Membrane transporters allow the selective transport of otherwise poorly permeable solutes across the cell membrane and thus, play a key role in maintaining cellular homeostasis in all kingdoms of ...life. Importantly, these proteins also serve as important drug targets. Over the last decades, major progress in structural biology methods has elucidated important structure-function relationships in membrane transporters. However, structures obtained using methods such as X-ray crystallography and high-resolution cryogenic electron microscopy merely provide static snapshots of an intrinsically dynamic, multi-step transport process. Therefore, there is a growing need for developing new experimental approaches capable of exploiting the data obtained from the high-resolution snapshots in order to investigate the dynamic features of membrane proteins. Here, we present the basic principles of hydrogen-deuterium exchange mass-spectrometry (HDX-MS) and recent advancements in its use to study membrane transporters. In HDX-MS experiments, minute amounts of a protein sample can be used to investigate its structural dynamics under native conditions, without the need for chemical labelling and with practically no limit on the protein size. Thus, HDX-MS is instrumental for resolving the structure-dynamic landscapes of membrane proteins in their apo (ligand-free) and ligand-bound forms, shedding light on the molecular mechanism underlying the transport process and drug binding.
Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields. TTFields are a unique anti-mitotic treatment modality delivered in a continuous, noninvasive ...manner to the region of a tumor. It was previously postulated that by exerting directional forces on highly polar intracellular elements during mitosis, TTFields could disrupt the normal assembly of spindle microtubules. However there is limited evidence directly linking TTFields to an effect on microtubules. Here we report that TTFields decrease the ratio between polymerized and total tubulin, and prevent proper mitotic spindle assembly. The aberrant mitotic events induced by TTFields lead to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells. The effect of TTFields on cell viability and clonogenic survival substantially depends upon the cell division rate. We show that by extending the duration of exposure to TTFields, slowly dividing cells can be affected to a similar extent as rapidly dividing cells.
Human KCNH2 channels (hKCNH2, ether-à-go-go EAG-related gene, hERG) are best known for their contribution to cardiac action potential repolarization and have key roles in various pathologies. Like ...other KCNH family members, hKCNH2 channels contain a unique intracellular complex, consisting of an N-terminal eag domain and a C-terminal cyclic nucleotide-binding homology domain (CNBHD), which is crucial for channel function. Previous studies demonstrated that the CNBHD is occupied by an intrinsic ligand motif, in a self-liganded conformation, providing a structural mechanism for the lack of KCNH channel regulation by cyclic nucleotides. While there have been significant advancements in the structural and functional characterization of the CNBHD of KCNH channels, a high-resolution structure of the hKCNH2 intracellular complex has been missing. Here, we report the 1.5 Å resolution structure of the hKCNH2 channel CNBHD. The structure reveals the canonical fold shared by other KCNH family members, where the spatial organization of the intrinsic ligand is preserved within the β-roll region. Moreover, measurements of small-angle x-ray scattering profile in solution, as well as comparison with a recent NMR analysis of hKCNH2, revealed high agreement with the crystallographic structure, indicating an overall low flexibility in solution. Importantly, we identified a novel salt-bridge (E807-R863) which was not previously resolved in the NMR and cryo-EM structures. Electrophysiological analysis of charge-reversal mutations revealed the bridge's crucial role in hKCNH2 function. Moreover, comparison with other KCNH members revealed the structural conservation of this salt-bridge, consistent with its functional significance. Together with the available structure of the mouse KCNH1 intracellular complex and previous electrophysiological and spectroscopic studies of KCNH family members, we propose that this salt-bridge serves as a strategically positioned linchpin to support both the spatial organization of the intrinsic ligand and the maintenance of the intracellular complex interface.
Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100–300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether ...TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.
The membrane-bound sodium-calcium exchanger (NCX) proteins shape Ca
homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of ...the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen-deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α₁ and α₂ repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na⁺ or Ca
binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca
/cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins.
Members of the Ca2+/cation exchanger superfamily (Ca2+/CA) share structural similarities (including highly conserved ion-coordinating residues) while exhibiting differential selectivity for Ca2+, ...Na+, H+, K+, and Li+. The archaeal Na+/Ca2+ exchanger (NCX_Mj) and its mammalian orthologs are highly selective for Na+, whereas the mitochondrial ortholog (NCLX) can transport either Li+ or Na+ in exchange with Ca2+. Here, structure-based replacement of ion-coordinating residues in NCX_Mj resulted in a capacity for transporting either Na+ or Li+, similar to the case for NCLX. This engineered protein may serve as a model for elucidating the mechanisms underlying ion selectivity and ion-coupled alternating access in NCX and similar proteins.
Abstract
The human
cis
-prenyltransferase (h
cis
-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations ...in h
cis
-PT cause severe human diseases. Here, we reveal that h
cis
-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for h
cis
-PT dysfunction in retinitis pigmentosa. Together, our structure of the h
cis
-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.
Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' ...function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.
Na(+)/Ca(2+) exchanger (NCX) proteins extrude Ca(2+) from the cell to maintain cellular homeostasis. Since NCX proteins contribute to numerous physiological and pathophysiological events, their ...pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamics simulations and ion flux analyses, have assigned the ion binding sites for 3Na(+) and 1Ca(2+), which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca(2+)-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca(2+)-dependent regulation is ortholog, isoform, and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative, or no response to regulatory Ca(2+). The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca(2+)-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca(2+) (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium (45)Ca(2+) binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca(2+) binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca(2+) binding to CBD1 rigidifies local backbone segments of CBD2, but not of CBD1. The extent and strength of Ca(2+)-dependent rigidification at CBD2 is splice-variant dependent, showing clear correlations with phenotypes of matching NCX variants. Therefore, diverse NCX variants share a common mechanism for the initial decoding of the regulatory signal upon Ca(2+) binding at the interface of CBDs, whereas the allosteric message is shaped by CBD2, the dynamic features of which are dictated by the splicing segment.
The N-methyl-D-aspartate receptors (NMDARs; GluNRS) are glutamate receptors, commonly located at excitatory synapses. Mutations affecting receptor function often lead to devastating ...neurodevelopmental disorders. We have identified two toddlers with different heterozygous missense mutations of the same, and highly conserved, glycine residue located in the ligand-binding-domain of
GRIN2B
: G689C and G689S. Structure simulations suggest severely impaired glutamate binding, which we confirm by functional analysis. Both variants show three orders of magnitude reductions in glutamate EC
50
, with G689S exhibiting the largest reductions observed for
GRIN2B
(~2000-fold). Moreover, variants multimerize with, and upregulate, GluN2B
wt
-subunits, thus engendering a strong dominant-negative effect on mixed channels. In neurons, overexpression of the variants instigates suppression of synaptic GluNRs. Lastly, while exploring spermine potentiation as a potential treatment, we discovered that the variants fail to respond due to G689’s novel role in proton-sensing. Together, we describe two unique variants with extreme effects on channel function. We employ protein-stability measures to explain why current (and future) LBD mutations in GluN2B primarily instigate Loss-of-Function.
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