Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways
Cu , Zn superoxide dismutase ( SOD1 ) is an extensively studied metalloenzyme that has become a paradigm for understanding protein structure and function as well as folding and misfolding associated with disease ( Valentine et al . , 2005 ) . The mature enzymatically active form of this antioxidant protein ( Cu2Zn2SOD1S–S ) is a homodimer comprised of 153-residue monomer subunits , each of which binds 1 Cu and 1 Zn ion and contains one intra-subunit disulfide bond ( Valentine et al . , 2005 ) . Mutations in the sod1 gene encoding for this enzyme account for ∼20% of all familial ALS ( fALS ) cases , or about 2% of all ALS occurrences ( Robberecht and Philips , 2013 ) . Despite the link between SOD1 and fALS and the identification of over 150 disease causing mutants , the molecular events leading to disease remain poorly understood . Inclusions containing SOD1 have been observed in patients afflicted with both familial and sporadic ALS , suggesting that both forms of the disease may share a common pathway ( Matsumoto et al . , 1995; Rotunno and Bosco , 2013 ) . However , whether cytotoxicity arises from the aggregates or from soluble misfolded conformers further upstream remains unclear . In its metal-bound oxidized form SOD1 is exceptionally stable , with a melting temperature over 90°C ( Stathopulos et al . , 2006 ) and is highly resistant to aggregation ( Stathopulos et al . , 2003; Sheng et al . , 2012; Broom et al . , 2014 ) . In contrast , the most immature form of SOD1 , apoSOD12SH , that lacks metal ions and the disulfide bond has a much higher propensity to misfold and aggregate in vitro ( Furukawa and O'Halloran , 2005; Sheng et al . , 2012; Broom et al . , 2014 ) and has been hypothesized to be the primary cause of toxicity in vivo ( Zetterström et al . , 2013 ) . Studies of apoSOD12SH form the basis of the work described herein . Misfolded protein conformations can be notoriously difficult to characterize because the tools of structural biology are most suited to proteins which adopt only single conformations that are extensively populated . In contrast , misfolded conformers can be high-energy states that are only marginally populated and transiently sampled by the native protein under physiological conditions ( Chiti and Dobson , 2008 ) . In this case they cannot be observed directly using ‘traditional’ biophysical approaches . Recent methodological advances in NMR spectroscopy have rendered such previously ‘invisible’ states ( also referred to as excited states in what follows ) amenable to structural analysis so long as they exchange with a ‘visible’ ( ground ) state , are populated at a level of approximately 0 . 5% or higher and have lifetimes ranging from 0 . 5–20 ms ( Palmer et al . , 2000; Sekhar and Kay , 2013 ) . Here , we have used a combination of different spin relaxation NMR experiments to explore the energy landscape of apoSOD12SH and to elucidate the structural features of several invisible states that are populated via conformational fluctuations originating from the monomeric ground conformer of the protein . Analysis of the resulting data yields structural information about each of the excited states in the form of chemical shifts as well as the kinetics and thermodynamics of the exchange process ( Palmer et al . , 2000; Korzhnev et al . , 2010; Nikolova et al . , 2011; Neudecker et al . , 2012; Sekhar and Kay , 2013 ) . We detect four excited state conformers in equilibrium with native apoSOD12SH . Two of these , involving either the local folding of a helix or dimerization , lead to structural features that are found in the native state of the mature metallated protein . In contrast , the other two conformers correspond to non-native oligomers . Structural models generated from the NMR data highlight the native dimer interface and the electrostatic loop as key motifs in forming non-native intermolecular contacts .
Detailed structural studies have established that each monomer of homodimeric Cu2Zn2SOD1S–S forms an 8-stranded antiparallel Greek key β-barrel with two short helices , one each in the Zn-binding ( 49–80 ) and electrostatic loops ( 126–143 ) ( Figure 1A ) ( Valentine et al . , 2005 ) . As the name implies the Zn-binding loop contains the Zn binding site , one of the cysteines involved in intra-subunit disulfide bond formation , and a large number of residues that make contacts at the native dimer interface . Metal binding is additionally stabilized by residues in the electrostatic loop ( Strange et al . , 2003 ) . Zinc binding , disulfide bond formation , and dimerization limit the flexibility of the Zn-loop and markedly increase protein stability ( Furukawa and O'Halloran , 2005 ) . A large number of hydrogen bonding interactions in both loops are thought to stabilize the protein by providing a network of connections between the loops and the metal binding sites ( Parge et al . , 1992 ) . 10 . 7554/eLife . 07296 . 003Figure 1 . ApoSOD12SH is folded in solution . ( A ) Structure of the mature homodimeric form of SOD1 ( Cu2Zn2SOD1S–S ) ( pdb code: 1hl5 ) ( Strange et al . , 2003 ) . Each monomer comprises an 8-stranded β-barrel with two long loops , the Zn-loop ( red ) and the electrostatic loop ( blue ) , an intra-subunit disulfide bond ( cyan ) , and one bound Cu ( yellow ) and Zn ( magenta ) ion . Each of the two long loops contains a short helix . Highlighted are positions 85 ( green ) and 4 ( orange ) that are sites of mutations discussed in the text . ( B ) 1H-15N HSQC correlation map of pWT apoSOD12SH , 25°C ( 600 MHz 1H frequency ) . Chemical shift assignments of backbone amides are indicated on the plot . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 003 Wild-type ( WT ) SOD1 contains four Cys residues , C57 and C146 that form the stabilizing intra-subunit disulfide bond described above and non-conserved C6 and C111 that have been replaced by Ala and Ser , respectively ( Lepock et al . , 1990; Broom et al . , 2014 ) , in the studies we have performed . The 1H-15N correlation spectrum of C6A , C111S apoSOD12SH ( referred to as pWT apoSOD12SH in what follows ) is well resolved and characteristic of a folded protein ( Figure 1B ) . Protein backbone 1H , 15N , and 13C chemical shifts provide both structural ( Cavalli et al . , 2007; Shen et al . , 2008 ) and dynamical information ( Berjanskii and Wishart , 2005 ) , with the amplitude of dynamics at each amide site most often quantified through an order parameter squared , S2 ( Lipari and Szabo , 1982 ) , that can be calculated from the measured chemical shifts . Values of S2 range between 0–1 with the extreme values corresponding to cases of no motion ( 1 ) or completely isotropic dynamics ( 0 ) , respectively . A comparison of the residue-specific order parameters calculated from chemical shifts of the ground states of pWT apoSOD12SH and Cu2Zn2SOD1S–S ( chemical shifts for the latter form obtained from Banci et al . ( 2002a ) ) shows that both loops become disordered in apoSOD12SH ( Figure 2A ) , while residues involved in β strands remain rigid . An excellent correlation is obtained between predicted inter-strand NOE patterns ( Figure 2B ) based on the X-ray structure of Cu2Zn2SOD1S–S ( Strange et al . , 2003 ) and observed NOEs measured from spectra recorded on apoSOD12SH , establishing that the β-barrel remains intact in the immature form of the protein as well . This is further confirmed by measured values of amide proton temperature coefficients in regions where β strands are known to form in Cu2Zn2SOD1S–S . Values greater than −4 . 6 ppb/K ( red line ) denote amides that are hydrogen bonded in structure ( Cierpicki and Otlewski , 2001 ) , as observed for the great majority of the β strand protons in apoSOD12SH ( Figure 2C ) . 10 . 7554/eLife . 07296 . 004Figure 2 . Structural features of the ground state of immature apoSOD12SH . ( A ) Chemical shift-derived order parameters squared ( Berjanskii and Wishart , 2005 ) , S2 , for the mature Cu2Zn2SOD1S–S ( red; based on published chemical shifts [Banci et al . , 2002a] ) and the immature apoSOD12SH ( green ) forms . Secondary structural elements of Cu2Zn2SOD1S–S are depicted above the plot , with the electrostatic loop shown in blue and the Zn-loop in red . ( B ) Correlation between inter-strand NOE pairs observed experimentally for apoSOD12SH ( red ) and predicted based on the crystal structure of Cu2Zn2SOD1S–S ( Strange et al . , 2003 ) ( blue ) . The secondary structure of Cu2Zn2SOD1S–S is indicated on the sides of the plot . ( C ) Temperature coefficients of amide protons of apoSOD12SH known to be involved in inter-strand hydrogen bonds in the crystal structure of Cu2Zn2SOD1S–S ( Strange et al . , 2003 ) . A cutoff of −4 . 6 ppb/K , often used to distinguish between protected and unprotected amides , is shown in red ( Cierpicki and Otlewski , 2001 ) . ( D ) Correlation between 15N R1ρ values of apoSOD12SH at 3 . 5 mM ( x-axis ) and 2 . 0 mM ( red ) or 0 . 8 mM ( green ) ( y-axis ) . Only 15N nuclei in ordered regions of apoSOD12SH with Rex values smaller than 2 s−1 in CPMG measurements ( 600 MHz , 3 . 5 mM protein concentration ) were chosen for this analysis . Solid lines represent best fits of the data to an equation of the form y = mx + c and the slope in each case is indicated on the plot . Ratios of translational diffusion coefficients , D , of apoSOD12SH at the indicated concentrations , that report on differences in sample viscosities with protein concentration , are also indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 004 We have also established that under the conditions of our NMR experiments pWT apoSOD12SH is monomeric , as expected based on previous characterizations of this protein ( Arnesano et al . , 2004 ) . Figure 2D correlates 15N transverse relaxation rates measured at protein concentrations of 2 mM and 3 . 5 mM ( red line ) or 0 . 8 mM and 3 . 5 mM ( green ) , with each point corresponding to a separate amide position in the protein . Slopes of 0 . 87 and 0 . 