Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase

Klein, Tobias; Vajpai, Navratna; Phillips, Jonathan J.; Davies, Gareth; Holdgate, Geoffrey A.; Phillips, Chris; Tucker, J.A.; Norman, Richard A.; Scott, Andrew D.; Higazi, Daniel R.; Lowe, David C.; Thompson, Gary S.; Breeze, Alexander L.

doi:10.1038/ncomms8877

Cited by 63 publications

(59 citation statements)

References 66 publications

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“…1b). Our findings are also consistent with the presence of a “molecular brake” in an inactive state of FGFR KD and similar kinases52, a feature near the hinge region that could restrain two kinase lobes and also suppress the catalytically significant DFG flip in the A-loop by imposing a particularly high free-energy barrier20.…”

Section: Discussionsupporting

confidence: 87%

“…Experimental approaches to monitor the dynamic properties of protein kinases at high resolution have been based mainly on application of nuclear magnetic resonance (NMR)1415, however, this approach has been successfully applied only to a small number of protein kinases161718192021. Due to the difficulties in obtaining experimental data, molecular dynamics (MD) simulations have been performed more extensively, providing theoretical calculations and working models for dynamic behavior of kinases212223242526272829.…”

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confidence: 99%

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Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Perdios

Lowe

Saladino

et al. 2017

Sci Rep

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Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

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Section: Discussionsupporting

confidence: 87%

mentioning

confidence: 99%

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Perdios

Lowe

Saladino

et al. 2017

Sci Rep

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“…; Klein et al . ); therefore, the conformational state of the P‐loop was considered not to affect the association of these two drugs with FGFR kinases. It has also been reported that dovitinib and ponatinib come into contact with the gatekeeper residue.…”

Section: Discussionmentioning

confidence: 99%

“…The reduced affinity of the V561M mutant for PD173074 was caused not only by steric hindrance around the gatekeeper residues, but also by steric clash induced by the structural change around the P-loop region. Crystallographic analysis showed that dovitinib and ponatinib showed no appreciable contact with the Ploop (Lesca et al 2014;Tucker et al 2014;Bunney et al 2015;Klein et al 2015); therefore, the conformational state of the P-loop was considered not to affect the association of these two drugs with FGFR kinases. It has also been reported that dovitinib and ponatinib come into contact with the gatekeeper residue.…”

Section: Discussionmentioning

confidence: 99%

Biophysical characterization of drug‐resistant mutants of fibroblast growth factor receptor 1

et al. 2016

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Over-expression and aberrant activation of tyrosine kinases occur frequently in human cancers. Various tyrosine kinase inhibitors (TKIs) are under clinical use, but acquisition of resistance to these drugs is a major problem. Here, we studied the interaction between two drug-resistant mutants of fibroblast growth factor receptor 1 (FGFR1), N546K and V561M, and four ATP-competitive inhibitors, ponatinib, dovitinib, PD173074 and BGJ-398. Among these protein-drug systems, the only marked reduction in affinity was that of PD173074 for the V561M mutant. We also examined the interaction of these FGFR1 variants to AMP-PNP, a nonhydrolyzable analogue of ATP, and showed that N546K showed increased affinity for the ATP analogue as compared with the wild type. These findings will help to clarify the mechanism of drug resistance in mutant tyrosine kinases.

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“…One such method that can resolve local (near amino acid) perturbations in stability in arbitrarily large proteins is hydrogen/deuterium-exchange mass spectrometry (HDX-MS) (11)(12)(13)(14)(15)(16). Moreover, these measurements are made in solution without the need to trap specific conformers, which has been used to answer questions of epitope mapping (17), protein-drug binding (18), protein-protein interactions (19), aggregation (20), effects of mutation (12), and allosteric regulation (21).…”

Section: Introductionmentioning

confidence: 99%

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Kish

Smith

Subramanian

et al. 2019

Preprint

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Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. The sensitivity of these measurements discerned that the 250's loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. Significance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability we provide direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop. Results Fully-automated and flexible pulse-labeling hydrogen/deuterium-exchange mass spectrometry with millisecond precisionOur goal was to quantify the structural switch in solution, along with other coincident dynamic changes in structure between activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of GlyP. To enable this, we developed a fully automated broadband bottom-up HDX-MS approach capable of accurate quantitative assessment of peptide stability. The newly developed ms2min system design ( Figure 1A) offers certain significant advances over the other designs previously employed. Notably, it allows fully-automated, software-selection of mixing times over six orders of magnitude (ms to hours) with 1 ms time resolution, flexible labeling temperature control (0 -25 C), quench temperature control (0 C), on-line connection for 'bottom-up' workflows, two-way communication for reciprocal control of labeling, washing, digestion, desalting and chromatography, automated digestion column wash injection, intercalated blank injections and sample list scheduling of multiple runs. Precision of D-labeling.To evaluate the measurement variability and labeling precision of the system we determined the repeatability of the quench-flow labeling method. On three separate days we measured the D uptake of 50, 70 and 150 replicates of Bradykinin and Leucine enkephalin at 100 ms mixing time. The short labeling period close to the limit of quantification, wa...

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Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase

Cited by 63 publications

References 66 publications

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Biophysical characterization of drug‐resistant mutants of fibroblast growth factor receptor 1

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

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