Structural basis for KCNE3 modulation of potassium recycling in epithelia

The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”

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Role of the Fourth Transmembrane Segment in TRAAK Channel Mechanosensitivity

10.1101/463034 ◽

2018 ◽

Author(s):

Mingfeng Zhang ◽

Fuqiang Yao ◽

Chengfang Pan ◽

Zhiqiang Yan

Keyword(s):

Ion Channels ◽

Transmembrane Domain ◽

Mechanical Force ◽

Dynamic Interactions ◽

Functional Roles ◽

Physiological Processes ◽

Gating Mechanism ◽

Voltage Gated Ion Channels ◽

Voltage Sensing Domain

AbstractMechanosensitive ion channels such as Piezo, TRAAK, TRPs and OSCA are important transmembrane proteins that are involved in many physiological processes such as touch, hearing and blood pressure regulation. Unlike ligand-gated channels or voltage-gated ion channels, which have a canonical ligand-binding domain or voltage-sensing domain, the mechanosensitive domain and related gating mechanism remain elusive. TRAAK channels are mechanosensitive channels that convert a physical mechanical force into a flow of potassium ions. The structures of TRAAK channels have been solved, however, the functional roles of the structural domains associated with channel mechanosensitivity remains unclear. Here, we generated a series of chimeric mutations between TRAAK and a non-mechanosensitive silent TWIK-1 K2P channel. We found that the selectivity filter region functions as the major gate of outward rectification and found that lower part of fourth transmembrane domain (M4) is necessary for TRAAK channel mechanosensitivity. We further demonstrated that upper part of M4 can modulate the mechanosensitivity of TRAAK channel. Furthermore, we found that hydrophilic substitutions of W262 and F121 facing each other, and hydrophobic substitutions of Q258 and G124, which are above and below W262 and F121, respectively, greatly increase mechanosensitivity, which suggests that dynamic interactions in the upper part of M4 and PH1 domain are involved in TRAAK channel mechanosensitivity. Interestingly, these gain-of-function mutations are sensitive to cell-poking stimuli, indicating that cell-poking stimuli generate a low membrane mechanical force that opens TRAAK channels. Our results thus showed that fourth transmembrane domain of TRAAK is critical for the gating of TRAAK by mechanical force and suggested that multiple dynamic interactions in the upper part of M4 and PH1 domain are involved in this process.

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Faculty Opinions recommendation of Structural basis of p75 transmembrane domain dimerization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726273130.793520822 ◽

2016 ◽

Author(s):

Mark Bothwell

Keyword(s):

Transmembrane Domain ◽

Structural Basis

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Structural Basis for the C-Terminal Domain of Mycobacterium tuberculosis Ribosome Maturation Factor RimM to Bind Ribosomal Protein S19

Biomolecules ◽

10.3390/biom11040597 ◽

2021 ◽

Vol 11 (4) ◽

pp. 597

Author(s):

Haoran Zhang ◽

Qiuxiang Zhou ◽

Chenyun Guo ◽

Liubin Feng ◽

Huilin Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Ribosomal Protein ◽

Solution Structure ◽

Molecular Mechanisms ◽

Structural Model ◽

Ribosomal Subunit ◽

Structural Basis ◽

Hydrophobic Core ◽

Terminal Domain ◽

Ribosomal Protein S19

Multidrug-resistant tuberculosis (TB) is a serious threat to public health, calling for the development of new anti-TB drugs. Chaperon protein RimM, involved in the assembly of ribosomal protein S19 into 30S ribosomal subunit during ribosome maturation, is a potential drug target for TB treatment. The C-terminal domain (CTD) of RimM is primarily responsible for binding S19. However, both the CTD structure of RimM from Mycobacterium tuberculosis (MtbRimMCTD) and the molecular mechanisms underlying MtbRimMCTD binding S19 remain elusive. Here, we report the solution structure, dynamics features of MtbRimMCTD, and its interaction with S19. MtbRimMCTD has a rigid hydrophobic core comprised of a relatively conservative six-strand β-barrel, tailed with a short α-helix and interspersed with flexible loops. Using several biophysical techniques including surface plasmon resonance (SPR) affinity assays, nuclear magnetic resonance (NMR) assays, and molecular docking, we established a structural model of the MtbRimMCTD–S19 complex and indicated that the β4-β5 loop and two nonconserved key residues (D105 and H129) significantly contributed to the unique pattern of MtbRimMCTD binding S19, which might be implicated in a form of orthogonality for species-dependent RimM–S19 interaction. Our study provides the structural basis for MtbRimMCTD binding S19 and is beneficial to the further exploration of MtbRimM as a potential target for the development of new anti-TB drugs.

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Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion

Science Advances ◽

10.1126/sciadv.abf3178 ◽

2021 ◽

Vol 7 (10) ◽

pp. eabf3178

Author(s):

Yuhang Liu ◽

Kyle P. Heim ◽

Ye Che ◽

Xiaoyuan Chi ◽

Xiayang Qiu ◽

...

