NMR in Cellular Structural Biology: Mitochondrion CCS CCS from - - PowerPoint PPT Presentation
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
Zn,Cu-SOD
HAH1
MT
GSH
Ctr1/2 CCS
Cu+
ATP7A/B
Mitochondrion
Cox17
D1 D2 D3 D4 D5 D6
Golgi complex
Cu+
Zn,Cu-SOD
CCS
MT
IMS
GSH
Sco2 Matrix
Cox11
Cytoplasm
CCO Sco1
Cox172S-S SecPr SecPr
EMBO Global Exchange Course Singapore 6-13 December 2017
Integrating Atomic Resolution with the Cellular Context
No free copper ions in the cytoplasm
Nucleus Golgi Ctr
Regulators MNK/WLN
Amine Oxidase, Lysyl oxidase
Cu(I)
?
Cu(II) Cu(I)
Cox23 Cox23 Cox19Cox19
Mitocondria
Sco1 Sco2 Cox11 MT ? CCS SOD CCO
Ceruloplasmin
SOD1 CCS Hah1 MT Cox17 Cox17
GSH GSSG E° of cytosolic glutathione = -289 mV, corresponding to GSH and GSSG in vivo concentrations of 13 mM and 0.7 mM
monomeric apo hSOD1SH-SH
Copper binding
C57 C146
Zinc binding Disulfide bond formation Zn Cu SS bond
These post translational modifications affect the fold properties and monomer/dimer equilibrium
Cu Zn
dimeric (Cu2,Zn2) hSOD1SS Active enzyme: (2O2
O2+ H2O2)
SOD1: present in cytoplasm, mitochondrial IMS, nucleus, peroxisomes
Isotopically labelled proteins are overexpressed and directly
living human cells. Transfected HEK293T cells are used as a model system for human cells Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution. 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.
HS SH
1H
15N
Cys 146 Cys 111 Cys 6 SH Cys 57
1H 15N
apo-SOD1 E,Zn-SOD1 apo-SOD1
Isotopically labelled proteins are overexpressed and directly
living human cells. Transfected HEK293T cells are used as a model system for human cells Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution. 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.
+ Zn(II)
HS SH HS SH SH HS
1H
15N
Cys 146 Cys 111 Cys 6 SH Cys 57
1H 15N
E,Zn-SOD1 E,Zn-SOD1
Isotopically labelled proteins are overexpressed and directly
living human cells. Transfected HEK293T cells are used as a model system for human cells Maturation processes such as protein folding, post translational modifications (i.e. metal binding, disulfide bond formation) are followed at atomic resolution. 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.
SH SH S S S S
+ Cu(I) + Zn(II)
HS SH HS SH SH HS
1H
15N
Cys 146 Cys 111 Cys 6 SH S-S Cys 57
1H 15N
Cu,Zn-SOD1 Cu,Zn-SOD1
Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013
HS SH SH HS HSSH SH SH SH SH HS SH SH HS SH SH HS HS HSSH SH HS
Zn(II)
S S S S S S S S HS HS
CCS
SH SH S S S S
Cu(II) Zn(II) Cu(I)
E,E-SOD1SH E,Zn-SOD1SH E,Zn-SOD1S-S Cu(I),Zn-SOD1S-S
Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR, Nat Chem Biol, 2013
to mutations of SOD1.
scattered throughout the sequence.
promoting aggregation of the apo protein
Luchinat E, Barbieri L, Rubino JT, Kozyreva T, Cantini F, Banci L, Nat. Comm., 2014
Luchinat E, Barbieri L, Rubino JT, Kozyreva T, Cantini F, Banci L, Nat. Comm., 2014
Many SOD1 mutants do not bind zinc in the cell, and accumulate as an unstructured species, which does NOT evolve toward the native form This unstructured species DOES NOT bind zinc It could be a precursor of SOD1 aggregates
(A4V, I35T, G37R, G85R, G93A, I113T) I113T G93A
I113T#
I113T
The mutations do not affect zinc binding in vitro
Nucleus Golgi
Hah1
Ctr
Regulators MNK/WLN
Amine Oxidase, Lysyl oxidase
CCS
Cu(I)
?
Cu(II) Cu(I)
Cox17 SOD1 MT Cox23 Cox23 Cox19 Cox19
Mitocondria
Sco1 Sco2 Cox11 MT ? CCS SOD CCO
Ceruloplasmin
No free copper ions in the cytoplasm
Banci, Bertini, Ciofi-Baffoni, Karit, Kozyreva, Palumaa, Nature, 2010
The knowledge of the structures of the proteins and of their complexes allows the atomic level description of the transfer processes
NDOR1 ANAMORSIN GRX3 Cfd1 Nbp35 Nar1 CIAO1 MMS19 Holo Cytosolic Proteins Apo Cfd1 Nbp35 MIP18
e-
ANAMORSIN
MiA40
? ? ?
BOLA2
? ? ? ?
Structural properties of Anamorsin An essential protein for FeS cluster biosynthesis
Mia40 recognition site Intrinsically Disordered Domain N-terminal domain Folded domain “standard” approach Linker [2Fe-2S] cluster Highly paramagnetic with fast relaxation and no PCS The worst case!! Mia40 forms two disulfide bonds when in mitochondria This motif binds a cluster in the cytoplasm
Playing For or Against Paramagnetic Relaxation?
