Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation

Analytical ultracentrifugation methods were utilized to further characterize the acid denaturation pathways of wild-type, V30M, and L55P transthyretin (TTR) that generate intermediates leading to amyloid fibril formation and possibly the diseases senile systemic amyloidosis and familial amyloid polyneuropathy. Equilibrium and velocity methods were employed herein to characterize the TTR quaternary structural requirements for amyloid fibril formation. From neutral to slightly acidic conditions (pH 7.5-5.1), wild-type transthyretin (0.2-0.3 mg/mL, 100 mM KCl, 37 degrees C) exists as a tetramer and is incapable of fibril formation. Under more acidic conditions (pH 5 to 3.9), tetrameric wild-type TTR slowly dissociates to a monomer having an alternatively folded tertiary structure(s) that self-assembles at physiological concentration (0.2 mg/mL) into a ladder of quaternary structural intermediates of increasing molecular weight. These intermediates appear to be on the pathway of amyloid fibril formation, since they ultimately disappear when amyloid fibrils are observed. The V30M and L55P TTR variants exhibit similar acid denaturation pathways, with the exception that dissociation of the tetramer to the monomeric amyloidogenic intermediate occurs at a higher pH and to a much greater extent, allowing the quaternary structural intermediates to be readily observed by velocity methods. Partial denaturation and assembly of the monomeric amyloidogenic intermediate(s) occur at pH 5.4 for V30M and L55P TTR over a 72 h period, during which wild-type TTR maintains its normal tetrameric three-dimensional structure. Interestingly, the L55P and V30M familial amyloid polyneuropathy (FAP) associated variants form amyloid protofilaments at pH 7.5 (37 degrees C) after several weeks of incubation, suggesting that the activation barriers for TTR tetramer dissociation to the monomeric amyloidogenic intermediate are much lower for the FAP variants relative to wild-type TTR, which does not form amyloid or amyloid protofilaments under these conditions. This study establishes the key role of the monomeric amyloidogenic intermediate and its self-assembly into a ladder of quaternary structural intermediates for the formation of wild-type, V30M, and L55P transthyretin amyloid fibrils

Oligomerization properties of GCN4 leucine zipper e and g position mutants

Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of > 65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing lambda repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (> dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants

Nuclear import factors importin alpha and importin beta undergo mutually induced conformational changes upon association

A heterodimer of importin alpha and importin beta accomplishes the nuclear import of proteins carrying classical nuclear localization signals (NLS). The interaction between the two import factors is mediated by the IBB domain of importin alpha and involves an extended recognition surface as shown by X-ray crystallography. Using a combination of biochemical and biophysical techniques we have investigated the formation of the importin beta:IBB domain complex in solution. Our data suggest that upon binding to the IBB domain, importin beta adopts a compact, proteolytically resistant conformation, while simultaneously the IBB domain folds into an alpha helix. We suggest a model to describe how these dual mutually induced conformational changes may orchestrate the nuclear import of NLS cargo in vivo

The most pathogenic transthyretin variant, L55P, forms amyloid fibrils under acidic conditions and protofilaments under physiological conditions

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37 degrees C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wild-type TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the beta-sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases

The nucleation of monomeric parallel beta-sheet-like structures and their self-assembly in aqueous solution

The aromatic diacid residue 4,6-dibenzofuranbispropionic acid (1) was designed to nucleate a parallel beta-sheet-like structure in small peptides in aqueous solution via a hydrogen-bonded hydrophobic cluster. Even though a 14-membered ring hydrogen bond necessary for parallel beta-sheet formation is favored in simple amides composed of 1, this hydrogen bonding interaction does not appear to be sufficient to nucleate parallel beta-sheet formation in the absence of hydrophobic clustering between the dibenzofuran portion of 1 and the hydrophobic side chains of the flanking alpha-amino acids. The subsequence --hydrophobic residue-1-hydrophobic residue-- is required for folding in the context of a nucleated two-stranded parallel beta-sheet structure. In all cases where the peptidomimetics can fold into two diastereomeric parallel beta-sheet structures having different hydrogen bonding networks, these conformations appear to exchange rapidly. The majority of the parallel beta-sheet structures evaluated herein undergo linked intramolecular folding and self-assembly, affording a fibrillar beta-sheet quaternary structure. To unlink folding and assembly, asymmetric parallel beta-sheet structures incorporating N-methylated alpha-amino acid residues have been synthesized using a new solid phase approach. Residue 1 facilitates the folding of several peptides described within affording a monomeric parallel beta-sheet-like structure in aqueous solution, as ascertained by a variety of spectroscopic and biophysical methods, increasing our understanding of parallel beta-sheet structure

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state

A glimpse of a possible amyloidogenic intermediate of transthyretin

Studies have indicated that partially unfolded states occur under conditions that favor amyloid formation by transthyretin (TTR), as well as other amyloidogenic proteins. In this study, we used hydrogen exchange measurements to show that there is selective destabilization of one half of the beta-sandwich structure of TTR under such conditions. This provides more direct information about conformational fluctuations than previously available, and will facilitate design of future experiments to probe the intermediates critical to amyloid formation

Inhibiting transthyretin conformational changes that lead to amyloid fibril formation

Insoluble protein fibrils resulting from the self-assembly of a conformational intermediate are implicated as the causative agent in several severe human amyloid diseases, including Alzheimer's disease, familial amyloid polyneuropathy, and senile systemic amyloidosis. The latter two diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of the lysosome. Here we demonstrate that flufenamic acid (Flu) inhibits the conformational changes of TTR associated with amyloid fibril formation. The crystal structure of TTR complexed with Flu demonstrates that Flu mediates intersubunit hydrophobic interactions and intersubunit hydrogen bonds that stabilize the normal tetrameric fold of TTR. A small-molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a possible approach to treat amyloid diseases. Molecules such as Flu also provide the means to rigorously test the amyloid hypothesis, i.e., the apparent causative role of amyloid fibrils in amyloid disease