A dimerization motif for transmembrane α–helices

Lemmon, Mark A.; Treutlein, Herbert; Adams, Paul D.; Brünger, A.T.; Engelman, Donald M.

doi:10.1038/nsb0394-157

Cited by 315 publications

(272 citation statements)

References 41 publications

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“…The EGFR transmembrane domain has been previously described to be critically involved in the communication between the ECD and the intracellular TKD. Therefore, the possible regulatory function of the TMD has received substantial attention (2,8,(33)(34)(35)(36)(37)(38). Two GxxxG dimerization motifs present on both ends of the TMD are supposed to regulate the association of ligand-free and ligandbound dimers (2,8).…”

Section: Resultsmentioning

confidence: 99%

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Kaszuba

Grzybek

Orłowski

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

147

156

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The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.R eceptor tyrosine kinases (RTKs) are cell surface receptors that receive and transduce signals mediating a variety of critical cellular processes, including cell growth, migration, proliferation, differentiation, and apoptosis. Among the many RTKs, the most studied is the epidermal growth factor receptor (EGFR), not least because of its involvement in the development and progression of epidermoid cancers and its resulting importance as a target for antineoplastic therapies.Structurally, the EGFR consists of the ectodomain (ECD) (further subdivided into four subdomains, DI-IV), the transmembrane domain (TMD), and the intracellular tyrosine kinase domain (TKD). Ligand binding induces conformational transitions of the ECD that stabilize receptor dimerization, culminating in the activation of the intracellular TKD and subsequent propagation of the activation signal (1). To prevent receptor activation and signaling in the absence of ligand, the structurally tethered ECD of monomeric EGFR blocks the intrinsic capacity of the TMD and the intracellular TKD to dimerize (2). Ligand binding is believed to release the self-inhibitory tether and facilitate receptor oligomerization and activation (3-6). A detailed understanding of the structural regulation of the intact full-length receptors in their native membranes promises to reveal the molecular basis for receptor regulation (7); however, the methodological limitations associated with crystallizing transmembrane proteins, together with the high flexibility of the full-length receptor, have prevented high-resolution crystallographic analysis.To fill this gap, extensive atom-scale molecular dynamics (MD) simulations were recently performed to elucidate the structural dynamics of the EGFR in a two-component lipid bilayer (8). These studies suggest a large interfacial contact area between the membrane and the ecto-and intracellular domains of the unli...

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Section: Resultsmentioning

confidence: 99%

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Kaszuba

Grzybek

Orłowski

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

147

156

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“…In an analysis of glycophorin A TM domain dimerization (31), the effects of single point mutations were used to identify a dimerization interface that was later confirmed by multiple substitutions (67) and by the NMR structure of the TM domain dimer (37). From our limited mutagenesis data, we identify residues His 173 , Ala 176 , and Gly 180 as participating in the BNIP3 TM domain dimer interface, with residue Gly 178 being located away from the interface.…”

Section: Discussionmentioning

confidence: 99%

Sequence-specific Dimerization of the Transmembrane Domain of the “BH3-only” Protein BNIP3 in Membranes and Detergent

Sulistijo

Jaszewski

MacKenzie

2003

Journal of Biological Chemistry

110

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Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-x L . We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM selfassociation in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala 176 and Gly 180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His 173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.The Bcl-2 superfamily of proteins plays a central role in regulating mitochondria-mediated apoptosis; the Bax subfamily promotes apoptosis, whereas the Bcl-2 subfamily protects against apoptosis (reviewed in Ref. 1). Protein-protein interactions between Bax and Bcl-2 subfamily members help determine cell fate (2, 3) and have accordingly been the subject of intensive study. Four regions of sequence homology, the BH1 through BH4 domains, contribute to the structure and function of these proteins, and the BH3 domain is particularly implicated in heterodimerization events (4 -9). Peptide and small molecule inhibitors of these protein-protein interactions can modulate apoptosis, further demonstrating the functional and pharmaceutical importance of these contacts (10 -13).Differentiation and development in metazoans requires celland signal-specific inputs to a functional Bcl-2/Bax checkpoint. The pro-apoptotic "BH3-only" proteins, which show homology to the Bcl-2 superfamily through the BH3 domain alone (14), are expressed or activated in specific tissues and in response to certain stimuli, making them candidates for connecting diverse signaling pathways to the ubiquitous apoptosis effector machinery (15, 16). BNIP3 (Bcl-2/19-kDa interacting protein 3) is a BH3-only protein whose expression and pro-apoptotic activity is induced following hyp...

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“…Helices M1, M2, and M3 are associated with one another and stabilised, in part, by G xxx G-like helical packing motifs between M1 and M3 (Extended Data Fig. 6a) 15 . The non-covalent association is strong enough that M1 and M2 remain associated with the core of the enzyme following proteolytic cleavage of the M2–M3 loop (Extended Data Fig.…”

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confidence: 99%

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Diver

Pedi

Koide

et al. 2018

Nature

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The maturation of Ras GTPases, and ~200 other cellular CaaX proteins, involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CaaX motif, proteolytic cleavage of the aaX residues, and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalyzed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryotic-specific integral membrane enzyme of the endoplasmic reticulum (ER)2. ICMT is the only cellular enzyme known to methylate prenylcysteine substrates; methylation is important for their biological functions, including the membrane localisations and subsequent activities of Ras1, prelamin A3, and Rab4. ICMT inhibition has potential for combating progeria3 and cancer5–8. Here we present an X-ray structure of ICMT, at 2.3 Å resolution, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for its cytosolic methyl donor, S-adenosyl-L-methionine (AdoMet), and for prenylcysteine substrates, which are associated with the ER membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localisations together in a membrane environment – a relatively uncharacterized, but defining feature of many integral membrane enzymes.

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A dimerization motif for transmembrane α–helices

Cited by 315 publications

References 41 publications

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Sequence-specific Dimerization of the Transmembrane Domain of the “BH3-only” Protein BNIP3 in Membranes and Detergent

Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

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