Publications by authors named "Yanwu Yang"

29 Publications

  • Page 1 of 1

Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone-receptor activation.

Proc Natl Acad Sci U S A 2021 Jul;118(30)

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202;

Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin's "hinge opening" and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone's native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (-fluoro-phenylboronic acid at Gly) and internal diol (3,4-dihydroxybenzoate at Lys). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a "smart" insulin analog to mitigate hypoglycemic risk in diabetes therapy.
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http://dx.doi.org/10.1073/pnas.2103518118DOI Listing
July 2021

Alteration of brain structural connectivity in progression of Parkinson's disease: A connectome-wide network analysis.

Neuroimage Clin 2021 Jun 6;31:102715. Epub 2021 Jun 6.

Department of Neurobiology, Neurology and Geriatrics, Xuanwu Hospital, Capital Medical University, National Clinical Research Center for Geriatric Disease, Beijing, China. Electronic address:

Pinpointing the brain dysconnectivity in idiopathic rapid eye movement sleep behaviour disorder (iRBD) can facilitate preventing the conversion of Parkinson's disease (PD) from prodromal phase. Recent neuroimage investigations reported disruptive brain white matter connectivity in both iRBD and PD, respectively. However, the intrinsic process of the human brain structural network evolving from iRBD to PD still remains largely unknown. To address this issue, 151 participants including iRBD, PD and age-matched normal controls were recruited to receive diffusion MRI scans and neuropsychological examinations. The connectome-wide association analysis was performed to detect reorganization of brain structural network along with PD progression. Eight brain seed regions in both cortical and subcortical areas demonstrated significant structural pattern changes along with the progression of PD. Applying machine learning on the key connectivity related to these seed regions demonstrated better classification accuracy compared to conventional network-based statistic. Our study shows that connectome-wide association analysis reveals the underlying structural connectivity patterns related to the progression of PD, and provide a promising distinct capability to predict prodromal PD patients.
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http://dx.doi.org/10.1016/j.nicl.2021.102715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209844PMC
June 2021

Theoretical prediction on photoelectric and supramolecular properties of benzoquinone-tetrathiafulvalene macrocyclic molecules.

J Mol Model 2021 May 7;27(6):157. Epub 2021 May 7.

School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, Jiangsu, China.

Benzoquinone has the ability to serve as an electron acceptor, and tetrathiafulvalene has the ability to serve as an electron donor. Based on the facts above, this work creatively cycles the benzoquinone unit and the tetrathiafulvalene unit alternately into macrocyclic molecules, the cyclopolymers of benzoquinone-tetrafluorene (C[n]QTTF, n = 3~6). To explore their structure and properties, the M06-2X functional of density functional theory (DFT) with 6-311g(d) basis set was used to optimize the ground-state structures of C[n]QTTF. Based on the stable configurations of the ground states, the electronic structure property is analyzed systematically. The results show that these macrocyclic molecules have excellent electron transport capability and electrochemical activity. Then, the electron absorption spectra of each system are carried out by using time-dependent density functional theory (TD-DFT) at the M062X/6-311+G(d) level. It turns out that their maximum absorption wavelengths are all in the visible range. Further calculation suggests that C[n]QTTF can also be characterized with one-dimensional self-assembly, double-walled assembly, and the host-guest inclusion performance, based on which it gains a variety of supramolecular structures. In summary, the benzoquinone-tetrafluorofurene macrocyclic molecules predicted by DFT calculations may be of assistance to the potential applications in organic electronics and supramolecular chemistry.
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http://dx.doi.org/10.1007/s00894-021-04782-5DOI Listing
May 2021

Evolution of insulin at the edge of foldability and its medical implications.

Proc Natl Acad Sci U S A 2020 11 5;117(47):29618-29628. Epub 2020 Nov 5.

Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106;

Proteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and β-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue Tyr, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric Tyr insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of Tyr is similar to that of Phe, adjoining core cystine B19-A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased ∼20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of Phe among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the -hydroxyl group of Tyr hinders pairing of cystine B19-A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge-excluded due to β-cell dysfunction-suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein's fitness landscape underlies both a rare monogenic syndrome and "diabesity" as a pandemic disease of civilization.
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http://dx.doi.org/10.1073/pnas.2010908117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703552PMC
November 2020

Mesenchymal stem cells in experimental autoimmune encephalomyelitis model of multiple sclerosis: A systematic review and meta-analysis.

Mult Scler Relat Disord 2020 Sep 23;44:102200. Epub 2020 May 23.

Department of Neurosurgery, The Third Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China; National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, No. 37 Guoxue Lane, Wuhou District, Chengdu 610041, Sichuan, China; Department of Geriatric, Sichuan Science City Hospital, No. 64, Mianshan Road, Mianyang, Sichuan, China. Electronic address:

Background/aims: Mesenchymal stem cells (MSCs) transplantation has been considered a possible therapeutic method for Multiple Sclerosis (MS). However, no quantitative data synthesis of MSCs therapy for MS exists. We conducted a systematic review and meta-analysis to evaluate the effects of MSCs in experimental autoimmune encephalomyelitis (EAE) animal model of MS.

Methods: We identified eligible studies published from January 1980 to January 2017 by searching four electronic databases (PubMed, MEDLINE, Embase and Web of Science). The outcome was the effects of MSCs on clinical performance evaluated by the EAE clinical score.

Results: 36 preclinical studies including 675 animals in MSCs treatment group, and 693 animals in control group were included in this meta-analysis. We found that MSCs transplantation significantly ameliorated the symptoms and delayed the disease progression (SMD = -1.25, 95% CI: -1.45 to -1.05, P < 0.001). However, no significant differences in effect sizes were unveiled relative to clinical score standard (P = 0.35), type of MSCs (P = 0.35), source of MSCs (P = 0.06), MSCs dose (P = 0.44), delivery methods (P = 0.31) and follow up period (P = 0.73).

Conclusions: The current study showed that MSCs transplantation could ameliorate clinical performance in EAE animal model of MS. These findings support the further studies translate MSCs to treat MS in humans.
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http://dx.doi.org/10.1016/j.msard.2020.102200DOI Listing
September 2020

"Register-shift" insulin analogs uncover constraints of proteotoxicity in protein evolution.

J Biol Chem 2020 03 31;295(10):3080-3098. Epub 2020 Jan 31.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202. Electronic address:

Globular protein sequences encode not only functional structures (the native state) but also protein foldability, a conformational search that is both efficient and robustly minimizes misfolding. Studies of mutations associated with toxic misfolding have yielded insights into molecular determinants of protein foldability. Of particular interest are residues that are conserved yet dispensable in the native state. Here, we exploited the mutant proinsulin syndrome (a major cause of permanent neonatal-onset diabetes mellitus) to investigate whether toxic misfolding poses an evolutionary constraint. Our experiments focused on an invariant aromatic motif (Phe-Phe-Tyr) with complementary roles in native self-assembly and receptor binding. A novel class of mutations provided evidence that insulin can bind to the insulin receptor (IR) in two different modes, distinguished by a "register shift" in this motif, as visualized by molecular dynamics (MD) simulations. Register-shift variants are active but defective in cellular foldability and exquisitely susceptible to fibrillation Indeed, expression of the corresponding proinsulin variant induced endoplasmic reticulum stress, a general feature of the mutant proinsulin syndrome. Although not present among vertebrate insulin and insulin-like sequences, a prototypical variant ([Gly]insulin) was as potent as WT insulin in a rat model of diabetes. Although in MD simulations the shifted register of receptor engagement is compatible with the structure and allosteric reorganization of the IR-signaling complex, our results suggest that this binding mode is associated with toxic misfolding and so is disallowed in evolution. The implicit threat of proteotoxicity limits sequence variation among vertebrate insulins and insulin-like growth factors.
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http://dx.doi.org/10.1074/jbc.RA119.011389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062160PMC
March 2020

