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

Insight into the Architecture of the NuRD Complex: Structure of the RbAp48-MTA1 Subcomplex

The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex

Hexameric ring structure of human MCM10 DNA replication factor

The DNA replication factor minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein crucial for origin firing. During the transition from pre-replicative complexes to pre-initiation complexes, MCM10 recruitment to replication origins is required to provide a physical link between the MCM2–7 complex DNA helicase and DNA polymerases. Here, we report the molecular structure of human MCM10 as determined by electron microscopy and single-particle analysis. The MCM10 molecule is a ring-shaped hexamer with large central and smaller lateral channels and a system of inner chambers. This structure, together with biochemical data, suggests that this important protein uses its architecture to provide a docking module for assembly of the molecular machinery required for eukaryotic DNA replication

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

Insight into the architecture of the NuRD complex: Structure of the RbAp48-MTA1 subcomplex

The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRDby determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex