagnetic relaxation enhancement and intra- and inter-molecular NOEs constraints.

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“…As it does not, this must indicate that the hsc-70 LID domain is loosely tethered to the fluid-like nanodisc surface while bound. The situation is reminiscent of the delocalized interaction of DnaJ J-domain to DnaK (the Hsp70 of Escherichia coli) (19), cytochrome-cytochrome complexes (20), and nonspecific protein/DNA interactions (21), all of which are, like the hsc70/PS interaction, electrostatic in nature.…”

Section: Hsc-70mentioning

confidence: 99%

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy

Morozova¹,

Clement²,

Kaushik

et al. 2016

Journal of Biological Chemistry

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hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ؎ 0.1 m. This interaction is specific and involves a total of 4 -5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.hsc-70 (HSPA8) is a constitutively expressed molecular chaperone. The human hsp-70 chaperone family consists of 11 highly homologous members specific to different cellular compartments and organelles (1). hsc-70 resides in the cellular cytosol and nucleus and plays a central role in cellular proteostasis and protein trafficking.Mammalian hsc-70 consists of the following four structural domains: a 44-kDa nucleotide binding domain (NBD) 3 (residues 1-384) with ATPase activity; a 12-kDa substrate binding domain (SBD) that binds to exposed hydrophobic sequences in client proteins (residues 385-505); a 10-kDa helical LID domain (residues 506 -605); and a 5-kDa dynamically unstructured C-terminal domain (CTD) (residues 606 -646) (2). Binding of ATP in the NBD triggers a global conformational change that releases peptide/protein cargo from the SBD (3). Furthermore, hydrolysis of ATP closes the LID and greatly enhances client-SBD affinity (4). This hydrolysis cycle is important in the chaperone activity of hsc-70 as it allows, with the aid of different co-chaperones, iterative binding to clients resulting in protein (re)folding (5). Furthermore, hsc-70 is involved in recruitment of ubiquitin ligases (6), which leads to cargo polyubiquitination and subsequent hsc-70 trafficking to the proteasome or the endocytic pathway for cargo degradation (7).Altogether, hsc-70's multiple interactions allow this chaperone to play an important role in several cellular activities, including ribosomal quality control, protein refolding, proteasome-linked degradation, macroautophagy, endosomal microautophagy, chaperone-mediated autophagy, endoplasmic reticulum/Golgi and mitochondrial targeting, and vesicle clathrin uncoating (8 -17). These activities place hsc-70 as one of the master controllers of cellular proteostasis.To carry out several of these functions, hsc-70 does not only interact with partner proteins but with membrane lipids as ...

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An Ensemble of Rapidly Interconverting Orientations in Electrostatic Protein–Peptide Complexes Characterized by NMR Spectroscopy

Cited by 10 publications

References 63 publications

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Interactions of the Chemokine CCL5/RANTES with Medium-Sized Chondroitin Sulfate Ligands

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy

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