Automatic Brain Tumor Segmentation Method Based on Modified Convolutional Neural Network.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:998-1001

In the domain of brain diseases, it is difficult for image registration after some brain structures are severely deformed because of diseases. Fortunately, convolutional neural network have gained many promising results in semantic segmentation challenging tasks in recent years. To enhance the performance of automatic brain tumor segmentation, this paper presents a robust segmentation algorithm based on convolutional neural network, which achieved improvement of 3.84% in segmenting the enhancing tumor. Our network architecture is developed from the prevalent U-Net. We combined it with ResNet and modified it to maximize its performance in our brain tumor segmentation task. In this work, BraTS 2017 dataset was employed to train and test the proposed network. Data imbalance was dealt with using a weighted cross entropy loss function. The problem of overfitting was handled through data augmentation. The proposed method achieved averaged dice scores of 0.883, 0.781 and 0.748 for whole tumor, tumor core and enhancing tumor respectively in the validation set and 0.877, 0.774, 0.757 respectively in the testing set.
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http://dx.doi.org/10.1109/EMBC.2019.8857303DOI Listing
July 2019

Multivariate Analysis of White Matter Structural Networks of Alzheimer's Disease.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:1140-1143

The connectome-wide association studies exploring association between brain connectome and disease phenotypes have suffered from massive number of comparisons. In this paper, we propose to apply a multivariate distance-based analytic framework on brain white matter (WM) structural networks invaded by Alzheimer's disease (AD). Eighty-three subjects including patients with AD, amnestic mild cognitive impairment (aMCI) and healthy subjects were scanned with dMRI. By constructing WM structural network for each individual, we used both multivariate and traditional univariate statistical models to complimentarily analyze network pattern and fiber strength changes due to AD. WM connections linked with several brain structures were found significantly changed between AD group and normal controls. No significant findings were observed between aMCI group and normal controls. Our results demonstrate the sensitivity of the combined connectome-based analytic framework in detecting abnormalities of structural brain network.
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http://dx.doi.org/10.1109/EMBC.2018.8512553DOI Listing
July 2018

The Correlation Analysis Between DTI Network Parameters and AVLT Scale Scores of Alzheimer's Disease.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:1132-1135

Neuroimaging and neuropsychology are employed to investigate the pathological features and clinical characteristics of Alzheimer's disease (AD) in order to find a method for the precise treatment. Diffusion tensor imaging (DTI) provides a non-intrusive examination of cranial nerve diseases which can help us observe the microstructure of neuron fibers. Building the brain network provides a chance to reveal the significance of specific brain region and the relevance among different regions. In this study, we propose a completely novel method to analyze AD. First whole brain network is built on the basis of a novel segmentation atlas, and global graph theoretical parameters are calculated to evaluate the characteristic of whole brain. Then graph theoretical parameters of specific brain regions are extracted based on whole brain network. Finally neuropsychology scale are employed and we analyze the correlation between graph theoretical parameters of specific regions and scale scores. Our results illustrate the connection between neuroimaging data and neuropsychological scores, and provide a reasonable explanation for the potential connection between clinical performance and physiological brain lesions of AD patients.
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http://dx.doi.org/10.1109/EMBC.2018.8512460DOI Listing
July 2018

A Novel Multimodal MRI Analysis for Alzheimer's Disease Based on Convolutional Neural Network.