72 are in excellent agreement with expected values if the differences in protein concentration dependent relaxation are due to viscosity , since very similar ratios of NMR derived translational diffusion coefficients are obtained from measurements at different protein concentrations ( 0 . 87 and 0 . 76 ) . Note that concentration dependent oligomerization would lead to deviations between relaxation and diffusion ratios , since these measures scale differently with molecular size ( MW−1 and MW−1/3 for relaxation times and diffusion , respectively , where MW is the molecular weight of the assumed spherical particle [Cantor and Schimmel , 1980] ) . Notably , isotropic rotational correlation times were measured to be close to a factor of two different between pWT apoSOD12SH and Cu2Zn2SOD1S–S ( 11 . 6 ns and 18 . 2 ns , ‘Materials and methods’ ) , although an exact twofold difference would not be expected because of the distinct shapes of monomeric and dimeric SOD1 . In order to probe the energy landscape of pWT apoSOD12SH we first carried out chemical exchange saturation transfer ( CEST ) experiments ( Forsén and Hoffman , 1963; Fawzi et al . , 2011; Lauzon et al . , 2011; Vallurupalli et al . , 2012 ) . In this class of relaxation experiment , a series of spectra are recorded whereby a weak radiofrequency ( r . f . ) perturbation is applied over a range of different frequencies ( 1 per spectrum ) and the effects monitored by quantifying the intensity of ground state peaks . When the r . f . is positioned close to an invisible excited state resonance the effect of the perturbation will be transferred to the ground state peak via chemical exchange , leading to a decrease in the intensity of the observed ground state resonance . When the r . f . is applied near the resonance position of the ground state correlation there is attenuation as well , via a well-characterized saturation effect ( Carrington and McLachlan , 1967 ) . Thus , a plot of the intensity of the ground state peak as a function of the position of the r . f . field produces a CEST profile with a main dip at the chemical shift of the resonance belonging to the major conformation and a smaller dip at the position of the corresponding resonance frequency of the minor conformer . CEST experiments recorded on apoSOD12SH show the presence of additional states with chemical shifts significantly different from the native conformation ( Figure 3 and Figure 3—figure supplement 1 ) . In CEST profiles , the size of the minor dip is sensitive to both the fractional population of the excited state and its rate of exchange with the ground state ( Fawzi et al . , 2011; Vallurupalli et al . , 2012 ) . For exchange process I , the size of the minor dip increases with protein concentration ( Figure 3A and Figure 3—figure supplement 1 , green and red curves ) , implying an association or oligomerization event . Backbone 15N , 13C′ , and 13Cα chemical shifts of the excited state involved in this process , as measured by the positions of minor state dips in CEST profiles ( Figure 3A–C and Figure 3—figure supplement 1 ) , localize to elements of structure forming the native dimer in Cu2Zn2SOD1S–S and correlate well with published chemical shifts of Cu2Zn2SOD1S–S ( Figure 3D , E ) ( Banci et al . , 2002a ) . This establishes that pWT apoSOD12SH transiently forms native-like dimers . 10 . 7554/eLife . 07296 . 005Figure 3 . pWT apoSOD12SH transiently samples a native dimer conformation . ( A ) 15N-CEST profiles for G61 of pWT apoSOD12SH that reports on the dimerization process , red: 1 . 3 mM , green: 0 . 7 mM and G85R apoSOD12SH ( blue: 1 . 5 mM ) . The G85R mutation does not interfere with transient native dimer formation as indicated by the large minor state dip . Profiles plot the intensity of the ground state peak ( I ) as a function of the position of the perturbing B1 field ( 31 Hz ) , normalized to the corresponding intensity obtained from a spectrum recorded without the CEST element ( I0 ) ( Vallurupalli et al . , 2012 ) . 13C′- ( B ) and 13Cα- ( C ) CEST profiles for residues V5 and F50 , respectively . Correlations of 15N ( D ) , 13C′ ( E , red ) and 13Cα ( E , blue ) CEST-derived excited state chemical shifts , ( ϖ , ppm , y-axis ) of apoSOD12SH with the ground state shifts ( Banci et al . , 2002a ) of Cu2Zn2SOD1S–S ( x-axis ) for residues localized to regions involved in transient dimerization . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 00510 . 7554/eLife . 07296 . 006Figure 3—figure supplement 1 . Conformationally excited states of apoSOD12SH containing ‘native’ features as studied by 15N ( Vallurupalli et al . , 2012 ) , 13C′ ( Vallurupalli and Kay , 2013 ) , 13Cα ( Long et al . , 2014 ) , and 13CH3 ( Bouvignies et al . , 2014 ) CEST—process I . ( A , B ) 15N CEST profiles of residues reporting on transient native-like dimerization at two different concentrations of pWT apoSOD12SH ( red: 1 . 3 mM , green: 0 . 7 mM ) . Also shown are corresponding profiles from G85R apoSOD12SH ( blue , 1 . 5 mM ) , establishing that the mutation has little effect on process I . 13C′ , 13Cα and methyl-13C ( 13CH3 ) CEST profiles for pWT residues reporting on transient dimerization ( C–E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 00610 . 7554/eLife . 07296 . 007Figure 3—figure supplement 2 . Estimating excited state lifetimes and populations for processes I and II . ( A , B ) CEST profiles ( circles ) of G51 , N53 , T54 , and G61 reporting on the transient native dimerization process ( I ) measured at 2 B1 fields ( 31 and 59 Hz ) . Solid lines are fits of CEST data for each residue independently to a two-state model of exchange , including profiles from both B1 fields ( A ) or for each B1 field separately ( B ) . A range of excited state populations and lifetimes from 1 . 5–4 . 5% and 2 . 2–4 . 3 ms is obtained . ( C , D ) CEST profiles at 2 B1 fields ( 31 and 59 Hz ) and CPMG profiles at two static magnetic fields ( corresponding to 1H resonance frequencies of 600 and 800 MHz ) for T135 and T137 reporting on the local folding process ( II ) . Solid lines are fits of both CEST and CPMG data to a two-state model ( C ) or to a three-state model ( D ) of conformational exchange . ( E ) Lowest energy ( χred2 ∼2 ) physically meaningful three-state models describing process II , where N is the native state of apoSOD12SH , H is the transiently populated helical state and I is an intermediate not directly visible in CEST measurements , but required for data fitting . Each of the shapes is meant to represent the absence ( circle ) or presence of the helix ( triangle ) that is formed via process II , as established on the basis of 15N chemical shifts; note that very large 15N chemical shift changes ( ∼10 ppm ) are observed for helix formation for both T135 and T137 so that these shifts can be used as direct reporters of structure . Exchange parameters for each model are indicated on the plot , where listed exchange rates refer to the sums of forward and backwards processes ( for example , 1400 s−1 = kIN + kNI in the model on the left ) . Lifetimes ( τH ) of the excited state H , corresponding to the conformer with the native-like electrostatic loop helix , were calculated according to the relations τH = 1/koff , H , where koff , H = kHI = kex , HI × ( pI/ ( pH + pI ) ) for the on-pathway model ( left ) and koff , H = kHN = kex , HN × ( pN/ ( pH + pN ) ) for the off-pathway model ( right ) . Values of τH ranged between 12–14 ms , depending on the model used and are reported in the text ( Figure 9 ) as an average over the two models . Note that the on-pathway and off-pathway model above cannot be distinguished , as both models fit the data equally well . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 00710 . 7554/eLife . 07296 . 008Figure 3—figure supplement 3 . Excited state apoSOD12SH conformations that are observed in ground state , mature Cu2Zn2SOD1S–S are generated via metal-independent processes . ( A ) Results of metal analysis of NMR samples of apoSOD12SH in regular and Suprasil NMR tubes ( Wilmad Lab-glass catalogue number 535-PP-7SUP , synthetic glass without transition metals ) performed immediately after CEST measurements were completed . A comparison of CEST profiles for residues belonging to transient dimerization ( B ) and local folding ( C ) measured on samples in a regular NMR tube known from metal analysis to have traces of Zn and Cu ( green ) and in a Suprasil tube ( red ) devoid of metal . No differences were noted , suggesting that the processes are not affected by trace metal . ( D ) 1H-15N HSQC spectrum of apoSOD12SH ( ∼700 μM ) containing externally added Zn ( ∼140 μM ) . The resonances of the Zn-bound form ( asterisk ) are in slow exchange with apoSOD12SH . CEST profiles of apoSOD12SH nuclei reporting on transient dimerization ( E ) or local folding ( F ) derived from experiments recorded using Suprasil tubes with either no added metal ( red , ∼700 µM protein ) or added Zn ( green , ∼700 µM protein , ∼140 µM Zn ) . The close similarities of the profiles provide strong evidence that the processes are metal independent . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 008 In contrast , a second process , referred to as process II subsequently , is concentration-independent ( Figure 4A and Figure 4—figure supplement 1 ) , although 15N , 13C′ , and 13Cα chemical shifts of the associated excited state ( Figure 4A–C ) also correlate well with those from Cu2Zn2SOD1S–S ( Figure 4D , E ) . Residues belonging to process II are localized to a region in the electrostatic loop that is unstructured in apoSOD12SH yet forms a short helix in Cu2Zn2SOD1S–S ( Valentine et al . , 2005 ) . Process II is thus a transient local folding event involving formation of a helix from a disordered region in the ground state . This conclusion is further reinforced by the secondary structural propensity ( SSP ) score ( Marsh et al . , 2006 ) calculated from chemical shifts of the excited state for nuclei reporting on process II ( residues 130–140 ) . SSP scores for the ground and excited states computed from chemical shifts of the two interchanging conformers show that the helical content in this region of apoSOD12SH increases considerably ( by up to ∼50% ) in the excited state , establishing that process II involves local structuring of a helix ( Figure 4F ) . Notably both processes I and II are independent of each other since the ALS mutant G85R ( Cao et al . , 2008 ) eliminates II with little effect on I ( Figures 3A , 4A and Figure 3—figure supplement 1 , Figure 4—figure supplement 1 ) and both involve the partial transformation of apoSOD12SH into a mature native-like state , limiting the flexibility of the Zn-binding and electrostatic loops ( processes I , II respectively ) and sequestering key structural elements , such as strands β1 and β8 ( process I ) . Fits of the processes ( Figure 3—figure supplement 2 ) , as described in ‘Materials and methods’ , provide an estimate of the population and lifetime of each excited state ( 25°C ) , with values of 2% , 13 ms and 3% , 3 ms for the helix and the native dimer respectively at an overall protein concentration of 1 . 3 mM , along with a dimer dissociation constant of 85 ± 50 mM . 10 . 7554/eLife . 07296 . 009Figure 4 . An excited state of pWT apoSOD12SH with a native-like helix in the electrostatic loop . 15N- ( A ) , 13C′- ( B ) and 13Cα- ( C ) CEST profiles for T135 , E132 and N131 that report on transient helix folding . Correlations of 15N ( D ) , 13C′ ( E , red ) and 13Cα ( E , blue ) CEST-derived excited state chemical shifts , ( ϖ , ppm , y-axis ) of apoSOD12SH with the ground state shifts ( Banci et al . , 2002a ) of Cu2Zn2SOD1S–S ( x-axis ) for residues localized to regions involved in transient helix formation . Helix formation is eliminated by the G85R mutation ( A , blue ) . ( F ) Secondary Structural Propensity ( SSP ) ( Marsh et al . , 2006 ) scores computed from chemical shifts of nuclei in the ground and excited states and localized to residues 130–140 that are sensitive to process II . A value of 0 indicates a random coil conformation and a value of +1 corresponds to a fully formed helix . The helicity increases by up to ∼50% in the excited state , showing that process II involves local folding . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 00910 . 7554/eLife . 07296 . 010Figure 4—figure supplement 1 . Conformationally excited states of apoSOD12SH containing ‘native’ features as studied by 15N ( Vallurupalli et al . , 2012 ) , 13C′ ( Vallurupalli and Kay , 2013 ) , 13Cα ( Long et al . , 2014 ) , and 13CH3 ( Bouvignies et al . , 2014 ) CEST—process II . ( A , B ) 15N CEST profiles of residues reporting on transient local folding at two different concentrations of pWT apoSOD12SH ( red: 1 . 3 mM , green: 0 . 7 mM ) . Also shown are corresponding profiles from G85R apoSOD12SH ( blue , 1 . 5 mM ) , establishing that the mutation eliminates process II . 13C′ , 13Cα and methyl-13C ( 13CH3 ) CEST profiles for pWT residues reporting on transient local folding ( C–E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 010 Both local folding and transient dimerization are metal-independent processes ( Figure 3—figure supplement 3 ) that result in conformations observed in mature Cu2Zn2SOD1S–S . Thus the amino acid sequence can code for the organization of the electrostatic and the Zn-binding loops in a manner independent of metal binding . Our results establish that metal binding is not a requirement for loop organization and further that zinc binding may , in part , proceed through a conformational selection mechanism whereby zinc binds to excited state conformers that contain the transiently formed helix in the electrostatic loop . The negative end of this helix dipole , which has been postulated to stabilize the metal-bound conformation in SOD1 ( Cao et al . , 2008 ) , may also facilitate zinc ligation by guiding the metal to the binding pocket through electrostatic interactions . In this regard , it is noteworthy that the G85R mutant , which has a reduced affinity for zinc ( Valentine et al . , 2005 ) , does not transiently form this helix as established by the absence of minor state CEST dips for the helix forming residues ( Figure 4A blue , Figure 4—figure supplement 1 ) . Additional millisecond timescale processes have been characterized using Carr-Purcell-Meiboom-Gill ( CPMG ) relaxation dispersion NMR ( Carr and Purcell , 1954; Meiboom and Gill , 1958; Palmer et al . , 2000 ) that takes advantage of the increase in effective transverse relaxation rates of NMR spins ( R2 , eff ) resulting from stochastic fluctuations between protein conformations . In this experiment , transverse relaxation rates of NMR probes are modulated by applying a variable number of chemical shift refocusing pulses during a fixed time element ( Mulder et al . , 2001 ) . Application of increasing numbers of pulses decreases the effective chemical shift differences between exchanging sites , resulting in a decrease in the measured effective transverse relaxation rate . Thus , dispersion profiles , R2 , eff vs rate of application of refocusing pulses ( νCPMG ) , showing a decrease in R2 , eff with increasing νCPMG are a hallmark of chemical exchange , while flat profiles indicate either that there is no exchange or that it proceeds much faster than the rate of application of the pulses . 15N and 13CH3 CPMG experiments ( Lundström et al . , 2007b; Vallurupalli et al . , 2007 ) on apoSOD12SH allow characterization of two additional millisecond timescale processes , referred to as processes III and IV ( Figures 5A , 6A ) . The positions of 15N and methyl-13C spins identified as probes of processes III and IV are highlighted in monomers of SOD1 in Figures 5B , 6B , respectively . Both processes are concentration dependent ( Figures 5C , 6C and Figure 5—figure supplement 1 ) , as seen from the significant increase in contributions to transverse relaxation rates from chemical exchange , Rex , with increasing total protein concentration , and hence correspond to transient oligomer formation . These processes can be separated on the basis of their temperature dependencies with Rex values from III and IV increasing and decreasing , respectively , with increasing temperature ( Figures 5D , 6D and Figure 5—figure supplement 1 ) . In order to quantify the differences in kinetics and thermodynamics of these exchange events in more detail we have further analyzed the dispersion data assuming a simple monomer ( M ) —dimer ( D ) equilibrium model ( see ‘Materials and methods’ , ‘Separating probes of processes I–IV’ for details ) , yielding excited state populations and lifetimes of 3 . 3 ± 0 . 2% , 6 . 0 ± 0 . 6 ms and 2 . 1 ± 0 . 1% , 1 . 6 ± 0 . 1 ms for processes III and IV , respectively , at 25°C , 1 . 3 mM protein . The populations and lifetimes of all of the apoSOD12SH states characterized in the present study are listed in Table 1 . 10 . 7554/eLife . 07296 . 011Figure 5 . ApoSOD12SH transiently forms distinct non-native oligomers—Process III . Representative 15N ( red , orange ) and 13CH3 ( green ) CPMG profiles for residues belonging to processes III ( A ) . Each curve shows the effective 15N or 13C transverse relaxation rate , R2 , eff , plotted as a function of the rate of pulsing in CPMG trains ( Palmer et al . , 2000 ) . ( B ) Residues identified as reporting on process III via CPMG experiments ( see ‘Materials and methods’ ) are highlighted on the structure of a monomer of Cu2Zn2SOD1S–S . Concentration ( C , 25°C ) and temperature-dependence ( D , 2 mM protein concentration ) of Rex values ( the difference in relaxation rates recorded at the lowest and highest CPMG field strengths [Palmer et al . , 2000] ) obtained from 15N CPMG measurements on pWT apoSOD12SH . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 01110 . 7554/eLife . 07296 . 012Figure 5—figure supplement 1 . The two non-native oligomerization processes ( III and IV ) are distinct from one another . Rex values , corresponding to the differences in effective methyl 13C transverse relaxation rates measured at the lowest and highest CPMG pulsing frequencies ( Palmer et al . , 2000 ) , for residues in pWT apoSOD12SH reporting on process III ( A , C ) or process IV ( B , D ) . Two different concentrations ( panels A , B; green: 2 . 5 mM , magenta: 0 . 8 mM , 25°C ) or two different temperatures ( panels C , D; red: 35°C , blue: 25°C , 2 . 5 mM protein concentration ) are shown . Stereospecific assignments are not available for Leu or Val prochiral methyl groups . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 01210 . 7554/eLife . 07296 . 013Figure 6 . ApoSOD12SH transiently forms distinct non-native oligomers—Process IV . ( A–D ) As Figure 5 with the exception that the residues selected are those probing process IV . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 01310 . 7554/eLife . 07296 . 014Table 1 . Excited state populations ( pE ) and lifetimes ( τE ) for processes I–IVDOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 014ProcessIIIIIIIVpE ( % ) 3 ± 12 . 1 ± 0 . 13 . 3 ± 0 . 22 . 1 ± 0 . 1τE ( ms ) 3 . 0 ± 0 . 613 ± 16 . 0 ± 0 . 61 . 6 ± 0 . 1Uncertainties for process I are calculated as ±1 s . d . of the values obtained from single B1 field fits of 15N CEST profiles of G51 , N53 , T54 , and G61 ( Figure 3—figure supplement 2B ) . Values for process II are the mean and standard deviation of pE and τE obtained from the two best three-state models ( Figure 3—figure supplement 2E ) . The equations for calculating these values are listed in the legend for Figure 3—figure supplement 2 . Note that very similar values of pE and τE are obtained from the two models ( on- and off-pathway ) and it is not possible to distinguish between them on the basis of the NMR data . Uncertainties for exchange parameters describing processes III and IV are obtained from global two-state fits of CPMG profiles reporting on each process . All values are relevant for a 1 . 