Recent Advances in Biomolecular NMR Cytoplasm MT Zn,Cu-SOD GSH NMR in Cellular Structural Biology: Mitochondrion CCS CCS Zn,Cu-SOD IMS from Single Molecules to Pathways Cu + Cu + Cox11 MT Matrix CCO Cox17 2S-S Ctr1/2 HAH1 Cox17 Lucia Banci Sco1 D1 Sco2 GSH Magnetic Resonance Center (CERM) D2 D6 D5 D3 ATP7A/B University of Florence D4 Golgi complex SecPr SecPr
Recent Advances in Biomolecular NMR • I n cell NMR For studying biomolecules in a cellular context • Mechanistic Systems Biology To describe and understand biological processes at molecular level • Structural Vaccinlogy Rational vaccine design based on the structural knowldge of the antigene
Integrating a Cellular Approach with Atomic Resolution Living systems are complex: mixture of proteins, nucleic acids, other biomolecules, several cellular compartments,...etc A Systems Biology approach is needed. All the players involved in a given process have to be considered as well as their 3D structural and dynamical interactions determined. Proteins must be framed within their cellular context
Integrating Atomic Resolution with the Cellular Context Copper trafficking in human cells Cu(II) No free copper ions Cu(I) in the cytoplasm Golgi Amine Oxidase, MNK/WLN Lysyl oxidase Hah1 Ceruloplasmin Regulators CCS SOD1 Ctr Nucleus Cu(I) ? Cox23 Sco2 Sco1 GSH GSSG Cox23 SOD MT Cox17 CCS Cox17 CCO Cox11 Cox19 Cox19 ? MT Mitocondria E° o f cytosolic glutathione = -289 mV, corresponding to GSH and GSSG in vivo concentrations of 13 mM and 0.7 mM
Let’s start with a single process Maturation of Cu,Zn-SOD1 C57 Cu Zinc Copper binding binding Zn C146 monomeric Disulfide bond SS bond formation apo hSOD1 SH-SH SOD1: present in cytoplasm, mitochondrial IMS, nucleus, peroxisomes Zn dimeric (Cu 2 ,Zn 2 ) hSOD1 SS Cu Active enzyme: - + 2H + (2O 2 O 2 + H 2 O 2 ) These post translational modifications affect the fold properties and monomer/dimer equilibrium
In-cell NMR can monitor functional processes in live human cells Understanding intracellular processes at the molecular level requires a high resolution description. In-cell NMR provides atomic-level information on a protein in the cellular environment. Transfected HEK293T cells are used as a model system for human cells 15 N apo-SOD1 Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells. Cys 146 15 N HS SH SH Cys 57 Cys 111 1 H Cys 6 Maturation processes such as E,Zn-SOD1 protein folding, post translational apo-SOD1 modifications (i.e. metal binding, 1 H disulfide bond formation) are followed at atomic resolution.
In-cell NMR can monitor functional processes in live human cells Understanding intracellular processes at the molecular level requires a high resolution description. In-cell NMR provides atomic-level information on a protein in the cellular environment. Transfected HEK293T cells are used as a model system for human cells 15 N E,Zn-SOD1 Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells. + Zn(II) Cys 146 15 N HS SH SH HS SH SH HS Cys 57 Cys 111 1 H Cys 6 Maturation processes such as E,Zn-SOD1 protein folding, post translational modifications (i.e. metal binding, 1 H disulfide bond formation) are followed at atomic resolution.
In-cell NMR can monitor functional processes in live human cells Understanding intracellular processes at the molecular level requires a high resolution description. In-cell NMR provides atomic-level information on a protein in the cellular environment. Transfected HEK293T cells are used as a model system for human cells 15 N Cu,Zn-SOD1 Isotopically labelled proteins are overexpressed and directly observed by hi-res NMR in living human cells. + Zn(II) Cys 146 15 N HS SH SH HS S-S SH SH HS Cys 57 Cys 111 + Cu(I) 1 H Cys 6 SH S S SH S S Maturation processes such as Cu,Zn-SOD1 protein folding, post translational modifications (i.e. metal binding, 1 H disulfide bond formation) are followed at atomic resolution. Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol , 2013
Following SOD1 maturation steps in human cells HS SH Cu(I),Zn-SOD1 S-S SH SH HSSH HS SH HS SH SH HS SH SH HSSH S S S S S HS SH S SH S S HS SH HS SH SH CCS Zn(II) E,E-SOD1 SH HS S S HS Cu(I) Zn(II) S S E,Zn-SOD1 SH Cu(II) E,Zn-SOD1 S-S Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol , 2013