Annu Int Conf IEEE Eng Med Biol Soc 2018 Jul;2018:754-757

Recent years, Alzheimer's disease (AD) has become a significant threat to human health while the accurate screening and diagnosis of AD remain a tough problem. Multimodal Magnetic resonance imaging (MRI) can help to identify the variation of brain function and structure in a non-invasive way. Deep learning, especially the convolutional neural networks (CNN), can be utilized to automatically detect appropriate features for classification, which is well adapted for computer-aided AD screening and identification. This paper proposed a multimodal MRI analytical method based on CNN, which is also suitable for single type MRI data analysis. First, the human brain network connectivity matrix were extracted from multimodal MRI data, used as the input data for CNN. Then a novel CNN framework was proposed to process the network matrix and classify AD, amnestic mild cognitive impairment (aMCI) patients and normal controls (NC). The advantage of this method lies in that we combined multimodal MRI information through CNN convolution kernel, and achieved a higher classification accuracy. In our experiments, the comprehensive classification accuracy of AD, aMCI patients and NC was as high as 92.06% when using multimodal MRI data as input, which is effective enough to provide a reference for multimodal MRI data analysis.
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http://dx.doi.org/10.1109/EMBC.2018.8512372DOI Listing
July 2018

An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein.

J Biol Chem 2018 01 7;293(1):47-68. Epub 2017 Nov 7.

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106; Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106. Electronic address:

Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling , of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (Δ 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation is compatible with native duration of activity Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.
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http://dx.doi.org/10.1074/jbc.M117.808626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766902PMC
January 2018

Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding.

J Biol Chem 2018 01 7;293(1):69-88. Epub 2017 Nov 7.

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106; Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106; Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106. Electronic address:

Domain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.
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http://dx.doi.org/10.1074/jbc.M117.808667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766920PMC
January 2018

A study of feature extraction for Alzheimer's disease based on resting-state fMRI.

Annu Int Conf IEEE Eng Med Biol Soc 2017 Jul;2017:517-520

The Alzheimer's Disease (AD) has become a major threat of human health with its incidence rate ascending year by year. Early diagnosis of AD is very important for AD patients to keep life quality. The resting-state fMRI (rs-fMRI) which precisely reflects the brain changes on the resting state of individuals provides a quantitative approach, which has been introduced to distinguish AD patients from normal population. In this study, we proposed a method to find the most distinctive features identifying AD patients from rs-fMRI images. The ALFF and ReHo parameters based on pre-processed rs-fMRI data were extracted, and some key parameters of the brain functional network based on graph theory were calculated. Then we tested the recognition performance of different classifiers, and the best classification algorithm, that is, Support Vector Machine (SVM) with linear-kernel are selected. Finally through a recursive feature selection procedure, we got the most distinctive feature set. Additionally, this study also implies that there may be several changes in some particular ROIs of the brain during the AD development.
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http://dx.doi.org/10.1109/EMBC.2017.8036875DOI Listing
July 2017

How Social Communications Influence Advertising Perception and Response in Online Communities?

Front Psychol 2017 14;8:1349. Epub 2017 Aug 14.

Department of Marketing, Hang Seng Management CollegeHong Kong, China.

This research aims to explore how social communications of online communities affect users' perception and responses toward social media advertising. We developed a conceptual model based on the SBT, encapsulating 9 constructs and 10 hypothesis extracted from the extant social media advertising literature. Our research outcome proves that social communications can effectively boost users' behaviors to be in accordance with an online social community, thus facilitate their acceptance and responses toward social media advertising, with users' group intention as an intervening factor. From an operational standpoint, it's an effective way to build and maintain social bonds between users and the community by boosting social communications, supporting fluent interpersonal communications. In addition, managers of an online community should elaborate on users' group intentions to increase users' advertising acceptance and response.
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http://dx.doi.org/10.3389/fpsyg.2017.01349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557725PMC
August 2017

Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

J Biol Chem 2016 Oct 30;291(42):22173-22195. Epub 2016 Aug 30.

From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity.
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http://dx.doi.org/10.1074/jbc.M116.741959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063998PMC
October 2016

Protective hinge in insulin opens to enable its receptor engagement.

Proc Natl Acad Sci U S A 2014 Aug 4;111(33):E3395-404. Epub 2014 Aug 4.

Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia

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 B-chain 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-β2 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 receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.
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http://dx.doi.org/10.1073/pnas.1412897111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143003PMC
August 2014

Biophysical optimization of a therapeutic protein by nonstandard mutagenesis: studies of an iodo-insulin derivative.