3 mM protein concentration , 25°C . Figure 7A–C shows distinct temperature dependencies for the dimerization equilibrium constant , KMD , and for the underlying rate constants kMD , kDM for each process , further highlighting that exchange events III and IV are independent . Indeed , all 3 oligomerization processes are independent of each other , as CPMG profiles reflecting the native dimerization process ( I ) cannot be well-fit using exchange parameters from analysis of process III ( ‘Materials and methods’ ‘Separating probes of processes I–IV’ and Figure 7—figure supplement 1 ) , while processes I and IV have distinct temperature dependencies ( Figure 7—figure supplement 1 ) . Thus , exchange processes III and IV correspond to the formation of non-native oligomers . 10 . 7554/eLife . 07296 . 015Figure 7 . Processes III and IV are distinct . Variation in the logarithms of the equilibrium constant for dimer formation ( A , KMD ) , as well as the association ( B , kMD ) and dissociation ( C , kDM ) rate constants with inverse temperature for processes III ( closed magenta circles ) and IV ( open black circles ) obtained as described in ‘Materials and methods’ , ‘Separating probes of processes I–IV’ . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 01510 . 7554/eLife . 07296 . 016Figure 7—figure supplement 1 . The two transient non-native oligomerization processes ( III and IV ) observed in apoSOD12SH are distinct from native dimerization ( process I ) . ( A ) 15N CPMG profiles of G85R apoSOD12SH ( 600 MHz , 1 . 5 mM protein concentration , 25°C ) for residues reporting on native dimerization ( process I ) can be fit together to extract chemical shift differences , Δϖ , between the ground state ( monomer ) and the excited conformer ( native dimer ) , that correlate well with the values obtained from CEST measurements on the same mutant ( C , red ) . ( B ) These dispersion profiles cannot be fit together with those from residues reporting on non-native oligomerization process III and the resulting Δϖ values are in error ( C , blue ) . Thus , processes I and III are distinct from each other . ( D ) This is illustrated further by comparing calculated Rex values for residues selected as reporting on processes III ( green ) and I ( red ) using Equation 1 of ‘Materials and methods’ and the best fit exchange values and chemical shift differences . Note that a maximum Rex value of 2 . 8 s−1 can be obtained for a reporter of process I ( red ) ( see ‘Materials and methods’ ‘Separating probes of processes I–IV’ , black horizontal line ) , based on fits of 15N CPMG dispersion profiles recorded on 1 . 5 mMG85R apoSOD12SH ( 600 + 800 MHz , 25°C ) . This value is clearly much smaller than those calculated and measured for residues reporting on process III . ( E ) Comparison of pE ( fractional population of the excited state ) and kex ( sum of forward and reverse exchange rates ) values obtained from separate two-state fits of dispersion profiles reporting on processes I ( red ) or III ( green ) ( 1 . 5 mM G85R apoSOD12SH , 600 + 800 MHz , 25°C ) showing that each process can be well fit to distinct exchange parameters . The distributions shown were obtained from a bootstrap analysis , as described previously ( Efron and Tibshirani , 1986; Sekhar et al . , 2012 ) . ( F , G ) Variation of measured Rex values corresponding to the differences in effective 15N transverse relaxation rates measured at the lowest and highest CPMG pulsing rates for 2 mM pWT apoSOD12SH for residues sensitive to process IV ( panel F , repeated from Figure 6 for ease of comparison ) and process I ( G ) at 35°C ( red ) and 25°C ( blue ) . The temperature dependencies of processes I and IV unequivocally distinguish them . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 016 The presence of at least four distinct exchange processes in apoSOD12SH suggests that the underlying free energy landscape of this protein is rugged . Further , the pervasive nature of exchange in this system significantly complicates analysis of the CEST and CPMG data . This is especially the case because there is considerable overlap in the regions that report on the various processes , with residues associated with the native dimer interface participating in three of the four exchange events that have been characterized . Not surprisingly , therefore , in some cases the relaxation data show complex exchange behaviour . This does not necessarily imply that the four exchange events discussed here are coupled , but can also result from spin probes reporting on one process ‘sensing’ fluctuating fields from proximal exchange events . For example , CEST profiles of G51 , N53 , T54 , and G61 clearly establish that these residues report on native dimerization , with shift differences in excess of ∼4 ppm between ground and excited state conformers . Yet , for these residues , 15N CPMG relaxation dispersion profiles recorded on pWT samples could not be fit to models of two-site exchange . In contrast , the corresponding profiles from a G85R apoSOD12SH mutant , where process II is eliminated and process IV reduced , could be fit to the simplest two-state exchange model . Further , the CEST profiles for these residues in pWT and G85R apoSOD12SH are very similar , suggesting that process I has not been significantly affected via the mutation . A likely explanation is that the ‘seemingly more simple exchange behaviour’ in the G85R mutant is the result of the elimination of local field fluctuations from these additional processes . The inability to fit the dispersion data for these residues in the pWT protein is , thus , at least in part the result of fluctuating local magnetic fields that arise from a number of simultaneous proximal processes . Most certainly , the multiple exchange processes do complicate assignment of residues to distinct exchange events . A detailed description of the approach taken , involving studies at a number of different temperatures and protein concentrations , is provided in ‘Materials and methods’ ‘Separating processes I–IV’ . Figure 8 and Table 2 summarize the criteria used for separating residues into distinct exchange processes and Figure 8 further indicates what residues were subsequently used as constraints in structure calculations ( see below ) . While not all residues reporting on each exchange process could be identified , a sufficient number of probes could be selected for each process both to identify its nature and to enable the structural characterization of the resulting excited state conformer ( as described below ) . 10 . 7554/eLife . 07296 . 017Figure 8 . Summary of the criteria used for assigning exchange processes I–IV . More details can be found in ‘Materials and methods’ , ‘Separating probes of processes I–IV’ . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 01710 . 7554/eLife . 07296 . 018Table 2 . Distinguishing between exchange processes I–IVDOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 018I ( native dimer ) II ( local folding ) III ( non-native dimer 1 ) IV ( non-native dimer 2 ) I ( Native dimer ) ––––II ( Local folding ) Concentration dependence*–––III ( Non-native dimer 1 ) Rex values in G85R and pE/τE values from G85R†Concentration dependence§––IV ( Non-native dimer 2 ) Temperature dependence of CPMG Rex values‡Concentration dependence#Temperature dependence of CPMG Rexvalues¶–*Figure 3 , Figure 3—figure supplement 1 , Figure 4 and Figure 4—figure supplement 1 . †Figure 7—figure supplement 1 . ‡Figure 7—figure supplement 1 . §Figure 4 , Figure 4—figure supplement 1 , Figure 5 , Figure 5—figure supplement 1 . #Figure 4 , Figure 4—figure supplement 1 , Figure 6 , Figure 5—figure supplement 1 . ¶Figure 5 , Figure 6 , Figure 5—figure supplement 1 . Having established that apoSOD12SH transiently accesses at least two non-native oligomeric conformations , we next obtained structural models for these conformers by assuming that they are dimeric ( see ‘Materials and methods’ ‘Choice of dimer models for the association process’ ) and by using excited state chemical shifts as restraints in the biomolecular docking program HADDOCK ( Dominguez et al . , 2003 ) . Residues that were used as restraints in structure calculations are a subset of those reporting on each process as outlined in Figure 8 . They are listed in Table 3 and their positions in the structure of SOD1 are depicted in Figure 9—figure supplement 1A . As a first step and in order to validate the methodology the structure of the transient native-like dimer formed via process I was determined ( Supplementary File 1 ) and found to be superimposable with the dimeric holo-SOD crystal structure ( Valentine et al . , 2005 ) with a pair-wise all-atom RMSD of 0 . 9 Å from the crystal structure to the lowest energy HADDOCK derived model ( Figure 9A ) . 10 . 7554/eLife . 07296 . 019Table 3 . Residues reporting on processes I–IV†DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 019Process I‡Process II‡Process III§Process IV§V5*G130V5A6V7N131*A6I18K9E132*V7F45I17E133*L8V46E49S134*I17S68F50*T135*Q22G73G51*K136*V47G85D52*T137*H48N86N53*G138A60V87T54*N139*L67I104G61*A140*I112L106I113–G114S111G114–R115I113R115–L117V118V148–H120G141I149*–C146S142G150*–G147R143I151*–V148I151A152*–I149A152Q153–G150–––I151–––A152–––Q153–†Residues selected according to the criteria outlined in ‘Materials and methods’ ‘Separating probes of processes I–IV’ . Active residues used in the molecular docking program HADDOCK ( Dominguez et al . , 2003 ) are indicated in bold . A total of 28 and 14 restraints were used in structure calculations of symmetric dimers corresponding to excited states derived from processes I and III , respectively ( these numbers are doubled to indicate that a restraint from monomer A to B also pertains from B to A ) , with 7 restraints for the calculation of the excited state from process IV ( asymmetric ) . The energy funnels obtained in the structure calculations ( Figure 9—figure supplement 1 ) support the view that the structures have converged . For the non-native dimers , we have further evaluated the robustness of the calculations by repeating them after first removing 1 restraint at a time . Very similar ensembles to those illustrated in Figure 9—figure supplement 1 were obtained indicating that the structure calculation is not driven merely by a single restraint . For process III , we also obtain very similar structures if only restraints from 15N CPMG data are included . Moreover , we have run additional calculations for process IV starting with the NMR derived structure of monomeric C6A/C111S/Q133E/F50E/G51E apoSOD1S–S ( PDB accession code 1RK7 [Banci et al . , 2003] ) , in which the electrostatic and Zn-binding loops are disordered , rather than from a monomer of the native dimer X-ray structure ( PDB accession code 1HL5 [Strange et al . , 2003] ) . Very similar structures are obtained in all cases . ‡Residues labeled with * are those for which separate dips or shoulders corresponding to an excited state were observed in 15N , 13C′ or 13Cα CEST profiles . Recall that relatively small Δϖ values precluded the use of CEST for the study of processes III and IV . Note that a number of active residues are classified as reporting on both processes I and III; these correspond to residues that are at the native dimer interface ( and hence classified as reporters of process I , see ‘Separating probes of processes I–IV , Process I: Transient native-like dimerization’ ) and that have dispersion profiles and Rex values that are clearly sensitive to process III . §Rex > 5 s−1 ( 800 MHz ) for 15N ( 3 . 