Combining in-cell NMR with X-ray fluorescence microscopy In-cell NMR X-ray fluorescence Optical fluorescence E,Zn-SOD1 SH SOD1 nuclei Cu Zn 10 µm Correlation between the intracellular levels of SOD1 and the content of zinc Zn Zn Zinc bound to E,Zn-SOD1 Luchinat E, Gianoncelli A, Mello T, Galli A, Banci L, Chem Commun, 2015
Incomplete maturation of SOD1 fALS mutants - ALS: a motor neuron disease - 20% of familial cases is related to mutations of SOD1. - 165 mutations identified so far, scattered throughout the sequence. - Mutations are thought to cause defects in SOD1 maturation, promoting aggregation of the apo protein Luchinat E, Barbieri L, Rubino JT, Kozyreva T, Cantini F, Banci L, Nat. Comm., 2014
Maturation defects of fALS SOD1 mutants Many SOD1 mutants do not bind zinc in the cell, and accumulate as an unstructured species, which does NOT evolve toward the native form (A4V, I35T, G37R, G85R, G93A, I113T) The mutations do not affect zinc binding in vitro I113T# I113T G93A I113T This unstructured species DOES NOT bind zinc It could be a precursor of SOD1 aggregates Luchinat E, Barbieri L, Rubino JT, Kozyreva T, Cantini F, Banci L, Nat. Comm., 2014
A relatively small-scale, physiologically central system for systems biology: The mitochondrion Mitochondria derive from parassitic Gram-negative bacteria : they contain 1000 proteins but only 15 are produced in situ The large majority of mitochondrial proteins must be imported, including those involved in copper trafficking
Cox17 mitochondrial import IMS Reduced apoCox17 SH is unstructured SH SH SH SH SH SH SH SH SH SH SH apoCox17 3SH-SH Cox 17 is transporting + Mia40 Cu to CcO matrix cytosol OM IM Mia40 3S-S + Cox17 6SH Mia40 2S-S/2SH + Cox17 1S-S/4SH apoCox17 2S-S
Cox17 is unfolded in the cytoplasm detected in living cells The protein folding state depends on the cellular compartment Banci L, Bertini I, Cefaro C, Cenacchi L, Ciofi-Baffoni S, Felli I C, Gallo A, Gonnelli L, Luchinat E, Tokatlidis K, PNAS , 2010
The first step in Cox17 folding The hydrophobic cleft of Mia40 is Intermolecular Mia40- the interaction site for Cox17 Cox17 disulphide bond (detected by 13 C NMR on 13 C Cys ) Cox17 Structure of Cox17- Mia40 intermediate Mia40 Upon intermolecular S-S bond formation, the first helix of Cox17 is formed Then the first intramolecular S-S bond and the second helix are formed O 2 or glutathione can now rapidly form the second disulphide bond Banci, Bertini I, Cefaro C, Cenacchi L, Ciofi-Baffoni S, Felli I C, Gallo A, Gonnelli L, Eluchinat, Tokatlidis K, PNAS, 2010
Oxidative folding reaction between Mia40 and Cox17
Oxidative folding processes in IMS Mia40 acts as a chaperon Cytoplasm CHCH N A general folding process for C CHCH- fold proteins …and many more TOM IMS CHCH 1S-S Mia40 N C C N O 2 C CHCH N Mia40- CHCH H 2 O 2 C CHCH 2S-S N Matrix Protein fold state depends on the cellular compartment and is modulated by the protein redox state Banci L, Bertini I, Cefaro C, Cenacchi L, Ciofi S, Felli I C, Gallo A, Gonnelli L, Luchinat E, Tokatlidis K, PNAS , 2010
Steps in a mitochondrial pathway COX17 CcO copper chaperone Mia40 ALR Mitochondrial intermembrane Augmenter of Liver space Import Regeneration and Assembly protein 40 ALR regenerates the active import redox state of Mia40, i.e. with a disulfide bond in the CPC site
Structural model of the ALR/Mia40 complex based on NMR interaction data ALR: a FAD-dependent thiol oxidase It contains 4 SS bonds per subunit, 2 “active” and 2 ALR’ structural Hydrophobic interactions between Mia40 and the N-ter domain of ALR mediate efficient electron transfer from Mia40 to FAD in ALR ALR Mia40 N-terminus domain of ALR is unstructured Banci L, Bertini I, Calderone V, Cefaro C, Ciofi-Baffoni S, Gallo A, Tokatlidis K PNAS 2011
Electron shuttling mechanism ALR oxidized Mia40 2S-S Through 13 C NMR e - on 13 C Cys 2 + ALR reduced Mia40 3S-S C C C N 2 + +e - +e - N N ALR then transfers electrons to Cyt c Banci L, Bertini I, Calderone V, Cefaro C, Ciofi-Baffoni S, Gallo A, Tokatlidis K PNAS 2011
IMS protein import COX17 CcO copper chaperone e - e - ALR e - Mia40 CytC Augmenter of Mitochondrial intermembrane cytochrome c Liver space Import Regeneration and Assembly protein 40
Cu A assembly in the mitochondrion Cu(I)-Cox17 Cu Cu Cu Cu A Sco1 Cu B Sco2 CCO Matrix Cu Cu Cu IMS
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