Recent Advances in Biomolecular NMR Cytoplasm MT Zn,Cu-SOD GSH NMR in Cellular Structural Biology: Mitochondrion CCS CCS from Single Molecules to Pathways Zn,Cu-SOD IMS Cu + Cu + Cox11 MT Matrix CCO Cox17 2S-S Ctr1/2 HAH1 Lucia Banci Cox17 Sco1 D1 Sco2 GSH Magnetic Resonance Center (CERM) D2 D6 D5 D3 ATP7A/B University of Florence D4 SecPr Golgi complex SecPr EMBO Global Exchange Course Singapore 6-13 December 2017
Recent Advances in Biomolecular NMR • Mechanistic Systems Biology To describe and understand biological processes at molecular level • I n cell NMR For studying biomolecules in a cellular context • 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 in the cytoplasm Cu(I) Golgi Amine Oxidase, MNK/WLN Lysyl oxidase Hah1 Ceruloplasmin Regulators CCS SOD1 Ctr Nucleus Cu(I) ? Cox23 Sco2 Sco1 GSH GSSG Cox23 MT SOD Cox17 CCS Cox17 CCO Cox11 Cox19Cox19 ? 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 apo-SOD1 15 N 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 1 H modifications (i.e. metal binding, 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 E,Zn-SOD1 15 N 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 1 H modifications (i.e. metal binding, 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 Cu,Zn-SOD1 15 N 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 S SH S SH S S Maturation processes such as Cu,Zn-SOD1 protein folding, post translational 1 H modifications (i.e. metal binding, 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
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
Copper trafficking in human cells Cu(II) No free copper ions in the cytoplasm Cu(I) Golgi Amine Oxidase, MNK/WLN Lysyl oxidase Hah1 Ceruloplasmin SOD1 CCS Regulators Ctr Nucleus Cu(I) Sco2 ? Cox23 Sco1 Cox23 MT SOD Cox17 CCS Cox11 CCO Cox19 Cox19 ? MT Mitocondria
Towards systems biology of copper The knowledge of the structures of the proteins and of their complexes allows the atomic level description of the transfer processes Banci, Bertini, Ciofi-Baffoni, Karit, Kozyreva, Palumaa, Nature, 2010
System-wide understanding of biological processes requires an atomic-level description of the functional processes
Iron Sulfur Biogenesis in human cells ? BOLA2 GRX3 Cfd1 Cfd1 Nar1 ? ? Nbp35 Nbp35 CIAO1 MIP18 ANAMORSIN MMS19 ? ANAMORSIN Apo ? e - MiA40 Holo NDOR1 Cytosolic Proteins ? ?
Structural properties of Anamorsin An essential protein for FeS cluster biosynthesis Intrinsically Disordered Domain N-terminal domain This motif binds a cluster in the Mia40 cytoplasm recognition site Mia40 forms two disulfide bonds when in mitochondria [2Fe-2S] cluster Linker Folded domain Highly paramagnetic “ standard ” approach with fast relaxation and no PCS The worst case!!
Playing For or Against Paramagnetic Relaxation? Exploit the differences in longitudinal relaxation Inversion Recovery The effect of T 1 relaxation 10 ms 10 ms 20 ms 2 ms 50 ms 50 ms 100 ms 200 ms 500 ms 3-6 Å T 1 < 30ms 30 ms < 6-10 Å T 1 < 100 ms IR delays selected according to T 1 values 100 ms < 10-11 Å T 1 < 200 ms IR is combined with fast recycling times
Playing For or Against Paramagnetic Relaxation? Limit the negative effects of fast transverse relaxation Antiphase signal acquisition INEPT coherence transfer The effect of T 2 relaxation 1 no relax 100ms 10 10 ms ms 0.75 5 ms 0.5 2 ms 0.25 1 ms 0.5 ms 0 ppm 0 0.002 0.004 0.006 d 13 C t(s) We can detect peaks for signals Remove the reverse INEPT with Dn up to ca. 300 Hz Phasing AP in dispersion mode
Tailored 15 N HSQC of the [2Fe-2S]-domain of anamorsin 15 N- HSQC 15 N- IR-HSQC-AP 15 N 15 N 1 H 1 H 13 HN peaks, missing in standard 15 N HSQC, can be detected. 1 H T 1 values range from 5 to 30 ms Banci et al PNAS 2013, 2014; Ciofi-Baffoni, Gallo, Piccioli, J. Biomol NMR 2014
Paramagnetic-tailored 13 C-direct CACO of [2Fe-2S]- domain of anamorsin 13 C-CACO-IPAP 13 C-CACO-AP 13 C 13 C G252 S236 K244 T250 13 C 13 C 13 C signals, absent in standard 13 C-direct esperiments, are also observed via 13 C COSY and tailored CON Overall, about 10 additional 13 C resonances are detected
Structure of the [2Fe-2S] cluster binding region in anamorsin The “paramagnetic” 13 C The overall and 1 H T 1 s provide structure was then structural info around subjected to MD the FeS cluster trajectory in explicit water S236 G252 T250 K244 Blue - residues detected in the “diamagnetic” experiments Cyano - residues whose 13 C or 15 N signals were detected in paramagnetic-tailored 13 C or 15 N experiments
Anamorsin receives the [2Fe-2S] clusters from GRX3 in the cytoplasm The Trx domain of GRX3 is essential for protein-protein recognition Cluster site characterization complemented with EPR and No transfer Moessbauer data Banci, Ciofi-Baffoni, Gajda, Muzzioli, Peruzzini, Winkelmann, Nat. Chem Biol. 2015
… but in oxidative conditions, the BolA2-GRX3 complex is more efficient in anamorsin maturation The GRX3/BolA2 heterocomplex is more stable in oxidative conditions An alternative route to anamorsin maturation anamorsin Banci L, Camponeschi F, Ciofi-Baffoni S, Muzzioli R. J Am Chem Soc. 2015, Nuttle, X. et al. Nature 2016
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