5 mM ) and/or 13C-methyl ( 2 . 5 mM ) CPMG relaxation dispersion profiles . 10 . 7554/eLife . 07296 . 020Figure 9 . Monomeric apoSOD12SH is in equilibrium with mature and aberrantly associated conformations . ( A ) Superposition of ribbon diagrams of the lowest energy HADDOCK model of the native dimer excited state of pWT apoSOD12SH ( process I , blue ) and the X-ray structure of Cu2Zn2SOD1S–S ( pdb code: 1hl5 , green ) ( Strange et al . , 2003 ) . ( B ) Conformations transiently populated by apoSOD12SH ( blue ) , including states with structural features similar to the ground state of Cu2Zn2SOD1S–S ( ‘Mature conformations’ ) and aberrant oligomers ( ‘Non-native association’ ) . Structural models for the native dimer ( I ) and the symmetric ( III ) and asymmetric ( IV ) non-native dimers populated by apoSOD12SH as determined from the HADDOCK program ( Dominguez et al . , 2003 ) are shown along with the model that includes helix folding ( II ) taken directly as a monomer from the crystal structure of Cu2Zn2SOD1S–S ( Strange et al . , 2003 ) . The purple arrows indicate approximate rotations needed to transform magenta monomers of non-native dimer 1 to corresponding monomers of either the native dimer or non-native dimer 2 . In the latter case , two successive rotations about ( i ) an axis perpendicular to the plane of the paper and subsequently about ( ii ) a vertical axis are required . Note that the scheme shown here is the simplest one consistent with the spin relaxation data ( see ‘Discussion’ ) . ( C ) The A4V mutant severely reduces association via processes III and IV , as established by the significant decrease in the sizes of CPMG dispersion profiles . The top part of each panel shows CPMG profiles for pWT apoSOD12SH , with the corresponding curves for the A4V mutant indicated in the bottom . A small constant y-offset has been applied to some of the curves for clarity . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 02010 . 7554/eLife . 07296 . 021Figure 9—figure supplement 1 . Structural models for the oligomeric excited states of pWT apoSOD12SH from the biomolecular docking program HADDOCK ( Dominguez et al . , 2003 ) . ( A ) Active ( red ) and passive ( yellow ) residue restraints used to generate structural models of the native and non-native transiently formed dimers . The active residues are a subset of those with Rex ( 15N ( 3 . 5 mM ) or methyl-13C ( 2 . 5 mM ) ) > 5 s−1 , 800 MHz , 25°C for which main-chain or sidechain relative solvent accessibility values are larger than 40% . Passive residues are solvent accessible surface neighbours of the active residues . The active residues used to generate the native dimer ( process I ) in HADDOCK calculations are localized to a single region of pWT apoSOD12SH , as are those for non-native dimer 1 ( process III ) . L67 , indicated by * was consistently violated in docking calculations for non-native dimer 1 and was removed from the active restraint list . It appears that spin probes from L67 are not localized to the non-native dimer interface but rather report on a secondary process that depends on dimerization , such as a change in conformation . In contrast to residues associated with process III , active residues for process IV ( non-native dimer 2 ) do not cluster and can roughly be divided between a pair of sites , denoted by black and orange circles in ( A ) . ( B ) HADDOCK calculations for non-native dimer 2 converged well when the interface was enforced to derive from active residues in the ‘black’ circle in one monomer and from active residues from the ‘orange’ circle for the second , leading to an asymmetric dimer ( red , denoted by ‘distinct interfaces’ in the figure panel B ) . Notably , restraints from residues S68 and N86 ( residues indicated by * in A ) were consistently violated and these were subsequently removed from further calculations . Convergence was not achieved when the restraints derived from all active residues for each of the monomers were enforced without first separating them as described above and shown in the circles in A ( green , denoted by ‘single interface’ ) , with on average ∼80% of the restraints violated over the 200 final calculated structures and an average violation of 4 . 7 Å . By contrast , modelling Process IV as involving distinct interfaces ( i . e . , asymmetric dimer ) leads to less than 1 restraint violated on average , per structure , with an average distance of 0 . 4 Å per violation . Plots of the HADDOCK score vs RMSD ( to the lowest scored structure , left ) and a histogram of restraint violations ( right ) illustrate this . In these initial simulations both electrostatic and Zn-loops were kept fixed to the X-ray coordinates ( Strange et al . , 2003 ) , but were allowed to move in further calculations to generate the final set of structures , as described in ‘Materials and methods’ . ( C ) Ensembles of the native and non-native dimers including structures with the 10 lowest HADDOCK scores . Corresponding plots of HADDOCK score vs RMSD ( to the lowest scored structure ) for each of the dimers are shown below the structures . The larger RMSD spreads for the non-native dimers reflect to a large extent the fact that both electrostatic and Zn-loops were allowed to be flexible in the final set of calculations while the Zn-loop was fixed for the native dimer . Averaged over the lowest 10 members of the ensemble , 4 restraints were violated per structure with an average violation of 0 . 5 Å for the native dimer , 1 . 5 restraints per structure with an average violation of 0 . 7 Å for non-native dimer 1 and 1 restraint per structure with an average violation of 0 . 8 Å for non-native dimer 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 02110 . 7554/eLife . 07296 . 022Figure 9—figure supplement 2 . The asymmetric dimer from process IV can serve as a hub for the formation of larger oligomers . A pair of low energy dimers formed via process IV was docked via HADDOCK as described in ‘Materials and methods’ using restraints from process IV ( listed in Table 3 ) to form tetramers and the process repeated with the resulting tetramers to form octamers ( Monomer–Dimer—Tetramer–Octamer pathway ) . Alternatively , non-native dimer 2 from process IV can also recruit a native dimer ( left ) or a non-native dimer generated from process III to form tetrameric aggregates , as shown . These structures were generated from HADDOCK calculations where non-native dimer 2 was docked on to a native dimer molecule or a non-native dimer 1 molecule using restraints from process IV . DOI: http://dx . doi . org/10 . 7554/eLife . 07296 . 022 Using similar HADDOCK calculations structural models were generated for the non-native dimers as well ( Figure 9—figure supplement 1 , Supplementary Files 2 and 3 , see ‘Materials and methods’ ‘Building structural models of excited states using HADDOCK’ ) . These differ significantly from the mature dimeric form of Cu2Zn2SOD1S–S ( Figure 9B ) . The non-native dimer formed via process III ( referred to as non-native dimer 1 ) is symmetric and topologically related to the native dimer by a 180° rotation of one of the monomer units about an axis shown in Figure 9B . It is stabilized by an interface very similar to that of the native dimer , involving Zn-binding and β6-β7 loops , as well as strands β1 and β8 . Notably , the restraints used to model the excited state formed via this process ( Table 3 and Figure 9—figure supplement 1 ) localize to the native dimer interface on one side of the monomer , consistent with the symmetric dimer structural model . These restraints are a subset of those for process I , and it is reasonable to ask why the native structure is not generated in this case . This can be understood by noting that the native dimer interface involves contacts between residues from β1–β8 in one monomer and residues localized to the Zn-loop from a second , placing the corresponding β1–β8 residues in the second monomer far from those in the first . In contrast , since the restraint residues for process III include those from β1–β8 but not the Zn-loop , the process of satisfying the restraints ( i . e . , bringing restraint residues within a prescribed distance from each other ) places β1–β8 from both monomers of the dimer in proximity , leading to a structure where one of the monomers is rotated 180o compared to the native dimer . Unlike both dimeric structures formed via processes I and III , the second non-native dimer ( non-native dimer 2 , process IV ) is asymmetric , with the orientation of the monomers with respect to each other illustrated in Figure 9B . In this case the interface is formed by the β6–β7 and Zn loops as well as strands 1 and 8 from one monomer and by the electrostatic and Zn-binding loops from the second molecule . The asymmetry is to be expected based on the location of the input restraints ( Figure 9—figure supplement 1 ) , which are not localized to a particular region of the molecule , but spread into a number of clusters . These clusters of restraints cannot be simultaneously satisfied by a symmetric dimer , but can be readily accounted for by an asymmetric dimeric structure that brings one cluster on one monomer close to the remaining clusters on a second molecule . The structural models all point to an important role for strand β1 in the formation of excited states derived from processes I , III , and IV . This leads to the prediction that mutations in this region could disrupt association . To test this , we mutated A4 in β1 to V and recorded further CPMG experiments to evaluate its effects on the association processes . As expected , the effect is pronounced with both native ( I ) and non-native dimerization processes ( III ) completely abolished , while formation of the asymmetric dimer via process IV is considerably diminished ( Figure 9C ) .
