KCNE1 is a single span membrane protein that modulates the voltage-gated potassium channel KCNQ1 (K V 7.1) by slowing activation and enhancing channel conductance to generate the slow delayed rectifier current (I Ks ) that is critical for the repolarization phase of the cardiac action potential. Perturbation of channel function by inherited mutations in KCNE1 or KCNQ1 results in increased susceptibility to cardiac arrhythmias and sudden death with or without accompanying deafness. Here, we present the three-dimensional structure of KCNE1. The transmembrane domain (TMD) of KCNE1 is a curved α-helix and is flanked by intra-and extracellular domains comprised of α-helices joined by flexible linkers. Experimentally-restrained docking of the KCNE1 TMD to a closed state model of KCNQ1 suggests that KCNE1 slows channel activation by sitting on and restricting the movement of the S4-S5 linker that connects the voltage sensor to the pore domain. We postulate that this is an adhesive interaction that must be disrupted before the channel can be opened in response to membrane depolarization. Docking to open KCNQ1 indicates that the extracellular end of the KCNE1 TMD forms an interface with an intersubunit cleft in the channel that is associated with most known gain-of-function disease mutations. Binding of KCNE1 to this "gain-of-function cleft" may explain how it increases conductance and stabilizes the open state. These working models for the KCNE1/KCNQ1 complexes may be used to formulate testable hypotheses for the molecular bases of disease phenotypes associated with the dozens of known inherited mutations in KCNE1 and KCNQ1.KCNE1 (previously called minK) belongs to the KCNE family of single-span membrane proteins that modulate the activity of several voltage-gated K + channels, including KCNQ1 (K V 7.1). In cardiac myocytes KCNE1 forms obligate complexes with KCNQ1 to generate the

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells including biosynthesis, cell signaling, and oxidative protection. Herein, we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux, that selectively and potently targets the amino acid transporter ASCT2 (SLC1A5). Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively, contributed to anti-tumor responses in vitro and in vivo. Representing a new class of targeted therapy, this is the first study to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

The S100 calcium-binding proteins are implicated as effectors in calcium-mediated signal transduction pathways. The three-dimensional structure of the S100 protein calcyclin has been determined in solution in the apo state by NMR spectroscopy and a computational strategy that incorporates a systematic docking protocol. This structure reveals a symmetric homodimeric fold that is unique among calcium-binding proteins. Dimerization is mediated by hydrophobic contacts from several highly conserved residues, which suggests that the dimer fold identified for calcyclin will serve as a structural paradigm for the S100 subfamily of calcium-binding proteins.

The pikromycin (Pik)/methymycin biosynthetic pathway of Streptomyces venezuelae represents a valuable system for dissecting the fundamental mechanisms of modular polyketide biosynthesis, aminodeoxysugar assembly, glycosyltransfer, and hydroxylation leading to the production of a series of macrolide antibiotics, including the natural ketolides narbomycin and pikromycin. In this study, we describe four x-ray crystal structures and allied functional studies for PikC, the remarkable P450 monooxygenase responsible for production of a number of related macrolide products from the Pik pathway. The results provide important new insights into the structural basis for the C10/C12 and C12/C14 hydroxylation patterns for the 12-(YC-17) and 14-membered ring (narbomycin) macrolides, respectively. This includes two different ligand-free structures in an asymmetric unit (resolution 2.1 Å ) and two co-crystal structures with bound endogenous substrates YC-17 (resolution 2.35 Å ) or narbomycin (resolution 1.7 Å ). A central feature of the enzymesubstrate interaction involves anchoring of the desosamine residue in two alternative binding pockets based on a series of distinct amino acid residues that form a salt bridge and a hydrogen-bonding network with the deoxysugar C3 dimethylamino group. Functional significance of the salt bridge was corroborated by site-directed mutagenesis that revealed a key role for Glu-94 in YC-17 binding and Glu-85 for narbomycin binding. Taken together, the x-ray structure analysis, site-directed mutagenesis, and corresponding product distribution studies reveal that PikC substrate tolerance and product diversity result from a combination of alternative anchoring modes rather than an induced fit mechanism.Macrolide antibiotics comprise a large group of medicinal agents characterized by a macrocyclic lactone ring, to which one or more sugar residues are covalently linked. Despite considerable structural variation, macrolides represent a homogeneous group of therapeutic drugs with similar activity spectra and mode of action as anti-infective agents. The success of macrolide antibiotics is attributed to their propensity to bind to the large subunit of prokaryotic ribosomes and inhibit protein synthesis, thereby preventing bacterial growth (1, 2). The first generation macrolide introduced into clinical practice over 50 years ago was erythromycin. Since then, macrolide antibiotics have been further optimized, resulting in improved 14-, 15-, and 16-membered ring macrolides (second generation), acylides, and ketolides (third generation) (3).Most of the natural product macrolide antibiotics are produced by Streptomyces sp. and related bacteria, in which assembly of polyketides from simple carboxylic acid precursors is catalyzed by modular polyketide synthases. Over the past 15 years, advances in understanding the modular architecture of polyketide biosynthetic machinery has enabled development of metabolic engineering approaches for production of new antibiotics (4 -7). However, significant additional structural...