J Biol Chem 2014 Aug 3;289(34):23367-81. Epub 2014 Jul 3.

From the Departments of Biochemistry, Medicine, and Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106

Insulin provides a model for the therapeutic application of protein engineering. A paradigm in molecular pharmacology was defined by design of rapid-acting insulin analogs for the prandial control of glycemia. Such analogs, a cornerstone of current diabetes regimens, exhibit accelerated subcutaneous absorption due to more rapid disassembly of oligomeric species relative to wild-type insulin. This strategy is limited by a molecular trade-off between accelerated disassembly and enhanced susceptibility to degradation. Here, we demonstrate that this trade-off may be circumvented by nonstandard mutagenesis. Our studies employed Lys(B28), Pro(B29)-insulin ("lispro") as a model prandial analog that is less thermodynamically stable and more susceptible to fibrillation than is wild-type insulin. We have discovered that substitution of an invariant tyrosine adjoining the engineered sites in lispro (Tyr(B26)) by 3-iodo-Tyr (i) augments its thermodynamic stability (ΔΔGu 0.5 ± 0.2 kcal/mol), (ii) delays onset of fibrillation (lag time on gentle agitation at 37 °C was prolonged by 4-fold), (iii) enhances affinity for the insulin receptor (1.5 ± 0.1-fold), and (iv) preserves biological activity in a rat model of diabetes mellitus. (1)H NMR studies suggest that the bulky iodo-substituent packs within a nonpolar interchain crevice. Remarkably, the 3-iodo-Tyr(B26) modification stabilizes an oligomeric form of insulin pertinent to pharmaceutical formulation (the R6 zinc hexamer) but preserves rapid disassembly of the oligomeric form pertinent to subcutaneous absorption (T6 hexamer). By exploiting this allosteric switch, 3-iodo-Tyr(B26)-lispro thus illustrates how a nonstandard amino acid substitution can mitigate the unfavorable biophysical properties of an engineered protein while retaining its advantages.
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http://dx.doi.org/10.1074/jbc.M114.588277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156050PMC
August 2014

P450 enzyme-inducing and non-enzyme-inducing antiepileptic drugs for seizure prophylaxis after glioma resection surgery: a meta-analysis.

Seizure 2014 Sep 14;23(8):616-21. Epub 2014 May 14.

Department of Neurosurgery, West China Hospital, Si Chuan University, Chengdu 610041, China. Electronic address:

Purpose: The prognoses of seizure treatment with P450 enzyme-inducing and non-enzyme-inducing antiepileptic drugs after glioma resection surgery were investigated across several clinical studies. However, the results of these studies are inconsistent. We examined the relevant studies and conducted a meta-analysis of these two types of anti-epileptic drugs.

Methods: A bibliography search using the EMBASE, MEDLINE, ClinicalTrials.gov, and Cochrane Central Register of Controlled Trials databases was performed to identify potentially relevant articles and conference abstracts that investigated the effects of non-enzyme-inducing antiepileptic drugs (NEIAEDs) and enzyme-inducing antiepileptic drugs (EIAEDs) on the seizure prognoses of glioma patients.

Results: One RCT study and five observational studies were included. Pooled estimates of the relative risks (OR) and 95% confidence intervals (CI) were calculated. The pooled odds ratio for NEIAEDs vs. EIAEDs for patients with glioma was 1.12 (95% CI=0.70-2.10). The pooled odds ratio for NEIAEDs vs. EIAEDs for low-grade gliomas was 1.77 (95% CI=0.71-4.40). The pooled odds ratio for LEV vs. PHY was 1.459 (95% CI=0.731-2.910).

Conclusions: No significant difference between the efficacies of P450 enzyme-inducing and non-enzyme-inducing antiepileptic drugs for prophylactic late seizure treatment was observed. However, few RCTs were available, and the acquisition of further evidence through high-quality RCTs is highly recommended.
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http://dx.doi.org/10.1016/j.seizure.2014.04.016DOI Listing
September 2014

Ki-67 overexpression in WHO grade II gliomas is associated with poor postoperative seizure control.