Using NMR methods exquisitely sensitive to the presence of excited protein conformations ( Sekhar and Kay , 2013 ) , we have demonstrated that under physiologically relevant conditions immature monomeric apoSOD12SH has a rugged free energy landscape on which mature conformations resembling the native state of metallated SOD1 coexist with aberrant non-native oligomers . The high propensity of the exposed dimer interface and the flexible electrostatic loop to self-associate results in the formation of three distinct oligomeric forms which exist in equilibrium with the native state of apoSOD12SH . Structural models for the two non-native oligomers reported here may provide insight for the rational design of therapeutic agents to prevent the early stages of nascent SOD1 aggregation and ultimately formation of the cytotoxic species that leads to disease .
Genes encoding pWT SOD1 ( Getzoff et al . , 1992; Vassall et al . , 2011; Broom et al . , 2014 ) ( pseudo wild-type protein where nonconserved Cys 6 and Cys 111 are replaced by Ala and Ser , respectively ) or ALS-associated mutants G85R and A4V ( Valentine et al . , 2005 ) were inserted into the plasmid vector pHSOD1ASlacIq ( Getzoff et al . , 1992 ) , with protein expression in BL21 Escherichia coli cells . Suitably labeled proteins were grown to 0 . 6–0 . 7 OD600 in M9 minimal media ( Na2HPO4 [6 g/l] , KH2PO4 [3 g/l] , NaCl [0 . 5 g/l] , 15NH4Cl [0 . 5 g/l] , MgSO4 [2 mM] , 12C glucose [4 g/l for 15N-labeling] , 13C glucose [2 g/l for U-13C labeling] or 1-13C glucose [2 g/l for selective labeling of methyl groups ( Lundström et al . , 2007a ) ] , thiamine [0 . 0005 g/l] , CaCl2 [0 . 1 mM] with 100 mg/l ampicillin ) . Because Ile δ1 methyl groups are not efficiently 13C labeled when 1-13C glucose is used as the 13C source , methyl 13C , 3 , 3-D2α-ketobutyrate ( 50 mg/l ) was also added ( Gardner and Kay , 1997 ) approximately 1 hr prior to induction of protein overexpression . Protein expression was induced upon addition of 0 . 25 mM IPTG and the growth media were supplemented with CuSO4 and ZnSO4 ( 0 . 1 mM and 0 . 05 mM final concentrations , respectively ) at this time . Expression continued for ∼42 hr at 25°C after which cells were harvested by centrifugation . Purification of Cu2Zn2SOD1S–S was carried out using a modification of the procedure outlined in Getzoff et al . in which the DEAE column step was replaced by one involving a POROSHP2 column ( Getzoff et al . , 1992 ) . Subsequent generation of apo-protein and disulfide reduction were achieved following protocols described previously ( Vassall et al . , 2006 , 2011 ) . The metal content in samples was determined by Inductively Coupled Plasma Atomic Emission Spectroscopy . Reduction of the disulfide linking Cys 57 to Cys 146 ( apoSOD12SH ) was verified by making use of the large differences in 13Cβ chemical shift values of Cys residues in oxidized ( on average 40 . 7 ± 3 . 8 ppm ) and reduced ( 28 . 4 ± 2 . 4 ppm ) states ( Sharma and Rajarathnam , 2000 ) . The measured values for Cys 57 and Cys 146 of 28 . 1 ppm and 32 . 5 ppm , respectively , are consistent with reduced residues . Samples of isotope-labeled apoSOD12SH were prepared in buffer containing 20 mM HEPES pH 7 . 4 , 1 mM TCEP , 1 mM NaN3 , and 90% H2O/10% D2O ( NMR buffer ) , while a 0 . 8 mM sample of Cu2Zn2SOD1S–S was prepared in buffer containing 20 mM HEPES pH 7 . 4 , 5 mM isoascorbate and 90% H2O/10% D2O . 15N-labeled samples of apoSOD12SH ranged in concentration from 0 . 7 mM to 3 . 5 mM , the concentration of U-[13C , 15N] apoSOD12SH was 1 . 5 mM and the selectively methyl-13C labeled samples were prepared at concentrations of 2 . 5 mM and 0 . 8 mM . Suprasil NMR tubes ( Wilmad Lab-glass , cat . no . 535-PP-7SUP ) were used in recording all spectra for apoSOD12SH samples unless stated otherwise . After placing the apoSOD12SH sample inside the NMR tube , the tube was immediately purged with Ar and sealed before use . NMR spectra were acquired using 11 . 7 T ( 1H frequency of 500 MHz ) , 14 . 0 T ( 600 MHz ) or 18 . 8 T ( 800 MHz ) Varian INOVA spectrometers , with the 600 MHz spectrometer equipped with a cryogenically cooled probe . All NMR experiments were carried out at 25°C unless stated otherwise , with the temperature in the sample chamber measured using a thermocouple attached to a digital thermometer placed inside an NMR tube containing D2O . NMR data sets were processed with the NMRPipe ( Delaglio et al . , 1995 ) suite of programs and visualized using Sparky ( Goddard and Kneller , 2006 ) . Peak intensities were extracted using either NMRPipe or by fitting the respective peak lineshapes using FuDA ( http://pound . med . utoronto . ca/software ) . Experiments for resonance assignments were recorded at 600 MHz using a U-[13C , 15N] apoSOD12SH sample . Assignments of backbone and sidechain resonances were obtained using a combination of 2D 1H-15N HSQC and 1H-13C HSQC data sets as well as 3D HNCACB , CBCA ( CO ) NH , HNCO , HN ( CA ) CO , ( H ) CCA ( CO ) NH , and H ( CCACO ) NH experiments , described in detail previously ( Cavanagh et al . , 1995; Sattler et al . , 1999 ) . Assigned chemical shifts of pWT apoSOD12SH have been deposited to the Biological Magnetic Resonance Data Bank ( BMRB accession number 26570 ) . Assignments of 1HN and 15N resonances for G85R and A4V mutants were transferred from pWT using 15N NOESY-HSQC ( Zhang et al . , 1994 ) data sets . Translational diffusion coefficients of apoSOD12SH and Cu2Zn2SOD1S–S were determined using a 1D pulsed field gradient 15N-edited longitudinal encode-decode ( LED ) scheme ( Choy et al . , 2002 ) . The total diffusion delay used in the measurements was 150 ms , and encoding and decoding gradient durations were 1 ms each . Each dataset comprised 15 gradient strengths ranging between 4–60 G/cm with three duplicate points for error analysis . The amide region ( 7 . 8–9 . 4 ppm ) of each 1D 15N-edited spectrum was integrated as a single unit and the variation in the resulting intensity ( I ) with squared gradient strength ( G2 ) fit to a single exponential decay of the form , I = I0 exp ( −dG2 ) , to extract a diffusion decay rate ( d ) which is linearly proportional to the translational diffusion coefficient of the protein . In order to estimate the rotational correlation times of apoSOD12SH and Cu2Zn2SOD1S–S , 15N spin relaxation rates , R1 and R1ρ , and steady state 1H-15N heteronuclear NOE values were measured using pulse sequences described earlier ( Farrow et al . , 1994; Korzhnev et al . , 2002; Ferrage et al . , 2010 ) . 11 relaxation delays ranging between 10–600 ms and 10 delays between 3–80 ms were used for the R1 and R1ρ experiments , respectively . Three relaxation delays were repeated in each of the experiments for error analysis . Residue-specific R1 and R1ρ values were obtained from fits of peak intensities vs relaxation time to a single exponential decay function , while NOE ratios were ascertained directly from intensities in experiments recorded with ( 5 s relaxation delay followed by 7 s saturation ) and without ( relaxation delay of 12 s ) saturation . Errors in NOE values were calculated by propagating the error in the respective peak intensities . R1ρ values were converted to R2 rates using the relation , R1ρ = R2cos2θ + R1sin2θ , where θ = tan−1 ( δ/BSL ) , BSL is the spin lock field strength ( Hz ) , δ is the resonance offset of the spin in question ( Hz ) , and θ is the angle made by the effective field with the direction of BSL irradiation . Values of rotational correlation times were obtained from fits of ( R1 , R2 , NOE ) to standard expressions ( Abragam , 1961; Kay et al . , 1989 ) with spectral density functions as defined by the model free approach of Lipari and Szabo ( Lipari and Szabo , 1982 ) . Only 15N spins with Rex < 2 s−1 , as measured in 15N CPMG experiments recorded at 600 MHz ( 25°C , 3 . 5 mM protein concentration ) , were used in the analysis . Residues were included only if they are in ordered regions of the protein and additionally for apoSOD12SH if chemical shift-derived order parameters ( Figure 2A ) were larger than ∼0 . 6 . All fits used the software package Modelfree 4 . 15 ( Mandel et al . , 1995 ) and for simplicity isotropic diffusion tensors were assumed with residue specific parameters that include the order parameter squared , S2 , and a timescale describing fast motion , τe . Overall , 15 ( localized to 5 of the 8 strands ) and 112 residues were considered in the analyses for apoSOD12SH and Cu2Zn2SOD1S–S , respectively , and correlation times of 11 . 6 ns ( apoSOD12SH ) and 18 . 