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

The 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.

[1]  D. Svergun,et al.  A practical guide to small angle X‐ray scattering (SAXS) of flexible and intrinsically disordered proteins , 2015, FEBS letters.

[2]  Patricia Grob,et al.  Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor , 2015, Molecular and Cellular Biology.

[3]  D. Weis,et al.  Mapping Residual Structure in Intrinsically Disordered Proteins at Residue Resolution Using Millisecond Hydrogen/Deuterium Exchange and Residue Averaging , 2015, Journal of The American Society for Mass Spectrometry.

[4]  V. Uversky,et al.  Presence and utility of intrinsically disordered regions in kinases. , 2014, Molecular bioSystems.

[5]  P. Tompa,et al.  Introducing protein intrinsic disorder. , 2014, Chemical reviews.

[6]  J. Schlessinger,et al.  The EGFR family: not so prototypical receptor tyrosine kinases. , 2014, Cold Spring Harbor perspectives in biology.

[7]  John G. Koland,et al.  Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation , 2014, PLoS Comput. Biol..

[8]  Christopher Bystroff,et al.  The designability of protein switches by chemical rescue of structure: mechanisms of inactivation and reactivation. , 2013, Journal of the American Chemical Society.

[9]  M. J. Chalmers,et al.  Time Window Expansion for HDX Analysis of an Intrinsically Disordered Protein , 2013, Journal of The American Society for Mass Spectrometry.

[10]  D. Sept,et al.  Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface* ♦ , 2013, The Journal of Biological Chemistry.

[11]  K. Gajiwala EGFR: Tale of the C‐terminal tail , 2013, Protein science : a publication of the Protein Society.

[12]  Vladimir N Uversky,et al.  The most important thing is the tail: Multitudinous functionalities of intrinsically disordered protein termini , 2013, FEBS letters.

[13]  D. Weis,et al.  Analysis of disordered proteins using a simple apparatus for millisecond quench-flow H/D exchange. , 2013, Analytical chemistry.

[14]  Huan‐Xiang Zhou,et al.  Rate constants and mechanisms of intrinsically disordered proteins binding to structured targets. , 2012, Physical chemistry chemical physics : PCCP.

[15]  Derek J. Wilson,et al.  Measuring dynamics in weakly structured regions of proteins using microfluidics-enabled subsecond H/D exchange mass spectrometry. , 2012, Analytical chemistry.

[16]  P. Tompa,et al.  Intrinsic disorder in cell signaling and gene transcription , 2012, Molecular and Cellular Endocrinology.

[17]  D. Higgins,et al.  Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega , 2011, Molecular systems biology.

[18]  David D Weis,et al.  Mapping unstructured regions and synergistic folding in intrinsically disordered proteins with amide H/D exchange mass spectrometry. , 2011, Biochemistry.

[19]  Yoshikazu Ohta,et al.  Structural Analysis of the Mechanism of Inhibition and Allosteric Activation of the Kinase Domain of HER2 Protein , 2011, The Journal of Biological Chemistry.

[20]  Lars Konermann,et al.  Hydrogen exchange mass spectrometry for studying protein structure and dynamics. , 2011, Chemical Society reviews.

[21]  C. Ebel,et al.  Analytical Ultracentrifugation, a Useful Tool to Probe Intrinsically Disordered Proteins , 2010 .

[22]  F. White,et al.  EGFRvIV: a previously uncharacterized oncogenic mutant reveals a kinase autoinhibitory mechanism , 2010, Oncogene.

[23]  John Kuriyan,et al.  Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3 , 2009, Proceedings of the National Academy of Sciences.

[24]  R. Bose,et al.  Her4 and Her2/neu Tyrosine Kinase Domains Dimerize and Activate in a Reconstituted in Vitro System* , 2009, The Journal of Biological Chemistry.

[25]  James R Faeder,et al.  Toward a quantitative theory of intrinsically disordered proteins and their function , 2009, Proceedings of the National Academy of Sciences.

[26]  Yongqi Huang,et al.  Kinetic advantage of intrinsically disordered proteins in coupled folding-binding process: a critical assessment of the "fly-casting" mechanism. , 2009, Journal of molecular biology.

[27]  John A. Tainer,et al.  Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS) , 2009, Nature Methods.

[28]  H. Dyson,et al.  Prediction of the rotational tumbling time for proteins with disordered segments. , 2009, Journal of the American Chemical Society.

[29]  M. Bolognesi,et al.  Function and Structure of Inherently Disordered Proteins This Review Comes from a Themed Issue on Proteins Edited Prediction of Non-folding Proteins and Regions Frequency of Disordered Regions Protein Evolution Partitioning Unstructured Proteins and Regions into Groups Involvement of Inherently Diso , 2022 .

[30]  Christopher J. Oldfield,et al.  The unfoldomics decade: an update on intrinsically disordered proteins , 2008, BMC Genomics.

[31]  Christopher J. Oldfield,et al.  Intrinsically disordered proteins in human diseases: introducing the D2 concept. , 2008, Annual review of biophysics.

[32]  B. Wallace,et al.  Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. , 2008, Biopolymers.

[33]  Christopher J. Oldfield,et al.  Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners , 2008, BMC Genomics.

[34]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[35]  J. Ferrell,et al.  Mechanisms of specificity in protein phosphorylation , 2007, Nature Reviews Molecular Cell Biology.

[36]  Peter V. Konarev,et al.  ATSAS 2.1 – towards automated and web-supported small-angle scattering data analysis , 2007 .

[37]  Christopher J. Oldfield,et al.  Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins. , 2007, Journal of proteome research.

[38]  John Kuriyan,et al.  An Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor , 2006, Cell.

[39]  T. Hazlett,et al.  Structure and dynamics of the epidermal growth factor receptor C‐terminal phosphorylation domain , 2006, Protein science : a publication of the Protein Society.

[40]  Sonia Longhi,et al.  Assessing protein disorder and induced folding , 2005, Proteins.

[41]  Christopher J. Oldfield,et al.  Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling , 2005, Journal of molecular recognition : JMR.

[42]  Marc S. Cortese,et al.  Comparing and combining predictors of mostly disordered proteins. , 2005, Biochemistry.

[43]  Krystal J Alligood,et al.  A Unique Structure for Epidermal Growth Factor Receptor Bound to GW572016 (Lapatinib) , 2004, Cancer Research.

[44]  C. Redfield,et al.  Using nuclear magnetic resonance spectroscopy to study molten globule states of proteins. , 2004, Methods.

[45]  Lee Whitmore,et al.  DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data , 2004, Nucleic Acids Res..

[46]  M. Sliwkowski,et al.  Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex. , 2004, Cancer cell.

[47]  L. Iakoucheva,et al.  Intrinsic disorder in cell-signaling and cancer-associated proteins. , 2002, Journal of molecular biology.

[48]  V. Uversky,et al.  Denatured collapsed states in protein folding: Example of apomyoglobin , 2001, Proteins.

[49]  J. Schlessinger Cell Signaling by Receptor Tyrosine Kinases , 2000, Cell.

[50]  Benjamin A. Shoemaker,et al.  Speeding molecular recognition by using the folding funnel: the fly-casting mechanism. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[51]  P. Schuck,et al.  Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. , 2000, Biophysical journal.

[52]  M. Eisenstein,et al.  Analysis of lissencephaly-causing LIS1 mutations. , 1999, European journal of biochemistry.

[53]  M. Moran,et al.  Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation , 1997, Molecular and cellular biology.

[54]  O. Ptitsyn,et al.  Further evidence on the equilibrium "pre-molten globule state": four-state guanidinium chloride-induced unfolding of carbonic anhydrase B at low temperature. , 1996, Journal of molecular biology.

[55]  D. Svergun,et al.  CRYSOL : a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates , 1995 .

[56]  O. Ptitsyn,et al.  "Partly folded" state, a new equilibrium state of protein molecules: four-state guanidinium chloride-induced unfolding of beta-lactamase at low temperature. , 1994, Biochemistry.

[57]  S W Englander,et al.  Isotope effects in peptide group hydrogen exchange , 1993, Proteins.

[58]  Yawen Bai,et al.  Primary structure effects on peptide group hydrogen exchange , 1993, Biochemistry.

[59]  Zhongqi Zhang,et al.  Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation , 1993, Protein science : a publication of the Protein Society.

[60]  P. Wyatt Light scattering and the absolute characterization of macromolecules , 1993 .

[61]  W C Johnson,et al.  Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. , 1987, Analytical biochemistry.

[62]  W C Johnson,et al.  Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. , 1986, Analytical biochemistry.

[63]  N. Kallenbach,et al.  Hydrogen exchange and structural dynamics of proteins and nucleic acids , 1983, Quarterly Reviews of Biophysics.

[64]  C. Cantor,et al.  Biophysical Chemistry: Part II: Techniques for the Study of Biological Structure and Function , 1980 .

[65]  D. Svergun,et al.  Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering. , 2012, Molecular bioSystems.

[66]  Vladimir N Uversky,et al.  What does it mean to be natively unfolded? , 2002, European journal of biochemistry.

[67]  P. Romero,et al.  Sequence complexity of disordered protein , 2001, Proteins.

[68]  N. Sreerama,et al.  Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. , 2000, Analytical biochemistry.

[69]  T. Laue,et al.  Modern applications of analytical ultracentrifugation. , 1999, Annual review of biophysics and biomolecular structure.

[70]  Obradovic,et al.  Predicting Protein Disorder for N-, C-, and Internal Regions. , 1999, Genome informatics. Workshop on Genome Informatics.

[71]  Romero,et al.  Sequence Data Analysis for Long Disordered Regions Prediction in the Calcineurin Family. , 1997, Genome informatics. Workshop on Genome Informatics.

[72]  J. Schlessinger,et al.  Signaling by Receptor Tyrosine Kinases , 1993 .

[73]  For introductions and reviews , 1985 .

[74]  A. Hvidt,et al.  Hydrogen exchange in proteins. , 1966, Advances in protein chemistry.