Mutations in the human voltage-gated potassium channel KCNQ1 are associated with predisposition to deafness and various cardiac arrhythmia syndromes including congenital long QT syndrome, familial atrial fibrillation, and sudden infant death syndrome. In this work 3-D structural models were developed for both the open and closed states of human KCNQ1 to facilitate structurally-based hypotheses regarding mutation-phenotype relationships. The KCNQ1 open state was modeled using Rosetta in conjunction with Molecular Operating Environment software, and is based primarily on the recently-determined open state structure of rat K v 1.2 (S. B Long et al., 2005, Science 309, 897 −903). The closed state model for KCNQ1 was developed based on the crystal structures of bacterial potassium channels and the closed state model for K v 1.2 of Yarov-Yarovoy et al. (2006, Proc. Nat. Acad. Sci. 103, 7292−7207). Using the new models for KCNQ1, we generated a database for the location and predicted residue-residue interactions for more than 85 disease-linked sites in both open and closed states. These data can be used to generate structure-based hypotheses for disease phenotypes associated with each mutation. The potential utility of these models and the database is exemplified by the surprising observation that four of the five known mutations in KCNQ1 that are associated with gain-of-function KCNQ1 defects are predicted to share a common interface in the open state structure between the S1 segment of the voltage sensor in one subunit and both the S5 segment and top of the pore helix from another subunit. This interface evidently plays an important role in channel gating.KCNQ1 is a voltage-gated potassium channel that participates critically in human physiology and is subject to several heritable disease-linked mutations (2;3). The most common splice variant of KCNQ1, also known as K v LQT1 or K v 7.1, contains 676 residues and consists of a cytosolic N-terminal domain followed by the voltage sensor (S1-S4), a canonical pore domain (S5-P-S6) and a long cytosolic C-terminus (see Figure 1). The best-characterized physiological function of KCNQ1 relates to its expression in cardiac tissue, where it co-assembles with * To whom correspondence should be addressed: E-mail: jens.meiler@vanderbilt. Disruption of KCNQ1 function by dominant mutations causes more than 50% of genotypeknown cases of congenital long QT syndrome type 1 (LQTS1), a potentially fatal arrhythmic disorder(6-10). In the autosomal dominant form of the disease, Romano-Ward syndrome, mutant KCNQ1 exerts dominant-negative effects that cause loss-of-function. Recessive lossof-function mutations in KCNQ1 cause Jervell and Lange-Nielsen syndrome (JLNS), characterized by both long-QT syndrome and congenital deafness(8;9;11). Deafness in this syndrome highlights the important role that KCNQ1 plays in tissues outside the cardiovascular system including the kidney, stomach, and ear. The association with congenital hearing loss, for example, reflects an important role for K...

Long QT syndrome–associated mutations in KCNQ1 most often destabilize the protein, leading to mistrafficking and degradation.

The crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts were monitored in single-stranded and duplex oligodeoxynucleotides using NMR spectroscopy. In both instances, the cis and trans diastereomers of the alpha-CH3 and gamma-OH groups underwent slow exchange, with the trans diastereomers being favored. In single-stranded oligodeoxynucleotides, the aldehyde intermediates were not detected spectroscopically, but their presence was revealed through the formation of N-terminal conjugates with the tetrapeptide KWKK. When annealed into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCYCTAGC)-3' containing the 5'-CpG-3' sequence context (X = R- or S-alpha-CH3-gamma-13C-OH-PdG; Y = 15N2-dG) at pH 7, partial opening of the R- or S-alpha-CH3-gamma-13C-OH-PdG adducts to the corresponding N2-(3-oxo-1-methyl-propyl)-dG aldehydes was observed at temperatures below the T(m) of the duplexes. These aldehydes equilibrated with their geminal diol hydrates; higher temperatures favored the aldehydes. When annealed opposite T, the S-alpha-CH3-gamma-13C-OH-PdG adduct was stable. At 37 degrees C, an interstrand DNA cross-link was observed spectroscopically only for the R-alpha-CH3-gamma-OH-PdG adduct. Molecular modeling predicted that the interstrand cross-link formed by the R-alpha-CH3-gamma-OH-PdG adduct introduced less disruption into the duplex structure than did the cross-link arising from the S-alpha-CH3-gamma-OH-PdG adduct, due to differing orientations of the R- and S-CH3 groups. Modeling also predicted that the alpha-methyl group of the aldehyde arising from the R-alpha-CH3-gamma-OH-PdG adduct is oriented in the 3'-direction in the minor groove, facilitating cross-linking. In contrast, the alpha-methyl group of the aldehyde arising from the S-alpha-CH3-gamma-OH-PdG adduct is oriented in the 5'-direction within the minor groove, potentially hindering cross-linking. NMR revealed that for the R-alpha-CH3-gamma-OH-PdG adduct, the carbinolamine form of the cross-link was favored in duplex DNA with the imine (Schiff base) form of the cross-link remaining below the level of spectroscopic detection. Molecular modeling predicted that the carbinolamine linkage maintained Watson-Crick hydrogen bonding at both of the tandem C.G base pairs. Dehydration of the carbinolamine cross-link to an imine, or cyclization of the latter to form a pyrimidopurinone cross-link, required disruption of Watson-Crick hydrogen bonding at one or both of the cross-linked base pairs.

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.