Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal Bchain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe B24 to a 60°rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β 2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile A2 , Val A3 , Val B12 , Phe B24 , and Phe B25 ) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptorbound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.diabetes mellitus | signal transduction | receptor tyrosine kinase | metabolism | protein structure H ow insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24-B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3-6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residuespecific photo-cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ) 2 receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently...

The voltage-gated proton channel Hv1 plays a critical role in the fast proton translocation that underlies a wide range of physiological functions, including the phagocytic respiratory burst, sperm motility, apoptosis, and metastatic cancer. Both voltage activation and proton conduction are carried out by a voltage-sensing domain (VSD) with strong similarity to canonical VSDs in voltage-dependent cation channels and enzymes. We set out to determine the structural properties of membrane-reconstituted human proton channel (hHv1) in its resting conformation using electron paramagnetic resonance spectroscopy together with biochemical and computational methods. We evaluated existing structural templates and generated a spectroscopically constrained model of the hHv1 dimer based on the Ci-VSD structure at resting state. Mapped accessibility data revealed deep water penetration through hHv1, suggesting a highly focused electric field, comprising two turns of helix along the fourth transmembrane segment. This region likely contains the H + selectivity filter and the conduction pore. Our 3D model offers plausible explanations for existing electrophysiological and biochemical data, offering an explicit mechanism for voltage activation based on a one-click sliding helix conformational rearrangement.V oltage-gated proton channels (hHv1) represent a remarkable evolutionary adaptation of canonical voltage sensing domain (VSD). In hHv1, both voltage-sensing and ion (H + ) conduction are carried out by a single domain, and undergo a global conformational rearrangement (1, 2). In humans, hHv1 is widely expressed and required for a variety of physiological processes (3), including optimal reactive oxygen species production by NADPH oxidase (4-6), B-cell proliferation and differentiation (7), and regulation of human sperm motility (8). hHv1 folds as an antiparallel four-helix bundle with its fourth transmembrane segment (S4) containing three putative gating charges. Just as in the VSDs from ion channels and enzymes, the positively charged S4 moves outwardly in response to a depolarization (9). Beyond sensing the transmembrane voltage, S4 reorientation in hHv1 participates in the formation of a protonselective permeation pathway responsible for the generation of the proton currents that underlie its multiple physiological functions (10, 11).Despite high sequence similarity and a common structural blueprint, VSDs from ion channels and enzymes display a wide range of electrophysiological properties; these include large variations in effective gating charge (z = ∼0.9 to ∼3.6 e − ) and midpoints of activation (V 1/2 = ∼−150 to ∼+150 mV). Existing VSD crystal structures (12-16) have provided key data on the arrangement and orientation of the transmembrane helices S1-S4, while at the same time revealed a considerable structural heterogeneity-particularly, the number and positions of both the gating charges and their compensating countercharges in relation to the hydrophobic "plug" or "gasket" electrically separating the intra-and extrace...

The voltage-dependent motor protein Prestin (SLC26A5) is responsible for the electromotive behavior of outer hair cells (OHCs) and underlies the cochlear amplifier 1 . Knock out or impairment of Prestin causes severe hearing loss [2][3][4][5] . Despite Prestin's key physiological role in hearing, the mechanism by which mammalian Prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here, we determined the structure of dolphin Prestin in six distinct states using single particle cryo-electron microscopy. Our structural and functional data suggest that Prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Clor SO4 = ). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of Prestin, inhibits its function by immobilizing locking in a novel conformation. This suggests that the anion together with its coordinating fixed charges act as a dynamic voltage sensor. Analysis of all aniondependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein-membrane interface, suggesting a novel mechanism of area expansion. Visualization of Prestin's electromotility cycle distinguishes Prestin from closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at high resolution. Perozo lab for a healthy exchange of ideas and comments on the manuscript. We thank Dr. Peng Shi for sharing Tursiops Prestin plasmid. James Fuller, Joe Austin II, and Tera Lavoie at the University of Chicago Advanced Electron Microscopy Facility for microscope maintenance and training. N.B. would like to acknowledge the Biology of Inner ear course (BIE2019) and Gordon Conference (Auditory System Gordon Research Conference) for inspiring him to study hearing and Prestin.

"Derivative isolates" with 4- to 8-fold and 8- to 16-fold increases in MICs of vancomycin and teicoplanin, respectively, were selected from 2 susceptible clinical isolates of Staphylococcus aureus by serial incubation in low-level vancomycin. A protein of approximately 39 kDa was demonstrable in the cytoplasmic fraction and occasionally in the membrane fraction by SDS-PAGE of both derivatives. This protein was purified by DEAE chromatography, preparative SDS-PAGE, and electroelution. Derivative bacteria were larger on transmission electron microscopy, had thicker cell walls, and had changes in colony morphology on solid media. Further evidence for cell wall reorganization included loss of phage and capsular typing, decreased susceptibility to lysostaphin/lysozyme killing, and changes in condition for detection of optimal coagulase activity. The mechanism of decreased susceptibility to glycopeptide antibiotics among S. aureus derivative isolates is uncertain. The production of the approximately 39-kDa cytoplasmic protein and cell wall reorganization may mediate changed affinity of glycopeptide-peptidoglycan binding or impairment of glycopeptide access to its cell wall target.

Insulin-like growth factor I (IGF-I