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1.
Elife ; 92020 02 25.
Article in English | MEDLINE | ID: mdl-32096762

ABSTRACT

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.


Subject(s)
KCNQ1 Potassium Channel/physiology , Animals , Electrophysiology , Fluorometry , Humans , KCNQ1 Potassium Channel/chemistry , KCNQ1 Potassium Channel/metabolism , Magnetic Resonance Spectroscopy , Oocytes , Patch-Clamp Techniques , Protein Structure, Tertiary , Xenopus laevis
2.
Sci Adv ; 4(3): eaar2631, 2018 03.
Article in English | MEDLINE | ID: mdl-29532034

ABSTRACT

Mutations that induce loss of function (LOF) or dysfunction of the human KCNQ1 channel are responsible for susceptibility to a life-threatening heart rhythm disorder, the congenital long QT syndrome (LQTS). Hundreds of KCNQ1 mutations have been identified, but the molecular mechanisms responsible for impaired function are poorly understood. We investigated the impact of 51 KCNQ1 variants with mutations located within the voltage sensor domain (VSD), with an emphasis on elucidating effects on cell surface expression, protein folding, and structure. For each variant, the efficiency of trafficking to the plasma membrane, the impact of proteasome inhibition, and protein stability were assayed. The results of these experiments combined with channel functional data provided the basis for classifying each mutation into one of six mechanistic categories, highlighting heterogeneity in the mechanisms resulting in channel dysfunction or LOF. More than half of the KCNQ1 LOF mutations examined were seen to destabilize the structure of the VSD, generally accompanied by mistrafficking and degradation by the proteasome, an observation that underscores the growing appreciation that mutation-induced destabilization of membrane proteins may be a common human disease mechanism. Finally, we observed that five of the folding-defective LQTS mutant sites are located in the VSD S0 helix, where they interact with a number of other LOF mutation sites in other segments of the VSD. These observations reveal a critical role for the S0 helix as a central scaffold to help organize and stabilize the KCNQ1 VSD and, most likely, the corresponding domain of many other ion channels.


Subject(s)
KCNQ1 Potassium Channel/chemistry , KCNQ1 Potassium Channel/genetics , Long QT Syndrome/genetics , Mutation/genetics , Cell Membrane/drug effects , Cell Membrane/metabolism , HEK293 Cells , Humans , KCNQ1 Potassium Channel/metabolism , Leupeptins/pharmacology , Loss of Function Mutation/genetics , Magnetic Resonance Spectroscopy , Mutant Proteins/chemistry , Mutant Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Protein Domains , Protein Folding/drug effects , Protein Structure, Secondary , Proteolysis/drug effects
3.
Circ Cardiovasc Genet ; 10(5)2017 Oct.
Article in English | MEDLINE | ID: mdl-29021305

ABSTRACT

BACKGROUND: An emerging standard-of-care for long-QT syndrome uses clinical genetic testing to identify genetic variants of the KCNQ1 potassium channel. However, interpreting results from genetic testing is confounded by the presence of variants of unknown significance for which there is inadequate evidence of pathogenicity. METHODS AND RESULTS: In this study, we curated from the literature a high-quality set of 107 functionally characterized KCNQ1 variants. Based on this data set, we completed a detailed quantitative analysis on the sequence conservation patterns of subdomains of KCNQ1 and the distribution of pathogenic variants therein. We found that conserved subdomains generally are critical for channel function and are enriched with dysfunctional variants. Using this experimentally validated data set, we trained a neural network, designated Q1VarPred, specifically for predicting the functional impact of KCNQ1 variants of unknown significance. The estimated predictive performance of Q1VarPred in terms of Matthew's correlation coefficient and area under the receiver operating characteristic curve were 0.581 and 0.884, respectively, superior to the performance of 8 previous methods tested in parallel. Q1VarPred is publicly available as a web server at http://meilerlab.org/q1varpred. CONCLUSIONS: Although a plethora of tools are available for making pathogenicity predictions over a genome-wide scale, previous tools fail to perform in a robust manner when applied to KCNQ1. The contrasting and favorable results for Q1VarPred suggest a promising approach, where a machine-learning algorithm is tailored to a specific protein target and trained with a functionally validated data set to calibrate informatics tools.


