Publications by authors named "Emma J Petrie"

27 Publications

  • Page 1 of 1

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis.

Nat Commun 2021 04 13;12(1):2211. Epub 2021 Apr 13.

Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.
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http://dx.doi.org/10.1038/s41467-021-22400-zDOI Listing
April 2021

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

Nat Commun 2020 06 19;11(1):3150. Epub 2020 Jun 19.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
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http://dx.doi.org/10.1038/s41467-020-16819-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305203PMC
June 2020

Distinct pseudokinase domain conformations underlie divergent activation mechanisms among vertebrate MLKL orthologues.

Nat Commun 2020 06 19;11(1):3060. Epub 2020 Jun 19.

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.

The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL's conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the divergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, divergent regulatory mechanisms may exist among orthologous pseudoenzymes.
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http://dx.doi.org/10.1038/s41467-020-16823-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305131PMC
June 2020

MLKL trafficking and accumulation at the plasma membrane control the kinetics and threshold for necroptosis.

Nat Commun 2020 06 19;11(1):3151. Epub 2020 Jun 19.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

Mixed lineage kinase domain-like (MLKL) is the terminal protein in the pro-inflammatory necroptotic cell death program. RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption, although the precise choreography of events is incompletely understood. Here, we use single-cell imaging approaches to map the chronology of endogenous human MLKL activation during necroptosis. During the effector phase of necroptosis, we observe that phosphorylated MLKL assembles into higher order species on presumed cytoplasmic necrosomes. Subsequently, MLKL co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms. MLKL and tight junction proteins then steadily co-accumulate at the plasma membrane as heterogeneous micron-sized hotspots. Our studies identify MLKL trafficking and plasma membrane accumulation as crucial necroptosis checkpoints. Furthermore, the accumulation of phosphorylated MLKL at intercellular junctions accelerates necroptosis between neighbouring cells, which may be relevant to inflammatory bowel disease and other necroptosis-mediated enteropathies.
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http://dx.doi.org/10.1038/s41467-020-16887-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305196PMC
June 2020

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies.

Proc Natl Acad Sci U S A 2020 04 31;117(15):8468-8475. Epub 2020 Mar 31.

Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the "necrosome" and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.
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http://dx.doi.org/10.1073/pnas.1919960117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165463PMC
April 2020

Viral MLKL Homologs Subvert Necroptotic Cell Death by Sequestering Cellular RIPK3.

Cell Rep 2019 09;28(13):3309-3319.e5

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia. Electronic address:

Necroptotic cell death has been implicated in many human pathologies and is thought to have evolved as an innate immunity mechanism. The pathway relies on two key effectors: the kinase receptor-interacting protein kinase 3 (RIPK3) and the terminal effector, the pseudokinase mixed-lineage kinase-domain-like (MLKL). We identify proteins with high sequence similarity to the pseudokinase domain of MLKL in poxvirus genomes. Expression of these proteins from the BeAn 58058 and Cotia poxviruses, but not swinepox, in human and mouse cells blocks cellular MLKL activation and necroptotic cell death. We show that viral MLKL-like proteins function as dominant-negative mimics of host MLKL, which inhibit necroptosis by sequestering RIPK3 via its kinase domain to thwart MLKL engagement and phosphorylation. These data support an ancestral role for necroptosis in defense against pathogens. Furthermore, mimicry of a cellular pseudokinase by a pathogen adds to the growing repertoire of functions performed by pseudokinases in signal transduction.
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http://dx.doi.org/10.1016/j.celrep.2019.08.055DOI Listing
September 2019

The Structural Basis of Necroptotic Cell Death Signaling.

Trends Biochem Sci 2019 01 30;44(1):53-63. Epub 2018 Nov 30.

Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia. Electronic address:

The recent implication of the cell death pathway, necroptosis, in innate immunity and a range of human pathologies has led to intense interest in the underlying molecular mechanism. Unlike the better-understood apoptosis pathway, necroptosis is a caspase-independent pathway that leads to cell lysis and release of immunogens downstream of death receptor and Toll-like receptor (TLR) ligation. Here we review the role of recent structural studies of the core machinery of the pathway, the protein kinases receptor-interacting protein kinase (RIPK)1 and RIPK3, and the terminal effector, the pseudokinase mixed lineage kinase domain-like protein (MLKL), in shaping our mechanistic understanding of necroptotic signaling. Structural studies have played a key role in establishing models that describe MLKL's transition from a dormant monomer to a killer oligomer and revealing important interspecies differences.
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http://dx.doi.org/10.1016/j.tibs.2018.11.002DOI Listing
January 2019

Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis.

Nat Commun 2018 06 21;9(1):2422. Epub 2018 Jun 21.

The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species.
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http://dx.doi.org/10.1038/s41467-018-04714-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013482PMC
June 2018

The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis.

Cell Death Differ 2018 09 14;25(9):1567-1580. Epub 2018 Feb 14.

The Walter & Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.

The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL's pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) 'executioner' domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two 'brace' helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.
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http://dx.doi.org/10.1038/s41418-018-0061-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143630PMC
September 2018

Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin.

Sci Rep 2017 06 12;7(1):3294. Epub 2017 Jun 12.

Department of Biochemistry & Molecular Biology, The University of Melbourne, Victoria, Australia.

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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http://dx.doi.org/10.1038/s41598-017-03638-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468325PMC
June 2017

EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

Nat Microbiol 2017 01 13;2:16258. Epub 2017 Jan 13.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia.

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis. Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.
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http://dx.doi.org/10.1038/nmicrobiol.2016.258DOI Listing
January 2017

Insane in the membrane: a structural perspective of MLKL function in necroptosis.

Immunol Cell Biol 2017 02 17;95(2):152-159. Epub 2017 Jan 17.

Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Necroptosis (or 'programmed necrosis') is a caspase-independent cell death pathway that operates downstream of death receptors, including Tumour Necrosis Factor Receptor-1 (TNFR1), and the Toll-like receptors, TLR3 and TLR4. Owing to its immunogenicity, necroptosis has been attributed roles in the pathogenesis of several diseases, including inflammatory bowel disease and the tissue damage arising from ischaemic-reperfusion injuries. Only over the past 7 years has the core machinery of this pathway, the receptor-interacting protein kinase-3 (RIPK3) and the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), been defined. Our current understanding of the pathway is that RIPK3-mediated phosphorylation activates cytoplasmic MLKL, which is the most terminal known effector in the pathway, leading to MLKL's oligomerisation, translocation to, and permeabilisation of, the plasma membrane. Here, we discuss the insights gleaned from structural and biophysical studies of MLKL and highlight the known unknowns surrounding MLKL's mechanism of action and activation.
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http://dx.doi.org/10.1038/icb.2016.125DOI Listing
February 2017

The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis.

J Clin Invest 2016 11 17;126(11):4346-4360. Epub 2016 Oct 17.

Although necrosis and necroinflammation are central features of many liver diseases, the role of programmed necrosis in the context of inflammation-dependent hepatocellular death remains to be fully determined. Here, we have demonstrated that the pseudokinase mixed lineage kinase domain-like protein (MLKL), which plays a key role in the execution of receptor-interacting protein (RIP) kinase-dependent necroptosis, is upregulated and activated in human autoimmune hepatitis and in a murine model of inflammation-dependent hepatitis. Using genetic and pharmacologic approaches, we determined that hepatocellular necrosis in experimental hepatitis is driven by an MLKL-dependent pathway that occurs independently of RIPK3. Moreover, we have provided evidence that the cytotoxic activity of the proinflammatory cytokine IFN-γ in hepatic inflammation is strongly connected to induction of MLKL expression via activation of the transcription factor STAT1. In summary, our results reveal a pathway for MLKL-dependent programmed necrosis that is executed in the absence of RIPK3 and potentially drives the pathogenesis of severe liver diseases.
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http://dx.doi.org/10.1172/JCI87545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096909PMC
November 2016

The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

Nat Commun 2016 Apr 18;7:11344. Epub 2016 Apr 18.

