Folding of Cu, Zn superoxide dismutase and Familial Amyotrophic Lateral Sclerosis

Cu,Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.Comment: 16 pages, 5 figure

Computational methods for identifying a layered allosteric regulatory mechanism for ALS-causing mutations of Cu-Zn superoxide dismutase 1

The most prominent form of familial amyotrophic lateral sclerosis (fALS, Lou Gehrig's Disease) is caused by mutations of Cu-Zn superoxide dismutase 1 (SOD1). SOD1 maintains antioxidant activity under fALS causing mutations, suggesting that the mutations introduce a new, toxic, function. There are 100+ such known mutations that are chemically diverse and spatially distributed across the structure. The common phenotype leads us to propose an allosteric regulatory mechanism hypothesis: SOD1 mutants alter the correlated dynamics of the structure and differentially signal across an inherent allosteric network, thereby driving the disease mechanism at varying rates of efficiency. Two recently developed computational methods for identifying allosteric control sites are applied to the wild type crystal structure, 4 fALS mutant crystal structures, 20 computationally generated fALS mutants and 1 computationally generated non-fALS mutant. The ensemble of mutant structures is used to generate an ensemble of dynamics, from which two allosteric control networks are identified. One network is connected to the catalytic site and thus may be involved in the natural antioxidant function. The second allosteric control network has a locus bordering the dimer interface and thus may serve as a mechanism to modulate dimer stability. Though the toxic function of mutated SOD1 is unknown and likely due to several contributing factors, this study explains how diverse mutations give rise to a common function. This new paradigm for allostery controlled function has broad implications across allosteric systems and may lead to the identification of the key chemical activity of SOD1-linked ALS. Proteins 2011. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79415/1/22892_ftp.pd

Biophysical Mechanisms of Protein Aggregation

Protein aggregation related toxicity is implicated in a variety of neurodegenerative diseases including Alzheimer's, Huntington's, prion and Amyotrophic Lateral Sclerosis (ALS). The proteins or peptides known to aggregate in disease are unrelated in their amino acid sequence and native structure but form structurally similar aggregates - amyloids. Studies outlined in this dissertation were aimed at uncovering the underlying biophysical mechanisms of amyloid formation, and lay the groundwork to develop rational strategies to combat neurodegenerative diseases. More than 100 point mutations in the homodimeric metalloenzyme Cu, Zn superoxide (SOD1) dismutase are involved in a genetically inherited familial form of ALS (FALS). We have discovered a mechanism of in vitro SOD1 aggregation in which the native SOD1 dimer dissociates, metals are lost from the monomers and the resulting apo-monomers oligomerize in a rate-limiting step. Further, we have computationally estimated that a majority of FALS-associated point mutants in SOD1 (70 out of the 75 studied) decrease dimer stability and/or increase dimer dissociation propensity. Thus, we have proposed that the underlying biophysical basis of FALS-linked SOD1 aggregation is the mutation-induced increase in the propensity to form apo-monomers. To uncover the molecular determinants of SOD1 apo-monomer oligomerization, the rate-limiting step in aggregation, we have developed two complementary in silico approaches: (a) we have identified sequence fragments of SOD1 that have a high self-association propensity, and (b) we have performed molecular dynamics simulations of model SOD1 monomer and dimer folding and misfolding. In both cases, we have identified key residue-residue interactions in SOD1 responsible for maintaining fidelity to its native state. We have proposed that the disruption of one or more of these key interactions ("hot spots") is implicated in non-native oligomerization. To understand the effect of FALS mutations on the key interactions involved in maintaining native-state fidelity, we have studied the nanosecond dynamics of wild type SOD1 and 3 FALS-associated mutant apo-dimers and apo-monomers. We found that in wild type SOD1 the motions of the dimer interface are mechanically coupled to the motions of the structurally distal metal-coordinating loops of both monomeric subunits. We further found that the strain induced in the protein by dimer dissociation, point mutations, or by exposure to high temperature is transmitted to a specific hairpin in the protein, previously found to be implicated in maintaining fold fidelity. The altered dynamics of mutant SOD1 dimers and monomers provides structural insights into the flexibility required for oligomerization. Collectively, findings in this dissertation have enhanced our understanding of the complex mechanisms of protein aggregation. Mechanisms established and structural insights obtained herein may facilitate rational design of small molecules to prevent protein aggregation, hence provide a therapeutic intervention strategy in neurodegenerative diseases.Doctor of Philosoph

The impact of post-translational modifications on aggregation of Cu, Zn superoxide dismutase in amyotrophic lateral sclerosis