2 ns ( Cu2Zn2SOD1S–S ) were obtained . Note that the small number of apoSOD12SH residues selected for analysis reflects the widespread chemical exchange in this variant . All CEST experiments were recorded using previously published pulse schemes ( Vallurupalli et al . , 2012; Vallurupalli and Kay , 2013; Long et al . , 2014 ) on samples labeled with 15N ( 15N CEST ) or U-[15N , 13C] ( 13C′ , 13Cα and 13CH3 CEST ) . Relaxation delays ( TCEST ) of 350 ms ( 15N ) , 300 ms ( 13C′ ) , 125 ms ( 13Cα ) , and 250 ms ( 13CH3 ) were employed and CEST profiles were recorded using 1 or 2 B1 radiofrequency fields for 15N ( 31 and 59 Hz ) , 1 B1 field for 13Cα ( 27 Hz ) and 2 B1 fields for both13C′ ( 26 and 42 Hz ) and 13C-methyl ( 15 and 24 Hz ) spins . B1 field strengths were calibrated as reported earlier ( Vallurupalli et al . , 2012; Vallurupalli and Kay , 2013 ) . A series of 2D spectra were recorded with the B1 field stepped from 93 . 9–136 . 9 ppm for 15N ( 89 planes at B1 = 31 Hz and 54 planes at 59 Hz ) , 169–180 . 9 ppm for 13C′ ( 82 planes at 26 Hz and 50 planes at 42 Hz ) , 41 . 2–68 . 6 ppm for 13Cα ( 120 planes ) and 9 . 3–30 ppm for 13CH3 ( 139 planes at 15 Hz and 107 planes at 24 Hz ) . CEST profiles were generated as the ratio in intensities of peaks in spectra acquired with and without the TCEST period vs the position of the low power B1 field ( Fawzi et al . , 2011; Vallurupalli et al . , 2012 ) . Errors were determined from the scatter in the baseline of CEST profiles ( Bouvignies et al . , 2014 ) . Chemical shifts of the excited state were obtained directly from the CEST curves by fitting them to a sum of Gaussians using the ‘curvefit’ routine in Matlab ( The Mathworks Inc . , Natick , MA ) . In order to extract exchange parameters , CEST profiles were fit to two- or three-state exchange models using the home written program Chemex ( https://github . com/gbouvignies/chemex ) . Errors in fitted values were extracted using the covariance matrix method ( Taylor , 1997 ) . 15N CPMG data sets were recorded on U-15N , U-[15N , 13C] and selectively 13C-labeled samples produced with 1-13C glucose , while 13C methyl profiles were measured on selectively 13C-labeled samples only . CPMG experiments were generally , but not always , recorded at two static magnetic field strengths . Constant-time CPMG relaxation elements were used for both 15N ( Vallurupalli et al . , 2007 ) and 13CH3 ( Lundström et al . , 2007b ) experiments with relaxation delays ( TCPMG ) of 30 ms , approximately 20 different values of the CPMG pulsing frequency ranging from 33 . 3–1000 Hz for 15N , from 66 . 6–2000 Hz for 13CH3 and 3–5 duplicate points acquired for error analysis ( Korzhnev et al . , 2007 ) . Peak intensities in CPMG experiments were converted to effective transverse relaxation rates ( R2 , eff ) using the equation , R2 , eff = ( −1/TCPMG ) ln ( I/I0 ) , where I and I0 are peak intensities measured with and without the CPMG delay ( Korzhnev et al . , 2007 ) . Errors in intensities determined from duplicate points were propagated to obtain errors in R2 , eff ( Korzhnev et al . , 2007 ) . The variation in R2 , eff as a function of CPMG pulsing frequency ( so called relaxation dispersion profile ) was fit to a two-state model of exchange using in-house-written software CATIA ( http://pound . med . utoronto . ca/software ) , while Chemex was used for global fits of CEST and CPMG data to both two- and three-state exchange models . Both programs numerically propagate the Bloch–McConnell equations ( McConnell , 1958 ) relevant for a particular model of exchange . Errors in fitted values of populations , rate constants and chemical shift changes were determined using the covariance matrix method ( Taylor , 1997 ) . Transient dimerization ( process I ) transforms apoSOD12SH into a conformation which is very similar to the native ground state of metal-bound Cu2Zn2SOD1S–S while the local folding of the electrostatic loop , residues 130–140 ( process II ) , generates a helix which is found in the native dimeric Cu2Zn2SOD1S–S state . Because of the high affinity of SOD1 for Cu and Zn , ( dissociation constants smaller than ∼10−13 and ∼10−17 M , respectively [Crow et al . , 1997] ) it is necessary to establish that processes I and II are not ‘simply’ the result of apoSOD12SH binding to trace metals present in the sample . As illustrated in Figure 3—figure supplement 3 , a number of controls rule out that processes I and II are metal-dependent exchange events , reflecting rather the landscape of metal free apoSOD12SH and the propensity of this state to adopt ‘native-like’ conformations . We summarize the evidence that processes I and II are metal independent in the points below . After recording spectra of samples of pWT apoSOD12SH ( Suprasil tubes ) metal analysis was carried out via Inductively Coupled Plasma Atomic Emission Spectroscopy ( ICP-AES ) . No detectable levels of Cu or Zn could be found ( detection limits of 750 nM for Zn and 300 nM for Cu; Figure 3—figure supplement 3A ) . Among those elements tested ( Ag , Al , As , B , Ba , Be , Ca , Cd , Co , Cr , Cu , Fe , K , Mg , Mn , Mo , Na , Ni , Pb , Sb , Se , Si , Ti , Tl , V , and Zn ) only B ( 104 μM ) , Ca ( 2 . 25 μM ) , K ( 24 . 6 μM ) , and Na ( 17 . 3 mM ) could be detected , with Ca the only divalent metal . Its concentration is an order of magnitude smaller than what would be required to give rise to the excited state populations observed ( 2–3% ) , since for the protein concentration of 1 . 3 mM used in the analysis approximately 30 μM of metal would be necessary for the excited state to correspond to a Ca-bound state . This is further supported by the fact that very similar CEST profiles were obtained from samples that had been placed in regular glass NMR tubes where the Ca concentration was determined to be below the detection limit of ICP-AES ( Figure 3—figure supplement 3B , C ) . To further confirm that CEST profiles observed for residues reporting on processes I and II are not the result of direct Zn binding we added ∼140 μM Zn to a Zn and Cu-free apoSOD12SH sample in a Suprasil tube . For many residues , two sets of peaks are observed in 1H-15N HSQC spectra of the resultant sample due to the slow exchange between apoSOD12SH and the metal-bound form of the protein ( Figure 3—figure supplement 3D; representative peaks indicated with * ) . The CEST profiles from apoSOD12SH are , however , very similar to those recorded in the absence of metal ( Figure 3—figure supplement 3E , F ) . If the CEST profiles quantified were reporting on Zn binding the CEST dips would increase significantly to reflect an excited state population of ∼20% ( 140 μM Zn , 700 μM protein ) . The dips , however , do not change in size , confirming that the excited state is independent of Zn binding . Our results are consistent with expectations based on previous studies where the half-life for Zn release has been determined to be at least 11 hr ( Crow et al . , 1997 ) , orders of magnitude longer than is required for observation of chemical exchange via CEST where exchange rates of at least 50 s−1 are needed ( Vallurupalli and Kay , 2013 ) . Residue- and nucleus-specific Rex values obtained from 15N and 13C-methyl CPMG experiments were calculated as the difference between R2 , eff rates at the lowest and highest CPMG pulsing frequencies ( Palmer et al . , 2000 ) . In order to evaluate the protein concentration-dependence of Rex , data recorded at either 3 . 5 mM ( 15N ) or 2 . 5 mM ( 13CH3 ) , 800 MHz , were analyzed and residues with Rex > 5 s−1 selected . For this subset , ratios ( R ) of Rex values measured at protein concentrations of 3 . 5 mM and 0 . 7 mM ( 15N ) or 2 . 5 mM and 0 . 8 mM ( 13CH3 ) , 600 MHz , were calculated and the error in each R value , σR , obtained by propagating errors in Rex at the two concentrations . Residues for which R > 1 . 4 and R/σR ≥ 3 were classified as part of the concentration dependent group . The temperature-dependencies of CPMG profiles for residues with concentration-dependent Rex values were determined by examining Rex rates measured in either 2 mM ( 15N ) or 2 . 5 mM ( 13CH3 ) samples at 25°C and 35°C , 800 MHz . Residues with temperature-dependent Rex values were defined as those with D = |Rex ( 35°C ) − Rex ( 25°C ) | > 1 s−1 and D/σD ≥ 3 , where σD is the error in D obtained by propagating errors in Rex at 25° and 35°C . A total of 42 residues were selected from analysis of the protein concentration dependencies via these criteria and these were sorted into processes III ( 23 residues ) and IV ( 19 residues ) based on the temperature dependence criteria described above . CPMG and CEST data sets recorded on pWT apoSOD12SH and reporting on processes I and II could not be fit simultaneously to a two-state model of chemical exchange . ( In contrast , dispersion data from process III and IV were well fit to a two state model . ) As a result a three-state model was chosen in the analysis of processes I and II , with fits performed in several stages . First , a number of residues whose 15N CEST and CPMG profiles indicated large chemical shift differences between the ground and excited states were identified for each process ( Gly 51 , Asn 53 , Thr 54 and Gly 61 for process I and Thr 135 and Thr 137 for process II ) ; these residues were used in subsequent analyses to extract exchange parameters . Next , a complete set of three-state models was chosen making use of the fact that the chemical shifts for probes in two of the three states are known from CEST profiles , referred to as the ground ( N ) and the excited state ( H ) in what follows . Note that only two dips are observed in the CEST profiles , suggesting that the third state has similar chemical shifts to either N or H . There are , thus , three classes of linear three-state models that can be selected , depending on the relative position of the hidden intermediate ( I; not observed in the CEST profile in the ‘reaction scheme’ ) , as illustrated below . These include , N⇌kINkNII⇌kHIkIHH [1] , I⇌kNIkINN⇌kHNkNHH [2] , N⇌kHNkNHH⇌kIHkHII [3] , where I is on-pathway [1] or off-pathway and originating either from the ground [2] or the excited states [3] . In each of these three classes , the 15N chemical shift of I can be close to N or to H ( hence explaining the appearance of only two dips in each CEST trace ) , so that a total of six initial models was considered . The CEST and CPMG data were not of sufficiently high quality to distinguish the triangular model with all three states connected from the linear schemes described above , so the simplest linear 3-state schemes were selected . For each model , A⇌kBAkABB⇌kCBkBCC , the search in parameter space was carried out across 2 rate constants , kAB , ex = kAB + kBA and kBC , ex = kBC + kCB , and two populations ( corresponding to the sparsely populated states H and I , pH and pI in schemes [1]–[3] above ) . For transient dimerization ( process I ) , the grid consisted of 11 points in kAB , ex and kBC , ex logarithmically spaced to span the range 10–2016 s−1 , while the grid in pH and pI comprised 16 points linearly spaced between 0 . 