Timothy R. Dafforn

A KINETIC MECHANISM FOR THE POLYMERIZATION OF ALPHA 1-ANTITRYPSIN

The mutation in the Z deficiency variant of α1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that α1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of α1-antitrypsin were used to demonstrate the close association between protein stability and rate of α1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for α1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.

Pathogenic alpha(1)-antitrypsin polymers are formed by reactive loop-beta-sheet A linkage

α1-Antitrypsin is the most abundant circulating protease inhibitor and the archetype of the serine protease inhibitor or serpin superfamily. Members of this family may be inactivated by point mutations that favor transition to a polymeric conformation. This polymeric conformation underlies diseases as diverse as α1-antitrypsin deficiency-related cirrhosis, thrombosis, angio-edema, and dementia. The precise structural linkage within a polymer has been the subject of much debate with evidence for reactive loop insertion into β-sheet A or C or as strand 7A. We have used site directed cysteine mutants and fluorescence resonance energy transfer (FRET) to measure a number of distances between monomeric units in polymeric α1-antitrypsin. We have then used a combinatorial approach to compare distances determined from FRET with distances obtained from 2.9 × 106 different possible orientations of the α1-antitrypsin polymer. The closest matches between experimental FRET measurements and theoretical structures show conclusively that polymers of α1-antitrypsin form by insertion of the reactive loop into β-sheet A.

Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 microL), low-wavelength (down to 180 nm), low-pathlength (100 microm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-beta-sheet amyloid fibers of the Alzheimers derived protein Abeta and the long-chain assemblies of alpha1-antitrypsin polymers.

Polymerization of plasminogen activator inhibitor-1

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into β-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and β-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-A-sheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving β-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet.

Targeting a Surface Cavity of α1-Antitrypsin to Prevent Conformational Disease

Conformational diseases are caused by a structural rearrangement within a protein that results in aberrant intermolecular linkage and tissue deposition. This is typified by the polymers that form with the Z deficiency variant of α1-antitrypsin (Glu-342 → Lys). These polymers are retained within hepatocytes to form inclusions that are associated with hepatitis, cirrhosis, and hepatocellular carcinoma. We have assessed a surface hydrophobic cavity in α1-antitrypsin as a potential target for rational drug design in order to prevent polymer formation and the associated liver disease. The introduction of either Thr-114 → Phe or Gly-117 → Phe on strand 2 of β-sheet A within this cavity significantly raised the melting temperature and retarded polymer formation. Conversely, Leu-100 → Phe on helix D accelerated polymer formation, but this effect was abrogated by the addition of Thr-114 → Phe. None of these mutations affected the inhibitory activity of α1-antitrypsin. The importance of these observations was underscored by the finding that the Thr-114 → Phe mutation reduced polymer formation and increased the secretion of Z α1-antitrypsin from a Xenopus oocyte expression system. Moreover cysteine mutants within the hydrophobic pocket were able to bind a range of fluorophores illustrating the accessibility of the cavity to external agents. These results demonstrate the importance of this cavity as a site for drug design to ameliorate polymerization and prevent the associated conformational disease.

Physical characterization of serpin conformations.

The native serpin fold is characterized by being metastable. This thermodynamic characteristic is manifested in the conversion of the native state to other more stable conformations. Whilst this structural transition is required for proteinase inhibition and regulation of a range of biological phenomena, inappropriate structural changes can result in a number of disease states. Identification of these alternative conformations has been essential in our understanding of serpin structure and function. However, identifying these alternative forms is also important if we are not to misinterpret data due to the formation of these states during in vitro studies. The different physical properties of these alternative serpin conformational states make it possible to use a range of standard laboratory techniques to identify these structures. In this chapter, we will outline these general approaches that can be used routinely to identify the alternative serpin conformational states.

Murine serpin 2A is a redox-sensitive intracellular protein

Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on differentiation. When it is constitutively expressed in the multipotent haemopoietic cell line, FDCP-Mix, it causes a delay in differentiation and increased clonogenic potential. The serpin is also dramatically up-regulated on T-cell activation. It has an unusual reactive site Cys-Cys sequence, a unique C-terminal extension and lacks a typical cleavable N-terminal signal sequence. In spite of these features, the protein is not a member of the ovalbumin-serpin family, but is instead most closely related to human antichymotrypsin. We have shown that the serpin is intracellular with prominent nuclear localization. Transverse urea gradient gels and CD studies show that the protein undergoes the stressed-relaxed conformational change typical of inhibitory serpins. However, we have not detected complex-forming activity with a set of proteases. Thermal denaturation studies also show that the protein has decreased structural stability under reducing conditions, although it lacks disulphide bonds within the core of the molecule. Our results show that serpin 2A is an intracellular protein with the potential to mediate its biological effects via interaction with non-protease intracellular targets. Furthermore, the results presented suggest a model whereby the serpin interactions could be modulated by redox conditions or conformational change induced by cleavage of the reactive-site loop.

A cluster of familial Creutzfeldt-Jakob disease mutations recapitulate conserved residues in Doppel: a case of molecular mimicry?

Intrachromosomal deletions linking Dpl expression to the PrP promoter produce cerebellar degeneration that can be abrogated by the introduction of wild‐type PrP transgenes. Since Dpl‐like truncated forms of PrP are neuropathogenic in mice and likewise counterbalanced by expression of PrPC we asked whether naturally occurring mutant forms of human PrP have Dpl‐like attributes. Five PRNP missense mutations causing familial Creutzfeldt–Jakob disease (F‐CJD) map to a helical region found in both PrPC and Dpl and result in amino acids identical to conserved residues in Dpl. These F‐CJD alleles may cause mutant PrP to become a weak mimetic of Dpl structure and/or function.