Regulation and structural aspects of the human Na+/H+ exchanger NHE1: Roles of the N-terminal region and interactions with lipids

Jens Vogensen

Regulation and structural aspects of the human Na+/H+ exchanger NHE1: Roles of the N-terminal region and interactions with lipids

Jens Vogensen

Abstract

The plasma membrane Na+/H+ exchanger NHE1 (SLC9A1) is vital in regulating intracellular pH (pHi) in essentially all cells studied, and its dysregulation has been linked to cancer and cardiovascular diseases. A key open question in the field is that no atomic structures are available for any mammalian SLC9 family member. Based on homology modeling and biochemical analyses, the current dogma dictates that the topology of NHE1 is comprised of a short N-terminal tail, a 12 transmembrane helix (TM) domain, and a long cytoplasmic C-terminal tail. Conflicting model predictions and the possibility of a putative cleaved signal peptide in NHE1s N-terminus, combined with a lack of understanding of the interplay between NHE1 and the lipid membrane in which it is embedded, led us to reason that the topology of NHE1 in the membrane warrants further investigations. Between TM1 and TM2, NHE1 has a long extracellular loop (EL1), which is the site of N- and O-linked glycosylations. The proximal C-terminal cytoplasmic tail, including the lipid interacting domain (LID), interacts with anionic lipids and other regulatory factors, and is suggested as the proton sensor site. The overall aim of this thesis work was to provide new insight into the structure and regulation of NHE1 and its interaction with the lipid membrane, through investigations of: (1) the NHE1 N-terminus and the putative cleaved signal peptide; (2) the structural basis of the multiple interacting dynamics of the LID domain (3) interactions of NHE1 with cholesterol and sphingomyelin; and (4) the structure of NHE1 through cryo-EM. In Paper I, we focus on the structural and functional roles of the NHE1 N-terminal and proximal TM region. Here, we describe the roles of the region up to and including EL1 in the membrane localization and function of NHE1 and discover that NHE1 has an N-terminal cleaved signal peptide which is reliant on the helicity of the TM1. Further studies are required to precisely identify the molecular mechanism of cleavage. In Paper II, we investigate the LID subdomain. Through a combination of nucelar magnetic resonance (NMR) and molecular dynamics (MD) we elucidate the structural conformation of this domain. LID forms an α-helical hairpin structure, in which the C-terminal helix interacts with, and imbeds, in the membrane, while the N-terminal helix primarily interacts with the C-terminal helix. Disruption of this LID:membrane co-structure by mutagenesis drastically reduces the activity of NHE1 when expressed in AP-1 cells. We suggest that this close association of LID to the membrane serves as a multiple sensor site vital for NHE1 regulation and activity. In Paper III, we investigate lipid interactions of the TM domain of NHE1, with a specific interest in cholesterol and sphingomyelin. Lipidomic analysis of lipids co-purified with the TM part of NHE1 did not reveal specific interactions, suggesting that interactions between the NHE1 TM domain and lipids may be of relatively low affinity. Two cholesterol (CRAC)-interacting motifs were identified, in TM4 and TM12 respectively. Mutation of the CRAC motif in TM4 strongly reduced NHE1 activity, whereas mutation of that in TM12 reduced membrane localization, and both activity and membrane localization were abolished in double mutants lacking both CRAC motifs.. Finally, in the preliminary results on NHE1 structure, I highlight our efforts toward achieving the first atomic structure of a mammalian SLC9A. Collectively, the work carried out has expanded our understanding of key structural and functional elements of NHE1 and their roles in regulating NHE1 localization and function, and has established the foundation for additional future analyses of NHE1 structure and membrane interactions.

Original language	English

Publisher	Department of Biology, Faculty of Science, University of Copenhagen
Publication status	Published - 2019

Access to Document

PHD-Jens VogensenFinal published version, 73.2 MB

Cite this

@phdthesis{eded5712212c47c58a74bd7b4575479d,

title = "Regulation and structural aspects of the human Na+/H+ exchanger NHE1: Roles of the N-terminal region and interactions with lipids",