Subject(s)
Databases, Genetic , Genetic Variation , KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/metabolism , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Female , Humans , Long QT Syndrome/epidemiology , Male , Predictive Value of Tests , Protein Domains
4.
Mol Biol Cell ; 28(23): 3298-3314, 2017 Nov 07.
Article in English | MEDLINE | ID: mdl-28814505

ABSTRACT

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.


Subject(s)
Spindle Pole Bodies/metabolism , Spindle Pole Bodies/physiology , Bayes Theorem , Cell Cycle , Centrosome/metabolism , Computer Simulation , Crystallography, X-Ray/methods , Microtubule-Organizing Center/metabolism , Microtubules/metabolism , Mitosis , Nuclear Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/metabolism , Structure-Activity Relationship , X-Ray Diffraction/methods
5.
Biochim Biophys Acta Biomembr ; 1859(4): 586-597, 2017 Apr.
Article in English | MEDLINE | ID: mdl-27818172

ABSTRACT

Many years of studies have established that lipids can impact membrane protein structure and function through bulk membrane effects, by direct but transient annular interactions with the bilayer-exposed surface of protein transmembrane domains, and by specific binding to protein sites. Here, we focus on how phosphatidylinositol 4,5-bisphosphate (PIP2) and polyunsaturated fatty acids (PUFAs) impact ion channel function and how the structural details of the interactions of these lipids with ion channels are beginning to emerge. We focus on the Kv7 (KCNQ) subfamily of voltage-gated K+ channels, which are regulated by both PIP2 and PUFAs and play a variety of important roles in human health and disease. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.


Subject(s)
Epilepsy, Benign Neonatal/metabolism , Hearing Loss, Bilateral/metabolism , KCNQ1 Potassium Channel/chemistry , Long QT Syndrome/metabolism , Membrane Lipids/chemistry , Amino Acid Sequence , Binding Sites , Cell Membrane/chemistry , Cell Membrane/metabolism , Epilepsy, Benign Neonatal/pathology , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/metabolism , Hearing Loss, Bilateral/pathology , Humans , Hydrophobic and Hydrophilic Interactions , KCNQ1 Potassium Channel/deficiency , KCNQ1 Potassium Channel/metabolism , Long QT Syndrome/pathology , Membrane Lipids/metabolism , Models, Molecular , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol 4,5-Diphosphate/metabolism , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/deficiency , Protein Isoforms/metabolism , Protein Structure, Secondary
6.
Sci Adv ; 2(9): e1501228, 2016 09.
Article in English | MEDLINE | ID: mdl-27626070

ABSTRACT

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."


Subject(s)
Cystic Fibrosis/metabolism , KCNQ1 Potassium Channel/chemistry , Multiprotein Complexes/chemistry , Potassium Channels, Voltage-Gated/chemistry , Animals , Chloride Channels/chemistry , Computational Biology/methods , Cystic Fibrosis/pathology , Electrophysiological Phenomena , Epithelial Cells/chemistry , Epithelial Cells/metabolism , Humans , KCNQ1 Potassium Channel/metabolism , Multiprotein Complexes/metabolism , Potassium/chemistry , Potassium/metabolism , Potassium Channels, Voltage-Gated/metabolism , Protein Domains
7.
Proteins ; 84(1): 172-189, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26573747

ABSTRACT

Sarcomeric myosins have the remarkable ability to form regular bipolar thick filaments that, together with actin thin filaments, constitute the fundamental contractile unit of skeletal and cardiac muscle. This has been established for over 50 years and yet a molecular model for the thick filament has not been attained. In part this is due to the lack of a detailed molecular model for the coiled-coil that constitutes the myosin rod. The ability to self-assemble resides in the C-terminal section of myosin known as light meromyosin (LMM) which exhibits strong salt-dependent aggregation that has inhibited structural studies. Here we evaluate the feasibility of generating a complete model for the myosin rod by combining overlapping structures of five sections of coiled-coil covering 164 amino acid residues which constitute 20% of LMM. Each section contains ∼ 7-9 heptads of myosin. The problem of aggregation was overcome by incorporating the globular folding domains, Gp7 and Xrcc4 which enhance crystallization. The effect of these domains on the stability and conformation of the myosin rod was examined through biophysical studies and overlapping structures. In addition, a computational approach was developed to combine the sections into a contiguous model. The structures were aligned, trimmed to form a contiguous model, and simulated for >700 ns to remove the discontinuities and achieve an equilibrated conformation that represents the native state. This experimental and computational strategy lays the foundation for building a model for the entire myosin rod.


