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1. Flanking aromatic residue competition influences transmembrane peptide helix dynamics

   https://doi.org/10.1002/1873-3468.13926  

   McKay, Matthew J. ; Greathouse, Denise V. ; Koeppe, II, Roger E. ( September 2020 , FEBS Letters)

   To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW^4,20ALP23 (acetyl‐GGAW⁴A(LA)₆LAW²⁰AGA‐amide) were examined using deuterium NMR spectroscopy. It was found that L^5,19GW^4,20ALP23, a sequence isomer of the low to moderately dynamic GW^5,19ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F^4,5GW²⁰ALP23 restores a low level of dynamic averaging, similar to that of the F^4,5GW¹⁹ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.

    

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2. Protonation‐Driven Membrane Insertion of a pH‐Low Insertion Peptide

   https://doi.org/10.1002/ange.201605203  

   Hanz, Samuel Z. ; Shu, Nicolas S. ; Qian, Jieni ; Christman, Nathaniel ; Kranz, Patrick ; An, Ming ; Grewer, Christof ; Qiang, Wei ( August 2016 , Angewandte Chemie)

   The pH‐low insertion peptide (pHLIP) inserts into membranes and forms a transmembrane (TM) α‐helix in response to slight acidity, and has shown great potential for cancer diagnosis and treatment. As a lead, pHLIP is challenging to optimize because the mechanism of its pH‐dependent membrane interactions is not completely understood. Within pHLIP there are multiple D/E residues which could sense the pH change, the particular role played by each of them in the protonation‐driven insertion process is not clear. The precise location of the TM helix within the pHLIP sequence is also unknown. In this work, solid‐state NMR spectroscopy is used to address these central questions. Tracing backbone conformations revealed that the TM helix spans from A10 to D33 with a break at T19 to P20. Residue‐specific pK_avalues of D31, D33, D25, and D14 were determined to be 6.5, 6.3, 6.1, and 5.8, respectively, and define the sequence of protonations which lead to insertion. Furthermore, possible intermediate states which disrupt membranes at pH 6.4 were proposed based on tryptophan fluorescence quenching and NMR data.

    

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3. Structure and function of the calcium‐selective TRP channel TRPV6

   https://doi.org/10.1113/JP279024  

   Yelshanskaya, Maria V. ; Nadezhdin, Kirill D. ; Kurnikova, Maria G. ; Sobolevsky, Alexander I. ( March 2020 , The Journal of Physiology)

   Epithelial calcium channel TRPV6 is a member of the vanilloid subfamily of TRP channels that is permeable to cations and highly selective to Ca²⁺; it shows constitutive activity regulated negatively by Ca²⁺and positively by phosphoinositol and cholesterol lipids. In this review, we describe the molecular structure of TRPV6 and discuss how its structural elements define its unique functional properties. High Ca²⁺selectivity of TRPV6 originates from the narrow selectivity filter, where Ca²⁺ions are directly coordinated by a ring of anionic aspartate side chains. Divalent cations Ca²⁺and Ba²⁺permeate TRPV6 pore according to the knock‐off mechanism, while tight binding of Gd³⁺to the aspartate ring blocks the channel and prevents Na⁺from permeating the pore. The iris‐like channel opening is accompanied by an α‐to‐π helical transition in the pore‐lining transmembrane helix S6. As a result of this transition, the intracellular halves of the S6 helices bend and rotate by about 100 deg, exposing different residues to the channel pore in the open and closed states. Channel opening is also associated with changes in occupancy of the transmembrane domain lipid binding sites. The inhibitor 2‐aminoethoxydiphenyl borate (2‐APB) binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1‐S4 transmembrane helical bundle and shifts open‐closed channel equilibrium towards the closed state by outcompeting lipids critical for activation. Ca²⁺inhibits TRPV6 via binding to calmodulin (CaM), which mediates Ca²⁺‐dependent inactivation. The TRPV6‐CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head‐to‐tail arrangement. The CaM C‐terminal lobe plugs the channel through a unique cation‐π interaction by inserting the side chain of lysine K115 into a tetra‐tryptophan cage at the ion channel pore intracellular entrance. Recent studies of TRPV6 structure and function described in this review advance our understanding of the role of this channel in physiology and pathophysiology and inform new therapeutic design.image

    

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4. Studying Conformational Properties of Transmembrane Domain of KCNE3 in a Lipid Bilayer Membrane Using Molecular Dynamics Simulations

   https://doi.org/10.3390/membranes14020045  

   Moura, Anna Clara ; Asare, Isaac K. ; Cruz, Mateo Fernandez ; Aguado, Antonio Javier ; Tuck, Kaeleigh Dyan ; Campbell, Conner C. ; Scheyer, Matthew W. ; Obaseki, Ikponwmosa ; Alston, Steve ; Kravats, Andrea N. ; et al ( February 2024 , Membranes)