abstract = "The plasma membrane Na+/H+ exchanger NHE1 (SLC9A1) is vital in regulating intracellular pH (pHi) in essentially all cells studied, and its dysregulation has been linked to cancer and cardiovascular diseases. A key open question in the field is that no atomic structures are available for any mammalian SLC9 family member. Based on homology modeling and biochemical analyses, the current dogma dictates that the topology of NHE1 is comprised of a short N-terminal tail, a 12 transmembrane helix (TM) domain, and a long cytoplasmic C-terminal tail. Conflicting model predictions and the possibility of a putative cleaved signal peptide in NHE1s N-terminus, combined with a lack of understanding of the interplay between NHE1 and the lipid membrane in which it is embedded, led us to reason that the topology of NHE1 in the membrane warrants further investigations. Between TM1 and TM2, NHE1 has a long extracellular loop (EL1), which is the site of N- and O-linked glycosylations. The proximal C-terminal cytoplasmic tail, including the lipid interacting domain (LID), interacts with anionic lipids and other regulatory factors, and is suggested as the proton sensor site. The overall aim of this thesis work was to provide new insight into the structure and regulation of NHE1 and its interaction with the lipid membrane, through investigations of: (1) the NHE1 N-terminus and the putative cleaved signal peptide; (2) the structural basis of the multiple interacting dynamics of the LID domain (3) interactions of NHE1 with cholesterol and sphingomyelin; and (4) the structure of NHE1 through cryo-EM. In Paper I, we focus on the structural and functional roles of the NHE1 N-terminal and proximal TM region. Here, we describe the roles of the region up to and including EL1 in the membrane localization and function of NHE1 and discover that NHE1 has an N-terminal cleaved signal peptide which is reliant on the helicity of the TM1. Further studies are required to precisely identify the molecular mechanism of cleavage. In Paper II, we investigate the LID subdomain. Through a combination of nucelar magnetic resonance (NMR) and molecular dynamics (MD) we elucidate the structural conformation of this domain. LID forms an α-helical hairpin structure, in which the C-terminal helix interacts with, and imbeds, in the membrane, while the N-terminal helix primarily interacts with the C-terminal helix. Disruption of this LID:membrane co-structure by mutagenesis drastically reduces the activity of NHE1 when expressed in AP-1 cells. We suggest that this close association of LID to the membrane serves as a multiple sensor site vital for NHE1 regulation and activity. In Paper III, we investigate lipid interactions of the TM domain of NHE1, with a specific interest in cholesterol and sphingomyelin. Lipidomic analysis of lipids co-purified with the TM part of NHE1 did not reveal specific interactions, suggesting that interactions between the NHE1 TM domain and lipids may be of relatively low affinity. Two cholesterol (CRAC)-interacting motifs were identified, in TM4 and TM12 respectively. Mutation of the CRAC motif in TM4 strongly reduced NHE1 activity, whereas mutation of that in TM12 reduced membrane localization, and both activity and membrane localization were abolished in double mutants lacking both CRAC motifs.. Finally, in the preliminary results on NHE1 structure, I highlight our efforts toward achieving the first atomic structure of a mammalian SLC9A. Collectively, the work carried out has expanded our understanding of key structural and functional elements of NHE1 and their roles in regulating NHE1 localization and function, and has established the foundation for additional future analyses of NHE1 structure and membrane interactions. ",

author = "Jens Vogensen",

year = "2019",

language = "English",

publisher = "Department of Biology, Faculty of Science, University of Copenhagen",

}

TY - BOOK

T1 - Regulation and structural aspects of the human Na+/H+ exchanger NHE1: Roles of the N-terminal region and interactions with lipids

AU - Vogensen, Jens

PY - 2019

Y1 - 2019

N2 - The plasma membrane Na+/H+ exchanger NHE1 (SLC9A1) is vital in regulating intracellular pH (pHi) in essentially all cells studied, and its dysregulation has been linked to cancer and cardiovascular diseases. A key open question in the field is that no atomic structures are available for any mammalian SLC9 family member. Based on homology modeling and biochemical analyses, the current dogma dictates that the topology of NHE1 is comprised of a short N-terminal tail, a 12 transmembrane helix (TM) domain, and a long cytoplasmic C-terminal tail. Conflicting model predictions and the possibility of a putative cleaved signal peptide in NHE1s N-terminus, combined with a lack of understanding of the interplay between NHE1 and the lipid membrane in which it is embedded, led us to reason that the topology of NHE1 in the membrane warrants further investigations. Between TM1 and TM2, NHE1 has a long extracellular loop (EL1), which is the site of N- and O-linked glycosylations. The proximal C-terminal cytoplasmic tail, including the lipid interacting domain (LID), interacts with anionic lipids and other regulatory factors, and is suggested as the proton sensor site. The overall aim of this thesis work was to provide new insight into the structure and regulation of NHE1 and its interaction with the lipid membrane, through investigations of: (1) the NHE1 N-terminus and the putative cleaved signal peptide; (2) the structural basis of the multiple interacting dynamics of the LID domain (3) interactions of NHE1 with cholesterol and sphingomyelin; and (4) the structure of NHE1 through cryo-EM. In Paper I, we focus on the structural and functional roles of the NHE1 N-terminal and proximal TM region. Here, we describe the roles of the region up to and including EL1 in the membrane localization and function of NHE1 and discover that NHE1 has an N-terminal cleaved signal peptide which is reliant on the helicity of the TM1. Further studies are required to precisely identify the molecular mechanism of cleavage. In Paper II, we investigate the LID subdomain. Through a combination of nucelar magnetic resonance (NMR) and molecular dynamics (MD) we elucidate the structural conformation of this domain. LID forms an α-helical hairpin structure, in which the C-terminal helix interacts with, and imbeds, in the membrane, while the N-terminal helix primarily interacts with the C-terminal helix. Disruption of this LID:membrane co-structure by mutagenesis drastically reduces the activity of NHE1 when expressed in AP-1 cells. We suggest that this close association of LID to the membrane serves as a multiple sensor site vital for NHE1 regulation and activity. In Paper III, we investigate lipid interactions of the TM domain of NHE1, with a specific interest in cholesterol and sphingomyelin. Lipidomic analysis of lipids co-purified with the TM part of NHE1 did not reveal specific interactions, suggesting that interactions between the NHE1 TM domain and lipids may be of relatively low affinity. Two cholesterol (CRAC)-interacting motifs were identified, in TM4 and TM12 respectively. Mutation of the CRAC motif in TM4 strongly reduced NHE1 activity, whereas mutation of that in TM12 reduced membrane localization, and both activity and membrane localization were abolished in double mutants lacking both CRAC motifs.. Finally, in the preliminary results on NHE1 structure, I highlight our efforts toward achieving the first atomic structure of a mammalian SLC9A. Collectively, the work carried out has expanded our understanding of key structural and functional elements of NHE1 and their roles in regulating NHE1 localization and function, and has established the foundation for additional future analyses of NHE1 structure and membrane interactions.

