Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight ...different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.
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•FGFR3 TM domains associate in a left-handed parallel helical dimer via a heptad motif•The solved NMR structure likely corresponds to the FGFR3 basal phosphorylation state•Some, but not all, pathogenic mutations fall within the FGFR3 TM dimer interface•An FGFR3 signaling mechanism based on two alternative TM dimerization modes is proposed
The transmembrane domain (TMD) of FGFR3 is notorious for housing different mutations linked to diverse human pathologies. Bocharov et al. describe the dimerization of TMD and solve an NMR structure to provide testable hypotheses about the mechanism of signaling and the effects of pathogenic mutations on function.
Alzheimer’s disease is the most common type of neurodegenerative disease in the world. Genetic evidence strongly suggests that aberrant generation, aggregation, and/or clearance of neurotoxic ...amyloid-β peptides (Aβ) triggers the disease. Aβ accumulates at the points of contact of neurons in ordered cords and fibrils, forming the so-called senile plaques. Aβ isoforms of different lengths are found in healthy human brains regardless of age and appear to play a role in signaling pathways in the brain and to have neuroprotective properties at low concentrations. In recent years, different substances have been developed targeting Aβ production, aggregation, interaction with other molecules, and clearance, including peptide-based drugs. Aβ is a product of sequential cleavage of the membrane glycoprotein APP (amyloid precursor protein) by β- and γ-secretases. A number of familial mutations causing an early onset of the disease have been identified in the APP, especially in its transmembrane domain. The mutations are reported to influence the production, oligomerization, and conformational behavior of Aβ peptides. This review highlights the results of structural studies of the main proteins involved in Alzheimer’s disease pathogenesis and the molecular mechanisms by which perspective therapeutic substances can affect Aβ production and nucleation.
► We determined the left-handed dimeric structure of transmembrane domain of amyloid precursor protein. ► The dimerization mechanism of APP is important for understanding the molecular mechanism of ...production of amyloid-β peptide. ► Familial mutations of amyloid precursor protein can affect its lateral dimerization.
Some pathogenic mutations associated with Alzheimer’s disease are thought to affect structural-dynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln686-Lys726 was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy. The APP membrane-spanning α-helix Lys699-Lys724 self-associates in a left-handed parallel dimer through extended heptad repeat motif I702X3M706X2G709X3A713X2I716X3I720X2I723, whereas the juxtamembrane region Gln686-Val695 constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-β peptide.
APPjmtm and APPjmtmbind by comigration in gel electrophoresis (View interaction)
APPjmtm and APPjmtmbind by nuclear magnetic resonance (View interaction).
Integral membrane proteins are important components of a cell. Their structural and functional studies require production of milligram amounts of proteins, which nowadays is not a routine process. ...Cell-free protein synthesis is a prospective approach to resolve this task. However, there are few known membrane mimetics that can be used to synthesize active membrane proteins in high amounts. Here, we present the application of commercially available “Facade” detergents for the production of active rhodopsin. We show that the yield of active protein in lipid bicelles containing Facade-EM, Facade-TEM, and Facade-EPC is several times higher than in the case of conventional bicelles with CHAPS and DHPC and is comparable to the yield in the presence of lipid-protein nanodiscs. Moreover, the effects of the lipid-to-detergent ratio, concentration of detergent in the feeding mixture, and lipid composition of the bicelles on the total, soluble, and active protein yields are discussed. We show that Facade-based bicelles represent a prospective membrane mimetic, available for the production of membrane proteins in a cell-free system.
The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually ...large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.
Dysregulation of post‐translational modifications (PTMs) like phosphorylation is often involved in disease. NMR may elucidate exact loci and time courses of PTMs at atomic resolution and ...near‐physiological conditions but requires signal assignment to individual atoms. Conventional NMR methods for this base on tedious global signal assignment that may often fail, as for large intrinsically disordered proteins (IDPs). We present a sensitive, robust alternative to rapidly obtain only the local assignment near affected signals, based on FOcused SpectroscopY (FOSY) experiments using selective polarisation transfer (SPT). We prove its efficiency by identifying two phosphorylation sites of glycogen synthase kinase 3 beta (GSK3β) in human Tau40, an IDP of 441 residues, where the extreme spectral dispersion in FOSY revealed unprimed phosphorylation also of Ser409. FOSY may broadly benefit NMR studies of PTMs and other hotspots in IDPs, including sites involved in molecular interactions.
A new suite of 2D FOSY experiments focuses on one coupled nuclear spin system at a time, that is, a PTM site or a structural hotspot in general, with 3–5 known frequencies. With higher sensitivity and versatility than achievable by traditional experiments, FOSY solves the spectral dispersion problem and obtains only the relevant local NMR signal assignment in a few hours, as demonstrated for two phosphorylation sites in the 441‐residue IDP Tau.
The epidermal growth factor receptor (EGFR) family is an important class of receptor tyrosine kinases, mediating a variety of cellular responses in normal biological processes and in pathological ...states of multicellular organisms. Different modes of dimerization of the human EGFR transmembrane domain (TMD) in different membrane mimetics recently prompted us to propose a novel signal transduction mechanism based on protein–lipid interaction. However, the experimental evidence for it was originally obtained with slightly different TMD fragments used in the two different mimetics, compromising the validity of the comparison. To eliminate ambiguity, we determined the nuclear magnetic resonance (NMR) structure of the bicelle-incorporated dimer of the EGFR TMD fragment identical to the one previously used in micelles. The NMR results augmented by molecular dynamics simulations confirm the mutual influence of the TMD and lipid environment, as is required for the proposed lipid-mediated activation mechanism. They also reveal the possible functional relevance of a subtle interplay between the concurrent processes in the lipid and protein during signal transduction.
The human epidermal growth factor receptor (EGFR) of HER/ErbB receptor tyrosine kinase family mediates a broad spectrum of cellular responses transducing biochemical signals via lateral dimerization ...in plasma membrane, while inactive receptors can exist in both monomeric and dimeric forms. Recently, the dimeric conformation of the helical single-span transmembrane domains of HER/ErbB employing the relatively polar N-terminal motifs in a fashion permitting proper kinase activation was experimentally determined. Here we describe the EGFR transmembrane domain dimerization via an alternative weakly polar C-terminal motif A661xxxG665 presumably corresponding to the inactive receptor state. During association, the EGFR transmembrane helices undergo a structural adjustment with adaptation of inter-molecular polar and hydrophobic interactions depending upon the surrounding membrane properties that directly affect the transmembrane helix packing. This might imply that signal transduction through membrane and allosteric regulation are inclusively mediated by coupled protein–protein and protein–lipid interactions, elucidating paradoxically loose linkage between ligand binding and kinase activation.
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•EGFR TM domain dimerizes in the micellar environment via C-terminal A661xxxG665 motif•Resolved NMR structure of the dimer likely corresponds to inactive receptor state•Alternative TM domain packing of EGFR is governed by surrounding membrane properties•EGFR signaling and allosteric regulation are modulated by protein–lipid interactions
Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed ...that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.
Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors ...are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed α-helical bundle through their N-terminal double GG4-like motif T
648G
649X
2G
652A
653 and glycine zipper motif T
652X
3S
656X
3G
660, respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix–helix interaction to signal transduction.
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