   KCNE3 is a single-pass integral membrane protein that regulates numerous voltage-gated potassium channel functions such as KCNQ1. Previous solution NMR studies suggested a moderate degree of curved α-helical structure in the transmembrane domain (TMD) of KCNE3 in lyso-myristoylphosphatidylcholine (LMPC) micelles and isotropic bicelles with the residues T71, S74 and G78 situated along the concave face of the curved helix. During the interaction of KCNE3 and KCNQ1, KCNE3 pushes its transmembrane domain against KCNQ1 to lock the voltage sensor in its depolarized conformation. A cryo-EM study of KCNE3 complexed with KCNQ1 in nanodiscs suggested a deviation of the KCNE3 structure from its independent structure in isotropic bicelles. Despite the biological significance of KCNE3 TMD, the conformational properties of KCNE3 are poorly understood. Here, all atom molecular dynamics (MD) simulations were utilized to investigate the conformational dynamics of the transmembrane domain of KCNE3 in a lipid bilayer containing a mixture of POPC and POPG lipids (3:1). Further, the effect of the interaction impairing mutations (V72A, I76A and F68A) on the conformational properties of the KCNE3 TMD in lipid bilayers was investigated. Our MD simulation results suggest that the KCNE3 TMD adopts a nearly linear α helical structural conformation in POPC-POPG lipid bilayers. Additionally, the results showed no significant change in the nearly linear α-helical conformation of KCNE3 TMD in the presence of interaction impairing mutations within the sampled time frame. The KCNE3 TMD is more stable with lower flexibility in comparison to the N-terminal and C-terminal of KCNE3 in lipid bilayers. The overall conformational flexibility of KCNE3 also varies in the presence of the interaction-impairing mutations. The MD simulation data further suggest that the membrane bilayer width is similar for wild-type KCNE3 and KCNE3 containing mutations. The Z-distance measurement data revealed that the TMD residue site A69 is close to the lipid bilayer center, and residue sites S57 and S82 are close to the surfaces of the lipid bilayer membrane for wild-type KCNE3 and KCNE3 containing interaction-impairing mutations. These results agree with earlier KCNE3 biophysical studies. The results of these MD simulations will provide complementary data to the experimental outcomes of KCNE3 to help understand its conformational dynamic properties in a more native lipid bilayer environment.

    

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5. Structural Modeling of Cytokine-Receptor-JAK2 Signaling Complexes Using AlphaFold Multimer

   https://doi.org/10.1021/acs.jcim.3c00926  

   Pogozheva, Irina D. ; Cherepanov, Stanislav ; Park, Sang-Jun ; Raghavan, Malini ; Im, Wonpil ; Lomize, Andrei L. ( September 2023 , Journal of Chemical Information and Modeling)

   Homodimeric class 1 cytokine receptors include the erythropoietin (EPOR), thrombopoietin (TPOR), granulocyte colony-stimulating factor 3 (CSF3R), growth hormone (GHR), and prolactin receptors (PRLR). These cell-surface single-pass transmembrane (TM) glycoproteins regulate cell growth, proliferation, and differentiation and induce oncogenesis. An active TM signaling complex consists of a receptor homodimer, one or two ligands bound to the receptor extracellular domains and two molecules of Janus Kinase 2 (JAK2) constitutively associated with the receptor intracellular domains. Although crystal structures of soluble extracellular domains with ligands have been obtained for all the receptors except TPOR, little is known about the structure and dynamics of the complete TM complexes that activate the downstream JAK-STAT signaling pathway. Three-dimensional models of five human receptor complexes with cytokines and JAK2 were generated here using AlphaFold Multimer. Given the large size of the complexes (from 3220 to 4074 residues), the modeling required a stepwise assembly from smaller parts with selection and validation of the models through comparisons with published experimental data. The modeling of active and inactive complexes supports a general activation mechanism that involves ligand binding to a monomeric receptor followed by receptor dimerization and rotational movement of the receptor TM α-helices causing proximity, dimerization, and activation of associated JAK2 subunits. The binding mode of two eltrombopag molecules to TM α-helices of the active TPOR dimer was proposed. The models also help elucidating the molecular basis of oncogenic mutations that may involve a non-canonical activation route. Models equilibrated in explicit lipids of the plasma membrane are publicly available. 

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