Department of Biochemistry &Molecular Biology, The University of Melbourne, Victoria 3010, Australia.

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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http://dx.doi.org/10.1038/ncomms11344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837482PMC
April 2016

Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein.

Chemistry 2016 Jan 27;22(3):1146-51. Epub 2015 Nov 27.

Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, 3010, Australia.

The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N(α) -deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca(2+) ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.
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http://dx.doi.org/10.1002/chem.201503599DOI Listing
January 2016

In a Class of Their Own - RXFP1 and RXFP2 are Unique Members of the LGR Family.

Front Endocrinol (Lausanne) 2015 7;6:137. Epub 2015 Sep 7.

Department of Biochemistry and Molecular Biology, University of Melbourne , Parkville, VIC , Australia ; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, VIC , Australia.

The leucine-rich repeat-containing G protein-coupled receptors (LGRs) family consists of three groups: types A, B, and C and all contain a large extracellular domain (ECD) made up of the structural motif - the leucine-rich repeat (LRR). In the LGRs, the ECD binds the hormone or ligand, usually through the LRRs, that ultimately results in activation and signaling. Structures are available for the ECD of type A and B LGRs, but not the type C LGRs. This review discusses the structural features of LRR proteins, and describes the known structures of the type A and B LGRs and predictions that can be made for the type C LGRs. The mechanism of activation of the LGRs is discussed with a focus on the role of the low-density lipoprotein class A (LDLa) module, a unique feature of the type C LGRs. While the LDLa module is essential for activation of the type C LGRs, the molecular mechanism for this process is unknown. Experimental data for the potential interactions of the type C LGR ligands with the LRR domain, the transmembrane domain, and the LDLa module are summarized.
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http://dx.doi.org/10.3389/fendo.2015.00137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561518PMC
October 2015

Determinants of oligosaccharide specificity of the carbohydrate-binding modules of AMP-activated protein kinase.

Biochem J 2015 Jun;468(2):245-57

*Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.

AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The β-subunit exists in two isoforms (β1 and β2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (β1- and β2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. β2-CBM binds linear carbohydrates with 4-fold greater affinity than β1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of β2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-β-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of β1- and β2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of β2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of β2-CBM. In contrast, the α1,6 branch could not be observed in the β1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to β1- and β2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific β2-isoform may have greater affinity for partially degraded glycogen.
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http://dx.doi.org/10.1042/BJ20150270DOI Listing
June 2015

Investigation of interactions at the extracellular loops of the relaxin family peptide receptor 1 (RXFP1).

J Biol Chem 2014 Dec 28;289(50):34938-52. Epub 2014 Oct 28.

the Department of Biochemistry and Molecular Biology, and

Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists.
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http://dx.doi.org/10.1074/jbc.M114.600882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263891PMC
December 2014

Mapping key regions of the RXFP2 low-density lipoprotein class-A module that are involved in signal activation.

Biochemistry 2014 Jul 10;53(28):4537-48. Epub 2014 Jul 10.

Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, ‡Florey Institute of Neuroscience and Mental Health, and §School of Chemistry, University of Melbourne , Parkville, Victoria 3010, Australia.