Exploit the differences in longitudinal relaxation
30 ms < 6-10 Å T1 < 100 ms 100 ms < 10-11 Å T1< 200 ms 500 ms
Inversion Recovery
The effect of T1 relaxation 200 ms 100 ms 50 50 ms ms 20 ms 10 10 ms ms 2 ms IR delays selected according to T1 values 3-6 Å T1< 30ms
IR is combined with fast recycling times
0.25 0.5 0.75 1 0.002 0.004 0.006 t(s)
no relax
100ms 10 10 ms ms 5 ms 2 ms 1 ms 0.5 ms
Playing For or Against Paramagnetic Relaxation?
Limit the negative effects of fast transverse relaxation
The effect of T2 relaxation
INEPT coherence transfer
We can detect peaks for signals with Dn up to ca. 300 Hz
ppm
d 13C Remove the reverse INEPT Phasing AP in dispersion mode
Antiphase signal acquisition
Tailored 15N HSQC of the [2Fe-2S]-domain of anamorsin
1H 15N 15N- IR-HSQC-AP 15N 15N- HSQC 1H
13 HN peaks, missing in standard 15N HSQC, can be detected.
1H T1 values range from 5 to 30 ms
Banci et al PNAS 2013, 2014; Ciofi-Baffoni, Gallo, Piccioli, J. Biomol NMR 2014
Paramagnetic-tailored 13C-direct CACO of [2Fe-2S]- domain of anamorsin
13C 13C 13C 13C 13C-CACO-IPAP 13C-CACO-AP
T250 G252 S236 K244
13C signals, absent in standard 13C-direct esperiments, are also observed
via 13C COSY and tailored CON Overall, about 10 additional 13C resonances are detected
Structure of the [2Fe-2S] cluster binding region in anamorsin
Blue - residues detected in the “diamagnetic” experiments Cyano - residues whose 13C or 15N signals were detected in paramagnetic-tailored 13C or 15N experiments
T250 K244 G252 S236
The “paramagnetic” 13C and 1H T1s provide structural info around the FeS cluster
The overall structure was then subjected to MD trajectory in explicit water
Banci, Ciofi-Baffoni, Gajda, Muzzioli, Peruzzini, Winkelmann, Nat. Chem Biol. 2015
The Trx domain of GRX3 is essential for protein-protein recognition
Cluster site characterization complemented with EPR and Moessbauer data
…but in oxidative conditions, the BolA2-GRX3 complex is more efficient in anamorsin maturation
Banci L, Camponeschi F, Ciofi-Baffoni S, Muzzioli R. J Am Chem Soc. 2015, Nuttle, X. et al. Nature 2016
The GRX3/BolA2 heterocomplex is more stable in oxidative conditions An alternative route to anamorsin maturation
anamorsin
Anamorsin tightly interacts with Ndor1 through its flexible, unstructured linker The [2Fe-2S]-CIAPIN1 domain transiently interacts with the FMN domain
Anamorsin Ndor1 (closed conformation) Ndor1 (open conformation) Anamorsin Anamorsin
NADPH FAD FMN
[2Fe-2S]- CIAPIN1 domain
Banci , Bertini, Calderone, Ciofi-Baffoni, Giachetti, Jaiswal, Mikolajczyk, Piccioli, Winkelmann PNAS, 2013
FMN-binding domain of Ndor1 Flexible linker N-terminal domain
FMNH2
Banci , Bertini, Calderone, Ciofi-Baffoni, Giachetti, Jaiswal, Mikolajczyk, Piccioli, Winkelmann PNAS, 2013
An integrated approach to investigate electrons and FeS cluster transfer processes
ESI-MS Para-NMR Solution NMR, X-ray, Protein docking Biophysics: EPR, UV-vis, Mössbauer CD …
Banci et al. PNAS 2014 Banci, Ciofi-Baffoni, Bill et al. JBIC 2013 Banci, Ciofi-Baffoni et al. PNAS 2013 Banci, Ciofi-Baffoni et al. Chem Biol. 2011
Eukaryotic Fe/S assembly machineries
FF parameters determination
NDOR1 ANAMORSIN GRX3 Cfd1 Nbp35 Nar1 CIAO1 MMS19 Holo Cytosolic Proteins Apo Cfd1 Nbp35 MIP18
e-
ANAMORSIN
MiA40
? ? ?
BOLA2
Cultivate microorganism Genome-based approaches Pan-genome approach Determination of antigens structure
past present
Chemical shift mapping
1H-15N HSQC-TROSY
1H-15N HSQC-TROSY
mixture
Interaction between fHbpC and a fAb portion
The data show that mAb502 recognizes a conformational epitope within a well-defined area of the immunodominant C-terminal domain of fHbp.
1H-15N HSQC-TROSY of
fHbpC alone
1H-15N HSQC-TROSY-
CRINEPT
Residues predicted by immunological data and confirmed by NMR NMR data suggested the involvement of
Model of the complex between fHbpC & fAb portion of mAb502
Residues of fHbpC involved in binding to mAb502 mapped onto the full length protein structure
Structure of antigen fHbp Heavy chain of monoclonal antibody Mab502 Light chain of monoclonal antibody Mab502 Fab region
antibody
Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011 fHbp is very effective in inducing protective immunity eliciting antibodies but has different sequence in different strains of MenB
Structure-based design of a vaccine against Mengingococcus B
Scarselli, Cantini, Banci, Rappuoli et al., Science Transl. Med. 2011 By knowing the structural properties
all the variants, a chimera antigen was produced which elicits complete protective immunity
1200 MHz ( 1200 MHz (2018?) 2018?)
11 NMR spectrometers + 1 Relaxometer, The largest available magnetic field range (0.01 – 950 MHz)
Pulsed EPR
e-Research Infrastructure