5 and 23% . For the local folding process II , the grid in pH × pI × kAB , ex × kBC , ex consisted of 10 × 10 × 16 × 16 points between 0 . 5 and 14% in pH and pI ( linear grid ) and 10 and 11 , 529 s−1 in rates ( logarithmic grid ) . At each grid point χred2 was calculated by fitting CEST and CPMG profiles on a residue-specific basis using Chemex . Values of kAB , ex , kBC , ex , pH and pI along with ΔϖNH ( that can be obtained directly from the positions of the major and minor dips in the CEST profiles ) were kept fixed in each fit , while ΔϖNI was allowed to vary along with the relaxation rates R2N , R2H , R2I , and R1N . Here , ΔϖNH and ΔϖNI are the chemical shift differences for the corresponding residues in the ground state ( N ) and either the excited ( H ) or hidden intermediate ( I ) states , respectively , while R2N , R2H , and R2I are transverse relaxation rates of the ground , excited and hidden states respectively and R1N is the longitudinal relaxation rate of N . For each three state-model and for each residue the grid search protocol described above generated a χred2 surface as a function of kAB , ex , kBC , ex , pH , and pI . The surfaces for all residues of a specific process were then added and the top 10 values of kAB , ex , kBC , ex , pH , and pI ( i . e . , smallest χred2 ) for each of the six models were used as starting points in the next stage of χred2 minimizations . Here , all parameters were allowed to vary and the CEST and CPMG data for the residues indicated above associated with a particular process fit globally to extract final ‘optimal’ values of kAB , ex , kBC , ex , pH , pI , ΔϖNH , and ΔϖNI and the best models for describing the data . As described in ‘Separating probes of processes I–IV’ below high quality fits of the combined CEST/CPMG data using this approach could be obtained for residues reporting on process II but not I . In the analysis of CEST and CPMG relaxation data as a prelude to obtaining structural models of the oligomers formed via processes I , III and IV we have made the simplest assumption that in all cases dimeric structures are generated . This is justified as follows: ( i ) the chemical shifts of the excited state conformer formed from process I correlate very well with shifts of mature , native Cu2Zn2SOD1S–S published previously ( Strange et al . , 2003 ) ( Figure 3 ) , providing very strong evidence that process I corresponds to dimerization of apoSOD12SH monomers . ( ii ) Residues involved in the generation of the excited state of process III localize to a very specific region of structure ( Figure 9—figure supplement 1 ) . Formation of a symmetric dimer with each monomer contributing the same interfacial region can account for the shift changes . More complex models of oligomerization need not be invoked , but if they are then additional interfaces would have to be involved . This is not consistent with the localization of chemical shift changes to only a specific region of structure . ( iii ) Residues involved in the interface of the oligomer generated from process IV localize to two distinct regions and the chemical shift perturbations can be explained by the formation of an asymmetric dimer , as the simplest model ( Figure 9—figure supplement 1 , Figure 10 ) . This does not prove that the structure is dimeric and as is shown in Figure 9—figure supplement 2 , more complex models involving additional subunits can be generated simply by extending the basic dimeric building block using the same interfaces as those involved in formation of the various dimers via processes I , III and IV . It should be noted that dimeric structures formed in all cases are able to satisfy the input restraints and that more complex models are not required to explain the data . Residues for which chemical shift changes could be measured in fits of CEST and CPMG experiments were used to generate structural models of the excited states associated with processes I , III and IV . Residues belonging to process I with |ΔϖMD| > 0 . 3 ppm , as established from 15N , 13C′ or 13Cα CEST measurements , were chosen . All residues identified on the basis of the concentration- and temperature-dependence of their 15N or methyl-13C Rex values ( calculated as described above ) were used as ‘restraints’ in the calculation of excited states for processes III and IV . A list of residues is supplied in Table 3 ( see ‘Separating probes of processes I–IV’ above for selection criteria of residues to each of the different processes ) . The solvent accessible subsets of residues from the lists generated as described above were determined using the software package NACCESS ( Hubbard and Thornton , 1993 ) using 40% exposure of either backbone or sidechain atoms as the selection criterion; residues in this subset were chosen as ‘active’ in the HADDOCK docking protocol . One monomer from the Cu2Zn2SOD1S–S crystal structure ( Strange et al . , 2003 ) was chosen as the starting structure in the generation of excited state dimers , since the folds of apoSOD12SH and each of the monomers of the mature dimer are similar; both have intact β-barrels ( Figure 1 ) and differ predominantly in the arrangement and dynamics of the loops . The Zn-loop residues ( 49-80 ) and the electrostatic loop ( 126–143 ) were fixed to the coordinates of the crystal structure in initial trial runs , but subsequently defined as completely flexible segments to generate dimers for processes III and IV . For process I , only the electrostatic loop was allowed to be fully flexible because chemical shifts indicate that the Zn-loop becomes ordered upon forming the excited state dimer ( Figure 2 ) . In all HADDOCK runs , histidine protonation states were set using the software WhatIf ( Vriend , 1990 ) . Docking was done in three stages starting with an initial rigid body step , followed by semi-flexible simulated annealing and a final refinement in explicit water . 1000 structures were generated in the initial rigid-body docking step and the 200 lowest energy structures were chosen for subsequent annealing and refinement .
Amyotrophic lateral sclerosis ( ALS ) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu , Zn superoxide dismutase ( SOD1 ) . A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers . Here , we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 ( apoSOD12SH ) . Structural models of two of these establish that they possess features present in the mature dimeric protein . In contrast , the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions . Our results show that apoSOD12SH has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization .
Amyotrophic lateral sclerosis ( or ALS ) is a disease that affects the nerve cells in the brain and spinal cord , such that more and more of these cells die as the disease progresses . Since nerve cells direct muscle movement , people with ALS increasingly lose control of their muscles , and may therefore lose the ability to speak , eat , move , and breathe . Around one in five people who have an inherited form of ALS also have mutations in a gene that encodes an enzyme called SOD1 . This antioxidant enzyme detoxifies harmful chemicals that are the byproducts of normal cellular activity . The mature active form of this enzyme contains two SOD1 proteins , each of which binds one copper ion and one zinc ion . Each protein in a pair also contains a strong bond that helps to stabilize its three-dimensional structure . However , immature copies of this protein , which lack the strong bond and metal ions , often fold into the wrong shape . These misfolded proteins can clump together into clusters , and potentially lead to the development of ALS . Efforts to study misfolded proteins are limited by the fact that misfolding is a rare event . As a result , protein samples generally contain very small fractions of misfolded molecules and most techniques for investigating protein structure are ill-suited to probe the process of misfolding . Nuclear magnetic resonance spectroscopy ( or NMR for short ) is one technique that has been used to visualize flexible proteins , and protein folding and misfolding , down to the level of individual atoms . Recent improvements in NMR spectroscopy have opened up the possibility of studying protein structures that exist at levels as low as 1% of the sample . Now , Sekhar et al . have used NMR to document the dynamics of immature SOD1 proteins . The experiments show that the protein is flexible and readily switches back and forth between its ‘default’ shape and one of at least four different shapes; and at any one time , the fraction of molecules in solution that were in each of these shapes was around 1% . Two of the shapes have many of the same features as those in a mature SOD1 protein pair . But the other two shapes involve SOD1 proteins interacting in unusual ways , and may resemble the cluster-forming misfolded SOD1 proteins . By using the NMR data to create three-dimensional models of the SOD1 proteins , Sekhar et al . were able to identify two sites on immature SOD1 proteins involved in these unusual interactions that are inaccessible in mature enzymatically active SOD1 . These findings suggest that targeting these two sites with drugs could possibly block the formation of toxic versions of SOD1 early in the course of ALS , and thus prevent the progression of the disease .