Probing the folding pathway of a consensus serpin using single tryptophan mutants.

Conserpin is an engineered protein that represents the consensus of a sequence alignment of eukaryotic serpins: protease inhibitors typified by a metastable native state and a structurally well-conserved scaffold. Previously, this protein has been found to adopt a native inhibitory conformation, possess an atypical reversible folding pathway and exhibit pronounced resistance to inactivation. Here we have designed a version of conserpin, cAT, with the inhibitory specificity of α1-antitrypsin, and generated single-tryptophan variants to probe its folding pathway in more detail. cAT exhibited similar thermal stability to the parental protein, an inactivation associated with oligomerisation rather a transition to the latent conformation, and a native state with pronounced kinetic stability. The tryptophan variants reveal the unfolding intermediate ensemble to consist of an intact helix H, a distorted helix F and 'breach' region structurally similar to that of a mesophilic serpin intermediate. A combination of intrinsic fluorescence, circular dichroism, and analytical gel filtration provide insight into a highly cooperative folding pathway with concerted changes in secondary and tertiary structure, which minimises the accumulation of two directly-observed aggregation-prone intermediate species. This functional conserpin variant represents a basis for further studies of the relationship between structure and stability in the serpin superfamily.

Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells.

Amyloidogenic protein aggregation impairs cell function and is a hallmark of many chronic degenerative disorders. Protein aggregation is also a major event during acute injury; however, unlike amyloidogenesis, the process of injury-induced protein aggregation remains largely undefined. To provide this insight, we profiled the insoluble proteome of several cell types after acute injury. These experiments show that the disulfide-driven process of nucleocytoplasmic coagulation (NCC) is the main form of injury-induced protein aggregation. NCC is mechanistically distinct from amyloidogenesis, but still broadly impairs cell function by promoting the aggregation of hundreds of abundant and essential intracellular proteins. A small proportion of the intracellular proteome resists NCC and is instead released from necrotic cells. Notably, the physicochemical properties of NCC-resistant proteins are contrary to those of NCC-sensitive proteins. These observations challenge the dogma that liberation of constituents during necrosis is anarchic. Rather, inherent physicochemical features including cysteine content, hydrophobicity and intrinsic disorder determine whether a protein is released from necrotic cells. Furthermore, as half of the identified NCC-resistant proteins are known autoantigens, we propose that physicochemical properties that control NCC also affect immune tolerance and other host responses important for the restoration of homeostasis after necrotic injury.

Smoothing a rugged protein folding landscape by sequence-based redesign.

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.

Protein unfolding is essential for cleavage within the α-helix of a model protein substrate by the serine protease, thrombin.

Proteolysis has a critical role in transmitting information within a biological system and therefore an important element of biology is to determine the subset of proteins amenable to proteolysis. Until recently, it has been thought that proteases cleave native protein substrates only within solvent exposed loops, but recent evidence indicates that cleavage sites located within α-helices can also be cleaved by proteases, despite the conformation of this secondary structure being generally incompatible with binding into an active site of a protease. In this study, we address the mechanism by which a serine endopeptidase, thrombin, recognizes and cleaves a target sequence located within an α-helix. Thrombin was able to cleave a model substrate, protein G, within its α-helix when a suitable cleavage sequence for the enzyme was introduced into this region. However, structural data for the complex revealed that thrombin was not perturbing the structure of the α-helix, thus it was not destabilizing the helix in order to allow it to fit within its active site. This indicated that thrombin was only cleaving within the α-helix when it was in an unfolded state. In support of this, the introduction of destabilizing mutations within the protein increased the efficiency of cleavage by the enzyme. Our data suggest that a folded α-helix cannot be proteolytically cleaved by thrombin, but the species targeted are the unfolded conformations of the native state ensemble.

Enhanced molecular mobility of ordinarily structured regions drives polyglutamine disease.