The peptide hormone INSL3 and its receptor, RXFP2, have co-evolved alongside relaxin and its receptor, RXFP1. Both RXFP1 and RXFP2 are G protein-coupled receptors (GPCRs) containing the hallmark seven transmembrane helices in addition to a distinct ectodomain of leucine-rich repeats (LRRs) and a single low-density lipoprotein class-A (LDLa) module at the N-terminus. RXFP1 and RXFP2 are the only mammalian GPCRs known to contain an LDLa, and its removal does not perturb primary ligand binding to the LRRs; however, signaling is abolished. This presents a general mechanism whereby ligand binding induces a conformational change in the receptor to position the LDLa to elicit a signal response. Although the LDLa interaction site has not been identified, the residues important to the action have been mapped within the RXFP1 LDLa module. In this study, we comprehensively study the RXFP2 LDLa module. We determine its structure using nuclear magnetic resonance (NMR) and concurrently investigate the signaling of an RXFP2 with the LDLa removed (RXFP2-short), confirming that the LDLa is essential to signaling. We then replaced the LDLa with the second ligand binding module from the LDL receptor, LB2, creating the RXFP2-LB2 chimera. Unlike that in the equivalent RXFP1-LB2 chimera, signaling is rescued albeit modestly. Guided by the NMR structure, we dissected regions of the RXFP2 LDLa to identify specific residues that are important to signal activation. We determine that although the module is important to the activation of RXFP2, unlike the RXFP1 receptor, specific residues in the N-terminus of the domain are not involved in signal activation.
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http://dx.doi.org/10.1021/bi500797dDOI Listing
July 2014

Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

Front Endocrinol (Lausanne) 2013 11;4:171. Epub 2013 Nov 11.

Florey Department of Neuroscience and Mental Health, Florey Institute of Neuroscience and Mental Health , Melbourne, VIC , Australia ; Department of Biochemistry and Molecular Biology , Melbourne, VIC , Australia.

Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.
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http://dx.doi.org/10.3389/fendo.2013.00171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822782PMC
November 2013

The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

J Biol Chem 2013 Sep 7;288(39):28138-51. Epub 2013 Aug 7.

From the Florey Institute of Neuroscience and Mental Health and Florey Department of Neuroscience and Mental Health.

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.
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http://dx.doi.org/10.1074/jbc.M113.499640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784725PMC
September 2013

Recombinant RXFP1-LDL-A module does not form dimers.

Ital J Anat Embryol 2013 ;118(1 Suppl):4-6

The Relaxin receptor, RXFP1, is a complex G-protein coupled receptor (GPCR). It has a rhodopsin-like 7 transmembrane helix region and a large ecto-domain containing Leucine-rich repeats and a Low Desnsity Lipoprotein Class-A module at the N-terminus. RXFP1 and the closely related receptor for INSL3, RXFP2 are the only mammalian GPCRs to contain an LDL-A module. The LDL-A module has been shown to be essential for receptor signal activation. RXFP1, like other GPCRs, has been shown to form dimers however the interface upon association is currently unknown. As LDL-A modules are commonly found as repeats we hypothesized that the LDL-A module may associate at the dimer interface and play a role in receptor activation. To this end we analyzed the ability for the LDL-A module to oligomerise via Analytical Ultracentrifugation (AUC).
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May 2014

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22.

J Mol Biol 2011 Jan 16;405(3):804-18. Epub 2010 Nov 16.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia.

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.
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http://dx.doi.org/10.1016/j.jmb.2010.11.017DOI Listing
January 2011

AMPK beta subunits display isoform specific affinities for carbohydrates.

FEBS Lett 2010 Aug 14;584(15):3499-503. Epub 2010 Jul 14.

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.

AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.
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http://dx.doi.org/10.1016/j.febslet.2010.07.015DOI Listing
August 2010

Resolving the unconventional mechanisms underlying RXFP1 and RXFP2 receptor function.

Ann N Y Acad Sci 2009 Apr;1160:67-73

Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia.

The receptors for relaxin and insulin-like peptide 3 (INSL3) are now well-characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G-protein-coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine-rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low-density lipoprotein class A (LDL-A) module at their N-terminus. Ligand-mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B-chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL-A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL-A module is currently unknown. The specific understanding of the mechanisms underlying ligand-mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.
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http://dx.doi.org/10.1111/j.1749-6632.2009.03949.xDOI Listing
April 2009

CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence.

J Exp Med 2008 Mar 10;205(3):725-35. Epub 2008 Mar 10.

The Protein Crystallography Unit, ARC Centre of Excellence in Structural and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A-HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a "lock and key" interaction is typical of innate receptor-ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.
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http://dx.doi.org/10.1084/jem.20072525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275392PMC
March 2008