Measuring Dynamics in Weakly Structured Regions of Proteins Using Microfluidics-Enabled Subsecond H/D Exchange Mass Spectrometry

Rob, Tamanna; Liuni, Peter; Gill, P.; Zhu, Shaolong; Balachandran, Naresh; Berti, Paul J.; Wilson, Derek J.

doi:10.1021/ac300365u

Cited by 94 publications

(151 citation statements)

References 48 publications

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“…Microfluidic Device Fabrication-The microfluidic device was constructed as described previously (32,33). Briefly, the microfluidic channel design was generated in CorelDraw X3 (Corel Co., Ottawa, Ontario, Canada) and lasered onto blank precut polymethyl methacrylate (PMMA) 4 substrate (8.9 ϫ 3.8 ϫ 0.6 cm; Professional Plastics, Fullerton, CA).…”

Section: Methodsmentioning

confidence: 99%

Conformational Dynamics and Allostery in Pyruvate Kinase

Donovan

Zhu

Liuni

et al. 2016

Journal of Biological Chemistry

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Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogendeuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an ␣-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ␤-strands within the (␤/␣) 8 -barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

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Section: Methodsmentioning

confidence: 99%

Conformational Dynamics and Allostery in Pyruvate Kinase

Donovan

Zhu

Liuni

et al. 2016

Journal of Biological Chemistry

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“…Hydrogen/Deuterium Exchange Mass Spectrometry of EGFR/ ErbB Family RTKs-HDX-MS is a highly useful technique to identify and study IDRs (32)(33)(34)(35)(36). HDX-MS provides kinetic information about the exchange rate of amide hydrogen atoms along the backbone of a protein for solvent deuterons.…”

Section: Disorder Predictions On Egfr Her2mentioning

confidence: 99%

“…This rate of hydrogen/deuterium exchange is affected by factors such as the presence of strong hydrogen bonds, secondary structure, and tertiary structure (37)(38)(39)(40). IDRs show very rapid hydrogen/ deuterium exchange rates on the order of milliseconds to seconds (32)(33)(34)(35)(36). HDX-MS provides local information about different regions of the protein and readily distinguishes folded, globular regions from IDRs.…”

Section: Disorder Predictions On Egfr Her2mentioning

confidence: 99%

Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the Epidermal Growth Factor Receptor (EGFR) and HER3 Receptor Tyrosine Kinases

Keppel

Sarpong

Murray

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeThe epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases includes oncogenes important in the progression of breast and other cancers, and they are targets for many drug development strategies. Each member of the ErbB family possesses a unique, structurally uncharacterized C-terminal tail that plays an important role in autophosphorylation and signal propagation. To determine whether these C-terminal tails are intrinsically disordered regions, we conducted a battery of biophysical experiments on the EGFR and HER3 tails. Using hydrogen/deuterium exchange mass spectrometry, we measured the conformational dynamics of intracellular half constructs and compared the tails with the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared with the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size exclusion chromatography with multiangle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering each provide evidence that the EGFR and HER3 C-terminal tails are intrinsically disordered with extended, non-globular structure in solution. The intrinsic disorder and extended conformation of these tails may be important for their function by increasing the capture radius and reducing the thermodynamic barriers for binding of downstream signaling proteins. The epidermal growth factor receptor (EGFR)3 /ErbB family of receptor tyrosine kinases (RTKs) contains four member proteins: EGFR/ErbB1, HER2/ErbB2/neu, HER3/ErbB3, and HER4/ErbB4. These RTKs carry out important signaling functions via the sequential process of ligand binding by the extracellular domain, homo-or heterodimerization, activation of their intracellular kinase domain, and recruitment of downstream signaling proteins. These RTKs are also important oncogenic drivers in many breast, lung, and other human cancers (1). Several structural biology studies on the ErbB family have been published, and this has helped advance drug development for HER2-positive breast cancer (2). Protein crystallography studies published in 2004 showed the structure of pertuzumab bound to the extracellular domain of HER2 and lapatinib bound to the kinase domain of EGFR (3, 4). Since that time, growing structural biology-based understanding of how EGFR, HER2, and HER3 function at the atomic level has dramatically reshaped our understanding of RTKs (1, 2). Despite these advances, there is a domain in each EGFR/ErbB family protein for which little structural biology information is available; this domain is the C-terminal tail (CTT) domain. The CTTs contain numerous autophosphorylation sites that are essential for recruiting downstream signaling proteins and initiating intracellular signaling (5, 6). The CTT can also contribute to autoinhibition of the kinase domain of RTK (7). The lack of available crystallographic information on the CTT region of EGFR/ErbB fami...

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“…Mass spectrometry lends itself well to measurement of deuterium incorporation [10]. Previously, we and others have found that H/D exchange measurements on disordered proteins are more useful when the exchange process is measured on the millisecond timescale [11][12][13][14]. In a conventional H/D exchange experiment, the relative rates of deuterium incorporation in different parts of the protein can be estimated by subjecting the deuterated protein samples to rapid proteolysis under conditions that preserve the deuterium label [15].…”

mentioning

confidence: 99%

Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

Keppel

Weis

2014

J. Am. Soc. Mass Spectrom.

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Abstract. Measurement of residual structure in intrinsically disordered proteins can provide insights into the mechanisms by which such proteins undergo coupled binding and folding. The present work describes an approach to measure residual structure in disordered proteins using millisecond hydrogen/deuterium (H/D) exchange in a conventional bottom-up peptide-based workflow. We used the exchange mid-point, relative to a totally deuterated control, to quantify the rate of H/D exchange in each peptide. A weighted residue-by-residue average of these midpoints was used to map the extent of residual structure at near single-residue resolution. We validated this approach both by simulating a disordered protein and experimentally using the p300 binding domain of ACTR, a model disordered protein already wellcharacterized by other approaches. Secondary structure elements mapped in the present work are in good agreement with prior nuclear magnetic resonance measurements. The new approach was somewhat limited by a loss of spatial resolution and subject to artifacts because of heterogeneities in intrinsic exchange. Approaches to correct these limitations are discussed.

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Measuring Dynamics in Weakly Structured Regions of Proteins Using Microfluidics-Enabled Subsecond H/D Exchange Mass Spectrometry

Cited by 94 publications

References 48 publications

Conformational Dynamics and Allostery in Pyruvate Kinase

Conformational Dynamics and Allostery in Pyruvate Kinase

Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the Epidermal Growth Factor Receptor (EGFR) and HER3 Receptor Tyrosine Kinases

Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging

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