Polyglutamine expansion is a hallmark of nine neurodegenerative diseases, with protein aggregation intrinsically linked to disease progression. Although polyglutamine expansion accelerates protein aggregation, the misfolding process is frequently instigated by flanking domains. For example, polyglutamine expansion in ataxin-3 allosterically triggers the aggregation of the catalytic Josephin domain. The molecular mechanism that underpins this allosteric aggregation trigger remains to be determined. Here, we establish that polyglutamine expansion increases the molecular mobility of two juxtaposed helices critical to ataxin-3 deubiquitinase activity. Within one of these helices, we identified a highly amyloidogenic sequence motif that instigates aggregation and forms the core of the growing fibril. Critically, by mutating residues within this key region, we decrease local structural fluctuations to slow ataxin-3 aggregation. This provides significant insight, down to the molecular level, into how polyglutamine expansion drives aggregation and explains the positive correlation between polyglutamine tract length, protein aggregation, and disease severity.

The Z mutation alters the global structural dynamics of α1-antitrypsin.

α1-Antitrypsin (α1AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α1AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α1AT compared to the wild type protein, M α1AT, and so it is unclear why Z α1AT has an increased polymerization propensity. We speculated that the loss of these interactions would make the native state of Z α1AT more dynamic than M α1AT and that this change renders the protein more polymerization prone. We have used hydrogen/deuterium exchange combined with mass spectrometry (HXMS) to determine the structural and dynamic differences between native Z and M α1AT to reveal the molecular basis of Z α1AT polymerization. Our HXMS data shows that the Z mutation significantly perturbs the region around the site of mutation. Strikingly the Z mutation also alters the dynamics of regions distant to the mutation such as the B, D and I helices and specific regions of each ß-sheet. These changes in global dynamics may lead to an increase in the likelihood of Z α1AT sampling a polymerogenic structure thereby causing disease.

Oxidation of an exposed methionine instigates the aggregation of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of "nucleocytoplasmic coagulation." Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a "linchpin" whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.

Conformational behavior and aggregation of ataxin-3 in SDS.

Spinocerebellar ataxia type 3 (SCA3) is one of nine polyglutamine (polyQ) diseases all characterized by the presence of intraneuronal inclusions that contain aggregated protein. Aggregation of ataxin-3, the causative protein of SCA3, has been well characterized in vitro, with both pathogenic and non-pathogenic length ataxin-3 undergoing fibrillogenesis. However, only ataxin-3 containing an expanded polyQ tract leads to SCA3. Therefore other cellular factors, not present in previous in vitro studies, may modulate aggregation during disease. The interactions between fibrillar species and cell membranes have been characterized in a number of amyloid diseases, including Huntington's Disease, and these interactions affect aggregation and toxicity. We have characterized the effects of the membrane mimetic sodium dodecyl sulfate (SDS) on ataxin-3 structure and aggregation, to show that both micellar and non-micellar SDS have differing effects on the two stages of ataxin-3 aggregation. We also demonstrate that fibrillar ataxin-3 binds phospholipids, in particular phosphorylated phosphotidylinositols. These results highlight the effect of intracellular factors on the ataxin-3 misfolding landscape and their implications in SCA3 and polyQ diseases in general are discussed.

A method for the incremental expansion of polyglutamine repeats in recombinant proteins.

The polyglutamine diseases are caused by the expansion of CAG repeats. A key step in understanding the disease mechanisms, at the DNA and protein level, is the ability to produce recombinant proteins with specific length glutamine tracts which is a time-consuming first step in setting up in vitro systems to study the effects of polyglutamine expansion. Described here is a PCR-based method for the amplification of CAG repeats, which we used to incrementally extend CAG length by 3-5 repeats per cycle. This method could be translated into various contexts where amplification of repeating elements is necessary.

The roles of helix I and strand 5A in the folding, function and misfolding of α1-antitrypsin.

α(1)-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α(1)-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α(1)-Antitrypsin has never been examined. We have therefore created a range of α(1)Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α(1)-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.

Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin.

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded ß-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury.

Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment.