Keyword(s):

Membrane Fusion ◽

Human Cytomegalovirus ◽

Transmembrane Domain ◽

Proximal Region ◽

Viral Envelope ◽

Vaccine Antigen ◽

Glycoprotein B ◽

Structural Basis ◽

Fusion Inhibitor ◽

Host Cell Membrane

Human cytomegalovirus (HCMV) causes congenital disease with long-term morbidity. HCMV glycoprotein B (gB) transitions irreversibly from a metastable prefusion to a stable postfusion conformation to fuse the viral envelope with a host cell membrane during entry. We stabilized prefusion gB on the virion with a fusion inhibitor and a chemical cross-linker, extracted and purified it, and then determined its structure to 3.6-Å resolution by electron cryomicroscopy. Our results revealed the structural rearrangements that mediate membrane fusion and details of the interactions among the fusion loops, the membrane-proximal region, transmembrane domain, and bound fusion inhibitor that stabilized gB in the prefusion state. The structure rationalizes known gB antigenic sites. By analogy to successful vaccine antigen engineering approaches for other viral pathogens, the high-resolution prefusion gB structure provides a basis to develop stabilized prefusion gB HCMV vaccine antigens.

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Structural basis for the dominant or recessive character of GLIALCAM mutations found in leukodystrophies

Human Molecular Genetics ◽

10.1093/hmg/ddaa009 ◽

2020 ◽

Vol 29 (7) ◽

pp. 1107-1120 ◽

Cited By ~ 2

Author(s):

Xabier Elorza-Vidal ◽

Efren Xicoy-Espaulella ◽

Adrià Pla-Casillanis ◽

Marta Alonso-Gardón ◽

Héctor Gaitán-Peñas ◽

...

Keyword(s):

Structural Model ◽

Clinical Phenotype ◽

Structural Basis ◽

Recessive Character ◽

Adhesion Protein ◽

Recessive Mutations ◽

Cross Links ◽

Interacting Surfaces ◽

Ig Domain ◽

Subcortical Cysts

Abstract Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of leukodystrophy characterized by white matter edema, and it is caused mainly by recessive mutations in MLC1 and GLIALCAM genes. These variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. In addition, dominant mutations in GLIALCAM have also been identified in a subtype of MLC patients with a remitting phenotype. This variant has been named MLC2B. GLIALCAM encodes for an adhesion protein containing two immunoglobulin (Ig) domains and it is needed for MLC1 targeting to astrocyte–astrocyte junctions. Most mutations identified in GLIALCAM abolish GlialCAM targeting to junctions. However, it is unclear why some mutations behave as recessive or dominant. Here, we used a combination of biochemistry methods with a new developed anti-GlialCAM nanobody, double-mutants and cysteine cross-links experiments, together with computer docking, to create a structural model of GlialCAM homo-interactions. Using this model, we suggest that dominant mutations affect different GlialCAM–GlialCAM interacting surfaces in the first Ig domain, which can occur between GlialCAM molecules present in the same cell (cis) or present in neighbouring cells (trans). Our results provide a framework that can be used to understand the molecular basis of pathogenesis of all identified GLIALCAM mutations.

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Structural Basis for Dimerization of the BNIP3 Transmembrane Domain,

Biochemistry ◽

10.1021/bi802245u ◽

2009 ◽

Vol 48 (23) ◽

pp. 5106-5120 ◽

Cited By ~ 39

Author(s):

Endah S. Sulistijo ◽

Kevin R. MacKenzie

Keyword(s):

Transmembrane Domain ◽

Structural Basis

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Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Molecular Endocrinology ◽

10.1210/me.2011-1160 ◽

2011 ◽

Vol 25 (10) ◽

pp. 1804-1818 ◽

Cited By ~ 37

Author(s):

K. Coopman ◽

R. Wallis ◽

G. Robb ◽

A. J. H. Brown ◽

G. F. Wilkinson ◽

...

Keyword(s):

Ligand Binding ◽

Structural Model ◽

Transmembrane Domain ◽

Transmembrane Helix ◽

Receptor Activation ◽

Agonist Affinity ◽

Camp Generation ◽

N Terminus ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

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Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

The Journal of General Physiology ◽

10.1085/jgp.201611616 ◽

2016 ◽

Vol 148 (3) ◽

pp. 227-237 ◽

Cited By ~ 31

Author(s):

Sun-Joo Lee ◽

Feifei Ren ◽

Eva-Maria Zangerl-Plessl ◽

Sarah Heyman ◽

Anna Stary-Weinzinger ◽

...

Keyword(s):

Transmembrane Domain ◽

Membrane Lipids ◽

Inward Rectifier ◽

Primary Site ◽

Structural Basis ◽

Channel Activity ◽

Anionic Phospholipid ◽

X Ray ◽

Anionic Phospholipids ◽

Kir Channel

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL−) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL−. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL− binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL− binding and PIP2 sensitivity.

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Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched

10.1101/352443 ◽

2018 ◽

Cited By ~ 3

Author(s):

Yunxiao Zhang ◽

David P. Bulkley ◽

Kelsey J. Roberts ◽

Yao Xin ◽

Daniel E. Asarnow ◽

...

Keyword(s):

Plasma Membrane ◽

Basal Cell Carcinoma ◽

Cell Carcinoma ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Extracellular Domain ◽

Structural Basis ◽

Cholesterol Transport ◽

Common Cause ◽

Common Receptor

AbstractHedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

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