AB - The plasma membrane Na+/H+ exchanger NHE1 (SLC9A1) is vital in regulating intracellular pH (pHi) in essentially all cells studied, and its dysregulation has been linked to cancer and cardiovascular diseases. A key open question in the field is that no atomic structures are available for any mammalian SLC9 family member. Based on homology modeling and biochemical analyses, the current dogma dictates that the topology of NHE1 is comprised of a short N-terminal tail, a 12 transmembrane helix (TM) domain, and a long cytoplasmic C-terminal tail. Conflicting model predictions and the possibility of a putative cleaved signal peptide in NHE1s N-terminus, combined with a lack of understanding of the interplay between NHE1 and the lipid membrane in which it is embedded, led us to reason that the topology of NHE1 in the membrane warrants further investigations. Between TM1 and TM2, NHE1 has a long extracellular loop (EL1), which is the site of N- and O-linked glycosylations. The proximal C-terminal cytoplasmic tail, including the lipid interacting domain (LID), interacts with anionic lipids and other regulatory factors, and is suggested as the proton sensor site. The overall aim of this thesis work was to provide new insight into the structure and regulation of NHE1 and its interaction with the lipid membrane, through investigations of: (1) the NHE1 N-terminus and the putative cleaved signal peptide; (2) the structural basis of the multiple interacting dynamics of the LID domain (3) interactions of NHE1 with cholesterol and sphingomyelin; and (4) the structure of NHE1 through cryo-EM. In Paper I, we focus on the structural and functional roles of the NHE1 N-terminal and proximal TM region. Here, we describe the roles of the region up to and including EL1 in the membrane localization and function of NHE1 and discover that NHE1 has an N-terminal cleaved signal peptide which is reliant on the helicity of the TM1. Further studies are required to precisely identify the molecular mechanism of cleavage. In Paper II, we investigate the LID subdomain. Through a combination of nucelar magnetic resonance (NMR) and molecular dynamics (MD) we elucidate the structural conformation of this domain. LID forms an α-helical hairpin structure, in which the C-terminal helix interacts with, and imbeds, in the membrane, while the N-terminal helix primarily interacts with the C-terminal helix. Disruption of this LID:membrane co-structure by mutagenesis drastically reduces the activity of NHE1 when expressed in AP-1 cells. We suggest that this close association of LID to the membrane serves as a multiple sensor site vital for NHE1 regulation and activity. In Paper III, we investigate lipid interactions of the TM domain of NHE1, with a specific interest in cholesterol and sphingomyelin. Lipidomic analysis of lipids co-purified with the TM part of NHE1 did not reveal specific interactions, suggesting that interactions between the NHE1 TM domain and lipids may be of relatively low affinity. Two cholesterol (CRAC)-interacting motifs were identified, in TM4 and TM12 respectively. Mutation of the CRAC motif in TM4 strongly reduced NHE1 activity, whereas mutation of that in TM12 reduced membrane localization, and both activity and membrane localization were abolished in double mutants lacking both CRAC motifs.. Finally, in the preliminary results on NHE1 structure, I highlight our efforts toward achieving the first atomic structure of a mammalian SLC9A. Collectively, the work carried out has expanded our understanding of key structural and functional elements of NHE1 and their roles in regulating NHE1 localization and function, and has established the foundation for additional future analyses of NHE1 structure and membrane interactions.

M3 - Ph.D. thesis

BT - Regulation and structural aspects of the human Na+/H+ exchanger NHE1: Roles of the N-terminal region and interactions with lipids

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -