Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.Peer reviewe

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.</p

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.</p

Investigation of the Structural and Functional Dynamics of the TIM23 Protein Import Machinery

Archival Abstract Submitte

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties

Acknowledgements: The authors thank I. Chen, A. Gunnarson, E. Rhoades, R. Kriwacki, A. Elazar, and B. Lang for reading and critical comments on the manuscript; A. Carter and C. Lau for helping with protein purification; E. Derivery, J. Watson, and H. McMahon for helping with GUV construction; and Y. Ohashi for kindly providing the yeast strain for imaging. The authors thank T. Boothby for providing the original plasmid hosting tardigrade proteins. They thank J. Lu, G. Slodkowicz, Z. Shi, and Y. Yagita for helpful discussions on statistical analysis and interpretation of the data. They also thank Diamond Light Source for Beamtime (proposal SM24985) and the staff of Beamlines B21 for assistance with SAXS data collection; S. Mclaughlin and LMB Biophysics Instrument Centre for assistance in CD and fluorescence data collection; J. Howe and the LMB light microscopy team for assistance in microscopy; M. Daly and the LMB cell sorting facility for assistance in flow cytometry; LMB Scientific Computing for providing computational resources for simulation; and LMB media and glass wash for helping to prepare media and plates.Funder: United Kingdom Research and Innovation; DOI: https://doi.org/10.13039/100014013A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein–membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties.

Acknowledgements: The authors thank I. Chen, A. Gunnarson, E. Rhoades, R. Kriwacki, A. Elazar, and B. Lang for reading and critical comments on the manuscript; A. Carter and C. Lau for helping with protein purification; E. Derivery, J. Watson, and H. McMahon for helping with GUV construction; and Y. Ohashi for kindly providing the yeast strain for imaging. The authors thank T. Boothby for providing the original plasmid hosting tardigrade proteins. They thank J. Lu, G. Slodkowicz, Z. Shi, and Y. Yagita for helpful discussions on statistical analysis and interpretation of the data. They also thank Diamond Light Source for Beamtime (proposal SM24985) and the staff of Beamlines B21 for assistance with SAXS data collection; S. Mclaughlin and LMB Biophysics Instrument Centre for assistance in CD and fluorescence data collection; J. Howe and the LMB light microscopy team for assistance in microscopy; M. Daly and the LMB cell sorting facility for assistance in flow cytometry; LMB Scientific Computing for providing computational resources for simulation; and LMB media and glass wash for helping to prepare media and plates.Funder: United Kingdom Research and Innovation; DOI: https://doi.org/10.13039/100014013A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life

Dynamic conformational changes of a tardigrade group-3 late embryogenesis abundant protein modulate membrane biophysical properties

Acknowledgements: The authors thank I. Chen, A. Gunnarson, E. Rhoades, R. Kriwacki, A. Elazar, and B. Lang for reading and critical comments on the manuscript; A. Carter and C. Lau for helping with protein purification; E. Derivery, J. Watson, and H. McMahon for helping with GUV construction; and Y. Ohashi for kindly providing the yeast strain for imaging. The authors thank T. Boothby for providing the original plasmid hosting tardigrade proteins. They thank J. Lu, G. Slodkowicz, Z. Shi, and Y. Yagita for helpful discussions on statistical analysis and interpretation of the data. They also thank Diamond Light Source for Beamtime (proposal SM24985) and the staff of Beamlines B21 for assistance with SAXS data collection; S. Mclaughlin and LMB Biophysics Instrument Centre for assistance in CD and fluorescence data collection; J. Howe and the LMB light microscopy team for assistance in microscopy; M. Daly and the LMB cell sorting facility for assistance in flow cytometry; LMB Scientific Computing for providing computational resources for simulation; and LMB media and glass wash for helping to prepare media and plates.Funder: United Kingdom Research and Innovation; DOI: https://doi.org/10.13039/100014013A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein–membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life

Regulation of Kinase Activity in the Caenorhabditis elegans EGF Receptor, LET-23

In the active HER receptor dimers, kinases play distinct roles; one is the catalytically active kinase and the other is its allosteric activator. This specialization enables signaling by the catalytically inactive HER3, which functions exclusively as an allosteric activator upon heterodimerization with other HER receptors. It is unclear whether the allosteric activation mechanism evolved before HER receptors functionally specialized. We determined the crystal structure of the kinase domain of the only EGF receptor in Caenorhabditis elegans, LET-23. Our structure of a non-human EGFR kinase reveals autoinhibitory features conserved in the human counterpart. Strikingly, mutations within the putative allosteric dimer interface abrogate activity of the isolated LET-23 kinase and of the full-length receptor despite these regions being only partially conserved with human EGFR. Our results indicate that ancestral EGFRs have built-in features that poise them for allosteric activation that could facilitate emergence of the catalytically dead, yet functional, orthologs