The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive. We report a comparative molecular dynamics investigation of the native states of wild-type and Z α1-AT, revealing a striking contrast between their structures and dynamics in the breach region at the top of ß-sheet A, which is closed in the wild-type simulations but open in the Z form. Our findings are consistent with experimental observations, notably the increased solvent exposure of buried residues in the breach region in Z, as well as polymerization via domain swapping, whereby the reactive center loop is rapidly inserted into an open A-sheet before proper folding of the C-terminal ß-strands, allowing C-terminal domain swapping with a neighboring molecule. Taken together, our experimental and simulation data imply that mutations at residue 342 that either stabilize an open form of the top of ß-sheet A or increase the local flexibility in this region, may favor polymerization and hence aggregation.

Molecular pathways to polyglutamine aggregation.

Over 100 human cellular proteins contain a repetitive polyglutamine tract, however, only nine ofthese proteins are associated with disease. In these proteins, the expanded polyQ tract perturbs the native conformation, resulting in an ordered aggregation process that leads to the formation of amyloid-like fibrils. The misfolding pathway involves the formation of prefibrillar oligomeric structures, which are proposed to be involved in cellular toxicity. Non-polyQ host protein regions modulate the misfolding pathway, suggesting an importance ofprotein context in aggregation. This chapter describes the current research regarding polyQ misfolding, with emphasis on the species populated during aggregation, suggesting an important role of protein context in modulating the aggregation pathway.

Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate.

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.

Intracellular production of recombinant serpins in yeast.

Yeast are a valuable system for recombinant serpin production due to their ability to synthesize large amounts of heterologous gene products as well as their expression of folding chaperones and lack of endogenous serpin genes. In this chapter, we describe a method for intracellular expression of cytoplasmic serpins in the yeast Pichia pastoris. We also give details on how this system can be exploited to produce polymer-forming mutants of secretory serpins.

Methods to measure the kinetics of protease inhibition by serpins.

The serpin molecule has evolved an unusual mechanism of inhibition, involving an exposed reactive center loop (RCL) and conformational change to covalently trap a target protease. Successful inhibition of the protease is dependent on the rate of serpin-protease association and the efficiency with which the RCL inserts into ß-sheet A, translocating the covalently bound protease and thereby completing the inhibition process. This chapter describes the kinetic methods used for determining the rate of protease inhibition (k(a)) and the stoichiometry of inhibition. These kinetic variables provide a means to examine different serpin-protease pairings, assess the effects of mutations within a serpin on protease inhibition, and determine the physiologically cognate protease of a serpin.

Structural change in ß-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease.

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α(1)-antitrypsin (α(1)AT) deficiency cases. It leads to increased polymerization of the serpin α(1)AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α(1)AT and analyzed their fluorescence properties. α(1)AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of ß-sheet A and Trp238 on ß-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α(1)AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α(1)AT compared to M α(1)AT. Our data reveal that the Z mutation results in a conformational change at the top of ß-sheet A but does not affect the structural integrity of ß-sheet B.

The rate of polyQ-mediated aggregation is dramatically affected by the number and location of surrounding domains.

The nine polyglutamine (polyQ) neurodegenerative diseases are caused in part by a gain-of-function mechanism involving protein misfolding, the deposition of ß-sheet-rich aggregates and neuronal toxicity. While previous experimental evidence suggests that the polyQ-induced misfolding mechanism is context dependent, the properties of the host protein, including the domain architecture and location of the polyQ tract, have not been investigated. Here, we use variants of a model polyQ-containing protein to systematically determine the effect of the location and number of flanking folded domains on polyQ-mediated aggregation. Our data indicate that when a pathological-length polyQ tract is present between two domains, it aggregates more slowly than the same-length tract in a terminal location within the protein. We also demonstrate that increasing the number of flanking domains decreases the polyQ protein's aggregation rate. Our experimental data, together with a bioinformatic analysis of all human proteins possessing polyQ tracts, suggest that repeat location and protein domain architecture affect the disease susceptibility of human polyQ proteins.