Seizure 2013 Dec 12;22(10):877-81. Epub 2013 Aug 12.

Department of Neurosurgery, West China Hospital of Sichuan university, 610041 Chengdu, China.

Purpose: Seizures are the most common initial symptom in patients with low-grade gliomas, and approximately 30% of these patients still suffer from epilepsy after gross-total resection of the tumour. We examined the relationship between the overexpression of ki-67 in WHO grade II gliomas and seizure control.

Methods: A series of 93 histologically confirmed WHO grade II glioma tissues were analysed through immunohistochemical staining for ki-67 expression. Follow-up visits regarding seizure control were scheduled at 12 months. The Engel classification was used to categorise patients' seizure status.

Results: Of the 93 patients analysed, 65 (66.3%) patients initially presented with seizures. A total of 36 patients were diagnosed with WHO grade II oligodendrogliomas, 29 patients had oligoastrocytomas and 28 patients had astrocytomas. Ki-67 was over-expressed in 15 patients. One year after surgery poor seizure control was observed in 11 of these patients. In contrast, low ki-67 expression (<10%) was found in 78 patients. Poor seizure control was observed in 36 patients (difference between ki-67 over- and low expression groups P = 0.002). Logistic regression analysis revealed that patients with gross-total resection achieved better seizure control while ki-67 overexpression and age below 38 years were poor seizure control factors explained of the variance of seizure outcome (OR: 0.382, 4.354 and 1.822, respectively).

Conclusions: In WHO grade II gliomas, Ki-67 is a molecular marker which predicts poor seizure control of glioma patients after the resection of the tumour. Gross-total resection, ki-67 overexpression and age below 38 years significantly affect seizure prognosis.
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http://dx.doi.org/10.1016/j.seizure.2013.08.004DOI Listing
December 2013

Deciphering the hidden informational content of protein sequences: foldability of proinsulin hinges on a flexible arm that is dispensable in the mature hormone.

J Biol Chem 2010 Oct 27;285(40):30989-1001. Epub 2010 Jul 27.

Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe(B1) in placental mammals) is variably positioned within crystal structures and exhibits (1)H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1-B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.
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http://dx.doi.org/10.1074/jbc.M110.152645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945590PMC
October 2010

An Achilles' heel in an amyloidogenic protein and its repair: insulin fibrillation and therapeutic design.

J Biol Chem 2010 Apr 27;285(14):10806-21. Epub 2010 Jan 27.

From the Departments of Biochemistry Case Western Reserve University, Cleveland, Ohio 44106, USA.

Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native monomer leads to aggregation-coupled misfolding. Whereas beta-cells are protected from proteotoxicity by hexamer assembly, fibrillation limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous (13)C NMR chemical shifts and rapid (15)N-detected (1)H-(2)H amide-proton exchange were observed in one of the three classical alpha-helices (residues A1-A8) of the hormone, suggesting a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these anomalies in accordance with its protective role, solid-state (13)C NMR studies suggest that the A-chain segment participates in a fibril-specific beta-sheet. Accordingly, we investigated whether helicogenic substitutions in the A1-A8 segment might delay fibrillation. Simultaneous substitution of three beta-branched residues (Ile(A2) --> Leu, Val(A3) --> Leu, and Thr(A8) --> His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton exchange in the A1-A8 segment remained rapid, (13)Calpha chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as Ile(A2) and Val(A3) define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin. Although (13)Calpha chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an "Achilles' heel" in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their therapeutic deployment in the developing world.
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http://dx.doi.org/10.1074/jbc.M109.067850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856287PMC
April 2010

Solution structure of proinsulin: connecting domain flexibility and prohormone processing.

J Biol Chem 2010 Mar 27;285(11):7847-51. Epub 2010 Jan 27.