Aberrant conformers of disease-linked proteins have been proposed as cytotoxic agents in several late-onset neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Mutations in the gene encoding Cu, Zn superoxide dismutase (SOD1) are present in a subset of familial ALS (FALS) cases; most of these mutations destabilize the protein, although typically by a small margin relative to SOD1's exceptionally high stability. Therefore, SOD1 with FALS-linked substitutions often misfolds and aggregates, adopting aberrant conformations that interact with numerous cellular components and disrupt their functioning, despite having a more stable folded state than would be expected for an aggregation-prone protein. This fact, together with the specific death of motor neurons late in life despite ubiquitous expression of mutant SOD1 since birth, implicates factors in the cellular environment as substantial contributors to the cytotoxicity of mutant SOD1 in FALS. One non-genetic factor likely to influence misfolding and aggregation of SOD1 in human tissue is its susceptibility to abundant post-translational modifications, including phosphorylation and numerous oxidative modifications. We find that reversible oxidative modification of Cys-111 by the glutathione tripeptide destabilizes the native SOD1 homodimer, increasing the equilibrium dissociation constant of the WT dimer from low nanomolar to the low micromolar range, and further destabilizes SOD1 containing a FALS-linked substitution within the dimer interface (A4V). Assessment of the effect of glutathionylation on dimer dissociation kinetics using surface plasmon resonance revealed that this modification causes minimal change in dimer dissociation rate; therefore, the increased Kd observed for glutathionylated WT and A4V SOD1 must result from slowed association of modified monomers. In addition to inducing dissociation of the native dimer, Cys-111 glutathionylation promotes the assembly of soluble non-native oligomers that contain an epitope specific to disease-relevant misfolded SOD1. Our findings suggest that soluble non-native SOD1 oligomers share structural similarity to pathogenic misfolded species found in ALS patients, and therefore represent potential cytotoxic agents and therapeutic targets in ALS. Furthermore, the induction of SOD1 misfolding and aggregation by glutathionylation represents a possible mechanism by which oxidative stress brought on by aging triggers the transition of SOD1 from its natively folded state to cytotoxic conformations.Doctor of Philosoph

IN SILICO MODELING THE EFFECT OF SINGLE POINT MUTATIONS AND RESCUING THE EFFECT BY SMALL MOLECULES BINDING

Single-point mutation in genome, for example, single-nucleotide polymorphism (SNP) or rare genetic mutation, is the change of a single nucleotide for another in the genome sequence. Some of them will result in an amino acid substitution in the corresponding protein sequence (missense mutations); others will not. This investigation focuses on genetic mutations resulting in a change in the amino acid sequence of the corresponding protein. This choice is motivated by the fact that missense mutations are frequently found to affect the native function of proteins by altering their structure, interaction and other properties and cause diseases. A particular disease is the Snyder-Robinson syndrome (SRS), which is an X-linked mental retardation found to be caused by missense mutations in human spermine synthase (SMS). In this thesis, a rational approach to predict the effects of missense mutations on SMS wild-type characteristics was carried. Following this work, a structure-based virtual screening of small molecules was applied to rescue the disease-causing effect by searching the small molecules to stabilize the malfunctioning SMS mutant dimer

Folding and Stability Studies on Amyotrophic Lateral Sclerosis-Associated apo Cu, Zn Superoxide dismutases

Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation

qPIPSA: Relating enzymatic kinetic parameters and interaction fields

BACKGROUND: The simulation of metabolic networks in quantitative systems biology requires the assignment of enzymatic kinetic parameters. Experimentally determined values are often not available and therefore computational methods to estimate these parameters are needed. It is possible to use the three-dimensional structure of an enzyme to perform simulations of a reaction and derive kinetic parameters. However, this is computationally demanding and requires detailed knowledge of the enzyme mechanism. We have therefore sought to develop a general, simple and computationally efficient procedure to relate protein structural information to enzymatic kinetic parameters that allows consistency between the kinetic and structural information to be checked and estimation of kinetic constants for structurally and mechanistically similar enzymes. RESULTS: We describe qPIPSA: quantitative Protein Interaction Property Similarity Analysis. In this analysis, molecular interaction fields, for example, electrostatic potentials, are computed from the enzyme structures. Differences in molecular interaction fields between enzymes are then related to the ratios of their kinetic parameters. This procedure can be used to estimate unknown kinetic parameters when enzyme structural information is available and kinetic parameters have been measured for related enzymes or were obtained under different conditions. The detailed interaction of the enzyme with substrate or cofactors is not modeled and is assumed to be similar for all the proteins compared. The protein structure modeling protocol employed ensures that differences between models reflect genuine differences between the protein sequences, rather than random fluctuations in protein structure. CONCLUSION: Provided that the experimental conditions and the protein structural models refer to the same protein state or conformation, correlations between interaction fields and kinetic parameters can be established for sets of related enzymes. Outliers may arise due to variation in the importance of different contributions to the kinetic parameters, such as protein stability and conformational changes. The qPIPSA approach can assist in the validation as well as estimation of kinetic parameters, and provide insights into enzyme mechanism