Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

Ma, Ning; Lippert, Lisa G.; Devamani, Titu; Levy, Benjamin; Lee, Sangbae; Sandhu, Manbir; Vaidehi, Nagarajan; Sivaramakrishnan, Sivaraj

doi:10.1021/acs.biochem.8b00729

Cited by 12 publications

(20 citation statements)

References 15 publications

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“…In contrast, the indole maleimide inhibitors, BimI and sotrastaurin, substantially inhibited this interaction. Interestingly, the effects of this panel of inhibitors on the kinase-regulatory domain interaction recapitulate the results from our previous report on the kinase-substrate peptide interaction ( 18 ). Owing to this correlation, we tested the effects of the inhibitor on the interaction between the kinase and a pseudosubstrate peptide (PS peptide) at the N-terminus of the regulatory domain.…”

Section: Resultssupporting

confidence: 86%

“…We have previously shown that ATP-competitive SMKIs can allosterically influence the kinase-substrate interaction in PKCα ( 18 ). Here, we used previously reported SPASM PKCα sensors ( 15 ) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 F ). While we have previously established that the allosteric effects of BimI on substrate binding is mediated by changes in conformation of the kinase domain activation loop ( 18 ), the structural basis for inhibition of C1a binding remained unclear.…”

Section: Resultsmentioning

confidence: 99%

“…However, there is currently no crystal structure of the full-length PKCα. In our previous work, we started from the crystal structure of the kinase domain of PKCα (PDB ID: 3IW4) and derived a model of the kinase domain interacting with PS using GNEIMO running replica exchange MD (REMD) simulations ( 18 , 30 ). We also used the crystal structure of the kinase domain of PKCα that includes the C1b domain from the crystal structure of the full-length PKCβII (PDB ID: 3PFQ) ( 31 ).…”

Section: Methodsmentioning

confidence: 99%

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Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

Lippert

Ritt

et al. 2021

Journal of Biological Chemistry

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The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

Lippert

Ritt

et al. 2021

Journal of Biological Chemistry

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“…Starting from three dimensional structures of a GPCR in different conformational states, the differences in the inter-residue edge strengths for the network models derived in the fully active versus inactive states of GPCRs were delineated [63]. [69,70], phosphatases [71] and DNA repair proteins [72]. This approach can be used on any receptor more dynamic compared with the other states.…”

Section: Static Three-dimensional Structure-based Analysis Of Allostementioning

confidence: 99%

Allosteric communication regulates ligand‐specific GPCR activity

Nivedha

Vaidehi

2021

The FEBS Journal

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Accepted Article This article is protected by copyright. All rights reserved G protein coupled receptors (GPCRs) are membrane bound proteins that are ubiquitously expressed in many cell types and take part in mediating multiple signaling pathways. GPCRs are dynamic proteins and exist in an equilibrium between an ensemble of conformational states such as inactive and fully active states. This dynamic nature of GPCRs is one of the factors that confers their basal activity even in the absence of any ligand mediated activation. Ligands selectively bind and stabilize a subset of the conformations from the ensemble leading to a shift in the equilibrium towards the inactive or the active state depending on the nature of the ligand. This ligand-selective effect is achieved through allosteric communication between the ligand binding site and G protein or -arrestin coupling site. Similarly, the G protein coupling to the receptor exerts the allosteric effect on the ligand binding region leading to increased binding affinity for agonists and decreased affinity for antagonists or inverse agonists. In this review, we enumerate the current state of our understanding of the mechanism of allosteric communication in GPCRs with a specific focus on the critical role of computational methods in delineating the residues involved in allosteric communication. Analyzing allosteric communication mechanism using molecular dynamics simulations have revealed (i) a structurally conserved mechanism of allosteric communication that regulates the G protein coupling, (ii) a rational structure-based approach to designing selective ligands and (iii) an approach to designing allosteric GPCR mutants that are either ligand and G protein or -arrestin selective.

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ER/K-link—Leveraging a native protein linker to probe dynamic cellular interactions

Gupte

Ritt

Sivaramakrishnan

2021

Linkers in Biomacromolecules

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Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

Cited by 12 publications

References 15 publications

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation

Allosteric communication regulates ligand‐specific GPCR activity

ER/K-link—Leveraging a native protein linker to probe dynamic cellular interactions

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