Subject(s)
Myosin Subfragments/chemistry , Amino Acid Sequence , Cardiomyopathies/genetics , Crystallography, X-Ray , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Mutation , Myosin Subfragments/genetics , Protein Conformation , Protein Stability , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Temperature
8.
Biophys J ; 109(7): 1472-82, 2015 Oct 06.
Article in English | MEDLINE | ID: mdl-26445448

ABSTRACT

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 µm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 µm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 µm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.


Subject(s)
Kinesins/metabolism , Microtubules/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Biological Transport/physiology , Dimerization , Escherichia coli , Kinesins/genetics , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Protein Conformation , Quantum Dots , Sequence Homology , Video Recording
9.
Proc Natl Acad Sci U S A ; 112(29): E3806-15, 2015 Jul 21.
Article in English | MEDLINE | ID: mdl-26150528

ABSTRACT

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.


Subject(s)
Amino Acids/chemistry , Cardiac Myosins/chemistry , Cardiac Myosins/metabolism , Myosin Heavy Chains/chemistry , Myosin Heavy Chains/metabolism , Myosin Subfragments/chemistry , Myosin Subfragments/metabolism , Amino Acid Sequence , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Pliability , Protein Stability , Protein Structure, Secondary , Repetitive Sequences, Amino Acid , Sarcomeres/metabolism , Sequence Deletion , Structure-Activity Relationship
10.
J Biol Chem ; 289(52): 36249-62, 2014 Dec 26.
Article in English | MEDLINE | ID: mdl-25381442

ABSTRACT

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.


Subject(s)
Acetyltransferases/chemistry , Bacterial Proteins/chemistry , Acetylation , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Lysine/chemistry , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Salmonella paratyphi B/enzymology , Streptomyces lividans/enzymology , Substrate Specificity
11.
Biochemistry ; 52(15): 2574-85, 2013 Apr 16.
Article in English | MEDLINE | ID: mdl-23520975

ABSTRACT

We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling, and X-ray crystallography, we determined that FtsB self-associates, and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an interhelical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo.


Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Amino Acid Sequence , Cell Cycle Proteins/genetics , Circular Dichroism , Conserved Sequence , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Glutamine/chemistry , Hydrogen Bonding , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Periplasm/chemistry , Periplasm/metabolism , Point Mutation , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
12.
Chem Phys Lett ; 549: 86-92, 2012 Oct 11.
Article in English | MEDLINE | ID: mdl-23554514

ABSTRACT

Femtosecond photodynamics of the reverse ( 15E Pfr→ 15Z Pr) reaction of the red/far-red phytochrome Cph1 from Synechocystis were resolved with visible broadband transient absorption spectroscopy. Multi-phasic dynamics were resolved and separated via global target analysis into a fast-decaying (260 fs) excited-state population that bifurcates to generate the isomerized Lumi-F primary photoproduct and a non-isomerizing vibrationally excited ground state that relaxes back into the 15E Pfr ground state on a 2.8-ps time scale. Relaxation on a 1-ms timescale results in the loss of red absorbing region, but not blue region, of Lumi-F, which indicates that formation of 15Z Pr occurs on slower timescales.

13.
Proc Natl Acad Sci U S A ; 106(6): 1784-9, 2009 Feb 10.
Article in English | MEDLINE | ID: mdl-19179399

ABSTRACT

Photochemical interconversion between the red-absorbing (P(r)) and the far-red-absorbing (P(fr)) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The P(r)-to-P(fr) transition commences with a rapid Z-to-E photoisomerization at the C(15)=C(16) methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C(15)-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted C=C stretch and N-H in-plane rocking modes within 500 fs, reveal that initial distortion of the C(15)=C(16) bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to P(r) in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C(15)-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C(15)=C(16) Z-to-E isomerization occurs on the excited-state surface.


Subject(s)
Bacterial Proteins/chemistry , Phytochrome/chemistry , Protein Kinases/chemistry , Spectrum Analysis, Raman/methods , Isomerism , Kinetics , Photochemistry , Photoreceptors, Microbial , Spectrum Analysis, Raman/instrumentation
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