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.

The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic beta-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed by {(1)H}-(15)N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit alpha-helical-like features. Relative to canonical alpha-helices, however, segmental (13)C(alpha/beta) chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.
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http://dx.doi.org/10.1074/jbc.C109.084921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832934PMC
March 2010

Structural basis of focal adhesion localization of LIM-only adaptor PINCH by integrin-linked kinase.

J Biol Chem 2009 Feb 30;284(9):5836-44. Epub 2008 Dec 30.

Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA.

The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK.PINCH complex (28 kDa, K(D) approximately 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.
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http://dx.doi.org/10.1074/jbc.M805319200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2645833PMC
February 2009

Doublesex and the regulation of sexual dimorphism in Drosophila melanogaster: structure, function, and mutagenesis of a female-specific domain.

J Biol Chem 2008 Mar 9;283(11):7280-92. Epub 2008 Jan 9.

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.

The DSX (Doublesex) transcription factor regulates somatic sexual differentiation in Drosophila. Female and male isoforms (DSX F and DSX M) are formed due to sex-specific RNA splicing. DNA recognition, mediated by a shared N-terminal zinc module (the DM domain), is enhanced by a C-terminal dimerization element. Sex-specific extension of this element in DSX F and DSX M leads to assembly of distinct transcriptional preinitiation complexes. Here, we describe the structure of the extended C-terminal dimerization domain of DSX F as determined by multidimensional NMR spectroscopy. The core dimerization element is well ordered, giving rise to a dense network of interresidue nuclear Overhauser enhancements. The structure contains dimer-related UBA folds similar to those defined by x-ray crystallographic studies of a truncated domain. Whereas the proximal portion of the female tail extends helix 3 of the UBA fold, the distal tail is disordered. Ala substitutions in the proximal tail disrupt the sex-specific binding of IX (Intersex), an obligatory partner protein and putative transcriptional coactivator; IX-DSX F interaction is, by contrast, not disrupted by truncation of the distal tail. Mutagenesis of the UBA-like dimer of DSX F highlights the importance of steric and electrostatic complementarity across the interface. Two temperature-sensitive mutations at this interface have been characterized in yeast model systems. One weakens a network of solvated salt bridges, whereas the other perturbs the underlying nonpolar interface. These mutations confer graded gene-regulatory activity in yeast within a physiological temperature range and so may provide novel probes for genetic analysis of a sex-specific transcriptional program in Drosophila development.
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http://dx.doi.org/10.1074/jbc.M708742200DOI Listing
March 2008

Structural and functional insights into PINCH LIM4 domain-mediated integrin signaling.

Nat Struct Biol 2003 Jul;10(7):558-64

Department of Pharmacology, School of Medicine, Case Western Reserve University, 10500 Euclid Avenue, Cleveland, Ohio 44102, USA.

PINCH is an adaptor protein found in focal adhesions, large cellular complexes that link extracellular matrix to the actin cytoskeleton. PINCH, which contains an array of five LIM domains, has been implicated as a platform for multiple protein-protein interactions that mediate integrin signaling within focal adhesions. We had previously characterized the LIM1 domain of PINCH, which functions in focal adhesions by binding specifically to integrin-linked kinase. Using NMR spectroscopy, we show here that the PINCH LIM4 domain, while maintaining the conserved LIM scaffold, recognizes the third SH3 domain of another adaptor protein, Nck2 (also called Nckbeta or Grb4), in a manner distinct from that of the LIM1 domain. Point mutation of LIM residues in the SH3-binding interface disrupted LIM-SH3 interaction and substantially impaired localization of PINCH to focal adhesions. These data provide novel structural insight into LIM domain-mediated protein-protein recognition and demonstrate that the PINCH-Nck2 interaction is an important component of the focal adhesion assembly during integrin signaling.
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http://dx.doi.org/10.1038/nsb938DOI Listing
July 2003

Mapping the ligand binding pocket in the cellular retinaldehyde binding protein.

