Assignment of backbone NMR resonances and secondary structural elements of a reduced monomeric mutant of copper/zinc superoxide dismutase

Banci, Lucia; Benedetto, Marco; Bertini, Ivano; Conte, Rebecca Del; Piccioli, Mario; Richert, Thierry; Viezzoli, Maria Silvia

doi:10.1002/(sici)1097-458x(199712)35:12<845::aid-omr183>3.0.co;2-o

Cited by 16 publications

(27 citation statements)

References 4 publications

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“…Escherichia coli TOPP1 strains were transformed with the obtained plasmids and with pPCuZnSODlI q . To get large amounts of enzymes, the protein was expressed in 2xYT complex medium (22); to obtain uniformly 15 N-and, when needed, 13 C-labeled proteins for the sake of NMR studies, 15 N-and 13 C-enriched M9 chemically defined medium was used as described (23). An exception was the case of wtS134N, which was expressed without addition of copper sulfate to the culture because some expression tests revealed a sharp decrease in the protein yield upon addition of such salt.…”

Section: Methodsmentioning

confidence: 99%

Fully Metallated S134N Cu,Zn-Superoxide Dismutase Displays Abnormal Mobility and Intermolecular Contacts in Solution

Banci

Bertini

D’Amelio

et al. 2005

Journal of Biological Chemistry

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S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metaldeficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.Over 100 different mutations in the superoxide dismutase 1(SOD1) 4 gene have been linked to the inherited form of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by progressive death of motor neurons and consequent paralysis. The individual mutations have been shown to exert their pathological effects by a gain of function mechanism, implying that the copper-zinc superoxide dismutase variant (Cu,Zn-SOD) expressed from the mutated gene has in some way become toxic. Recent evidence suggests that SOD1-linked ALS, like many other neurodegenerative diseases, is a protein misfolding disorder characterized by abnormal deposits of aggregated proteins in neural tissues (1-3). Implicated also in the toxicity of ALS-mutant SOD1 proteins are accelerated oxidative damage and inhibition of proteasome, chaperone, or mitochondrial function (2, 4, 5).Relatively large, insoluble proteinaceous inclusions are observed in neural tissue in ALS and other neurodegenerative diseases, but it has been proposed that, rather than causing the disease, these deposits of aggregated protein may be formed as a part of a defensive mechanism to sequester misfolded and oligomerized protein when excessive amounts accumulate. More recently it has been suggested that the toxic species are smaller, high molecular weight oligomerized proteins formed from abnormal contacts of misfolded proteins resulting in protofibrils, pores, or other toxic species (6 -10). High molecular weight forms of oligomerized mutant SOD1 have been observed in several studies using ALS-SOD1 transgenic mice (11-13).Many of the ALS-linked Cu,Zn-S...

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Section: Methodsmentioning

confidence: 99%

Fully Metallated S134N Cu,Zn-Superoxide Dismutase Displays Abnormal Mobility and Intermolecular Contacts in Solution

Banci

Bertini

D’Amelio

et al. 2005

Journal of Biological Chemistry

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“…Indeed, in the segment 50-59 of Q133M2SOD, five backbone HN signals were not assigned, probably due to line-broadening as a consequence of proton exchange with solvent due to their surface location. This is consistent with the analysis of the amide hydrogen-deuterium exchange behaviour previously reported [51]. The other loops are still disordered even in the dimeric form.…”

Section: Structure Constraints and Calculationsmentioning

confidence: 99%

The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization

Banci¹,

Bertini²,

Cramaro³

et al. 2002

Eur J Biochem

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The solution structure of homodimeric Cu 2 Zn 2 superoxide dismutase (SOD) of 306 aminoacids was determined on a 13 C, 15 N and 70% 2 H labeled sample. Two-thousand eighthundred and five meaningful NOEs were used, of which 96 intersubunit, and 115 dihedral angles provided a family of 30 conformers with an rmsd from the average of 0.78 ± 0.11 and 1.15 ± 0.09 Å for the backbone and heavy atoms, respectively. When the rmsd is calculated for each subunit, the values drop to 0.65 ± 0.09 and 1.08 ± 0.11 Å for the backbone and heavy atoms, respectively.The two subunits are identical on the NMR time scale, at variance with the X-ray structures that show structural differences between the two subunits as well as between different molecules in the unit cell. The elements of secondary structure, i.e. eight b sheets, are the same as in the X-ray structures and are well defined. The odd loops (I, III and V) are well resolved as well as loop II located at the subunit interface. On the contrary, loops IV and VI show some disorder. The residues of the active cavity are well defined whereas within the various subunits of the X-ray structure some are disordered or display different orientation in different X-ray structure determinations. The copper(I) ion and its ligands are well defined. This structure thus represents a well defined model in solution relevant for structure-function analysis of the protein. The comparison between the solution structure of monomeric mutants and the present structure shows that the subunit-subunit interactions increase the order in loop II. This has the consequences of inducing the structural and dynamic properties that are optimal for the enzymatic function of the wild-type enzyme. The regions 37-43 and 89-95, constituting loops III and V and the initial part of the b barrel and showing several mutations in familial amyotrophis lateral sclerosis (FALS)-related proteins have a quite extensive network of H-bonds that may account for their low mobility. Finally, the conformation of the key Arg143 residue is compared to that in the other dimeric and monomeric structures as well as in the recently reported structure of the CCS-superoxide dismutase (SOD) complex.

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“…A series of theoretical calculations (97)(98)(99) of the electrostatic attraction of the enzyme for 02-and experimental determinations (100)(101)(102) of the SOD activities of CuZnSODs containing chemical modification or mutagenic substitution of conserved amino acid residues that form the channel have demonstrated the essential role of electrostatic guidance in the enzymatic activity. Recent NMR studies on monomeric mutant CuZnSOD suggest further that changes and/or fluctuations in the channel structure may be important in determining the catalytic efficiency of those enzymes (103)(104)(105). On the basis of all of these experiments and calculations, it seems clear that the selectivity of the enzyme for reaction with superoxide and the efficiency of the disproportionation reaction catalyzed by the enzyme depends critically on the ability of the enzyme to maintain the precisely engineered channel undisturbed and that the electrostatic steering of 0~into the active site of enzyme is one particularly important feature of CuZnSOD that makes it an efficient biological catalyst of Oz-disproportionation.…”

Section: Substrate Recognitionmentioning

confidence: 99%

Models of Superoxide Dismutases

Cabelli¹,

Riley²,

Rodríguez³

et al. 2000

Biomimetic Oxidations Catalyzed by Transition Metal Complexes

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Assignment of backbone NMR resonances and secondary structural elements of a reduced monomeric mutant of copper/zinc superoxide dismutase

Cited by 16 publications

References 4 publications

Fully Metallated S134N Cu,Zn-Superoxide Dismutase Displays Abnormal Mobility and Intermolecular Contacts in Solution

Fully Metallated S134N Cu,Zn-Superoxide Dismutase Displays Abnormal Mobility and Intermolecular Contacts in Solution

The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization

Models of Superoxide Dismutases

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