Biochemistry and Pharmacology - Research Publications
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ItemTadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2018-05-11)Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.
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ItemPolymorphism in disease-related apolipoprotein C-II amyloid fibrils: a structural model for rod-like fibrils(WILEY, 2018-08)Human apolipoprotein (apo) C-II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid-free apoC-II readily self-assembles into twisted-ribbon amyloid fibrils but forms straight, rod-like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod-like fibril formation compared to their effects on twisted-ribbon fibril formation. For instance, the double mutant, K30D-D69K apoC-II, readily formed twisted-ribbon fibrils, while the rate of rod-like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod-like apoC-II fibrils, using hydrogen-deuterium exchange and NMR analysis showed exchange protection consistent with a core cross-β structure comprising the C-terminal 58-76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross-β structure. X-ray fibre diffraction data for aligned rod-like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted-ribbon fibrils and are predicted for a model involving two cross-β fibrils in an off-set antiparallel structure with four apoC-II units per rise of the β-sheet. This model is consistent with the mutational effects on rod-like apoC-II fibril formation. The lipid-dependent polymorphisms exhibited by apoC-II fibrils could determine the properties of apoC-II in renal amyloid deposits and their potential role in the development of cardiovascular disease.
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ItemUnravelling the Carbohydrate-Binding Preferences of the Carbohydrate-Binding Modules of AMP-Activated Protein Kinase(WILEY-V C H VERLAG GMBH, 2018-02-02)The β subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the β1- and β2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. β2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2-Thr101Δ/Val134Thr differs from that of β1-CBM, and mutant β1-Thr101ins/Thr134Val differs from that of β2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type β-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.
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ItemElucidation of relaxin-3 binding interactions in the extracellular loops of RXFP3.(Frontiers Media SA, 2013)Relaxin-3 is a highly conserved neuropeptide in vertebrate species and binds to the Class A G protein-coupled receptor (GPCR) RXFP3. Relaxin-3 is involved in a wide range of behaviors, including feeding, stress responses, arousal, and cognitive processes and therefore targeting of RXFP3 may be relevant for a range of neurological diseases. Structural knowledge of RXFP3 and its interaction with relaxin-3 would both increase our understanding of ligand recognition in GPCRs that respond to protein ligands and enable acceleration of the design of drug leads. In this study we have used comparative sequence analysis, molecular modeling and receptor mutagenesis to investigate the binding site of the native ligand human relaxin-3 (H3 relaxin) on the human RXFP3 receptor. Previous structure function studies have demonstrated that arginine residues in the H3 relaxin B-chain are critical for binding interactions with the receptor extracellular loops and/or N-terminal domain. Hence we have concentrated on determining the ligand interacting sites in these domains and have focused on glutamic (E) and aspartic acid (D) residues in these regions that may form electrostatic interactions with these critical arginine residues. Conserved D/E residues identified from vertebrate species multiple sequence alignments were mutated to Ala in human RXFP3 to test the effect of loss of amino acid side chain on receptor binding using a Eu-labeled relaxin-3 agonist. Finally data from mutagenesis experiments have been used in ligand docking simulations to a homology model of human RXFP3 based on the peptide-bound chemokine receptor 4 (CXCR4) structure. These studies have resulted in a model of the relaxin-3 interaction with RXFP3 which will inform further interrogation of the agonist binding site.
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ItemThe Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015-05-01)The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.
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ItemThe complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1(NATURE PUBLISHING GROUP, 2016-04)H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.
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ItemCharacterisation of a cell-free synthesised G-protein coupled receptor(NATURE PORTFOLIO, 2017-04-24)G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS1). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS1 produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS1 showed non-competitive binding to the peptide ligand neurotensin8-13 when compared to E. coli produced en2NTS1. 1H-13C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced 13CH3-methionine labelled en2NTS1. The results obtained, indicate CFPS produced en2NTS1 is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.
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Itemβ-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase(NATURE PORTFOLIO, 2016-12-21)The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.
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ItemDistinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin(NATURE PORTFOLIO, 2017-06-12)Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker's highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.
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ItemMulti-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis(CELL PRESS, 2019-01-25)The peptide hormone H2 relaxin has demonstrated promise as a therapeutic, but mimetic development has been hindered by the poorly understood relaxin receptor RXFP1 activation mechanism. H2 relaxin is hypothesized to bind to two distinct ECD sites, which reorientates the N-terminal LDLa module to activate the transmembrane domain. Here we provide evidence for this model in live cells by measuring bioluminescence resonance energy transfer (BRET) between nanoluciferase-tagged RXFP1 constructs and fluorescently labeled H2 relaxin (NanoBRET). Additionally, we validate these results using the related RXFP2 receptor and chimeras with an inserted RXFP1-binding domain utilizing NanoBRET and nuclear magnetic resonance studies on recombinant proteins. We therefore provide evidence for the multi-component molecular mechanism of H2 relaxin binding to RXFP1 on the full-length receptor in cells. Also, we show the utility of NanoBRET real-time binding kinetics to reveal subtle binding complexities, which may be overlooked in traditional equilibrium binding assays.