J Biol Chem 2003 Apr 20;278(14):12390-6. Epub 2003 Jan 20.

Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

Retinoid interactions determine the function of the cellular retinaldehyde binding protein (CRALBP) in the rod visual cycle where it serves as an 11-cis-retinol acceptor for the enzymatic isomerization of all-trans- to 11-cis-retinol and as a substrate carrier for 11-cis-retinol dehydrogenase (RDH5). Based on preliminary NMR studies suggesting retinoid interactions with Met and Trp residues, human recombinant CRALBP (rCRALBP) with altered Met or Trp were produced and analyzed for ligand interactions. The primary structures of the purified proteins were verified for mutants M208A, M222A, M225A, W165F, and W244F, then retinoid binding properties and substrate carrier functions were evaluated. All the mutant proteins bound 11-cis- and 9-cis-retinal and therefore were not grossly misfolded. Altered UV-visible spectra and lower retinoid binding affinities were observed for the mutants, supporting modified ligand interactions. Altered kinetic parameters were observed for RDH5 oxidation of 11-cis-retinol bound to rCRALBP mutants M222A, M225A, and W244F, supporting impaired substrate carrier function. Heteronuclear single quantum correlation NMR analyses confirmed localized structural changes upon photoisomerization of rCRALBP-bound 11-cis-retinal and demonstrated ligand-dependent conformational changes for residues Met-208, Met-222, Trp-165, and Trp-244. Furthermore, residues Met-208, Met-222, Met-225, and Trp-244 are within a region exhibiting high homology to the ligand binding cavity of phosphatidylinositol transfer protein. Overall the data implicate Trp-165, Met-208, Met-222, Met-225, and Trp-244 as components of the CRALBP ligand binding cavity.
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http://dx.doi.org/10.1074/jbc.M212775200DOI Listing
April 2003

Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions.

J Biol Chem 2003 Apr 20;278(14):12397-402. Epub 2003 Jan 20.

Department of Medical Biosciences, Umeå University, S-901 85 Umeå, Sweden.

Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.
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http://dx.doi.org/10.1074/jbc.M207300200DOI Listing
April 2003

Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites.

J Cell Sci 2002 Dec;115(Pt 24):4777-86

Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

PINCH, integrin-linked kinase (ILK) and calponin homology-containing ILK-binding protein (CH-ILKBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites we have undertaken a combined structural, mutational and cell biological analysis. Three-dimensional structure-based point mutations identified specific PINCH and ILK sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-ILK-CH-ILKBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-ILK-CH-ILKBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibited the PINCH-ILK-CH-ILKBP complex formation, suggesting that the PINCH-ILK-CH-ILKBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally, we provide evidence that the formation of the PINCH-ILK-CH-ILKBP complex, while necessary, is not sufficient for ILK localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures.
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http://dx.doi.org/10.1242/jcs.00166DOI Listing
December 2002

Mechanism of magnesium activation of calcium-activated potassium channels.

Nature 2002 Aug;418(6900):876-80

Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106-7207, USA.

Large-conductance (BK type) Ca(2+)-dependent K(+) channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing. BK channels are activated by membrane depolarization and intracellular Ca(2+) and Mg(2+) (refs 6-10). The energy provided by voltage, Ca(2+) and Mg(2+) binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways. Here we report a molecular investigation of a Mg(2+)-dependent activation mechanism. Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg(2+)-binding site in the RCK/Rossman fold domain -- an intracellular structural motif that immediately follows the activation gate S6 helix -- is responsible for Mg(2+)-dependent activation. Mutations that impair or abolish Mg(2+) sensitivity do not affect Ca(2+) sensitivity, and vice versa. These results indicate distinct structural pathways for Mg(2+)- and Ca(2+)-dependent activation and suggest a possible mechanism for the coupling between Mg(2+) binding and channel opening.
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http://dx.doi.org/10.1038/nature00941DOI Listing
August 2002
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