Local and Global Effects of a Cavity Filling Mutation in a Metastable Serpin

The native state of most globular proteins is the conformation with the lowest Gibbs free energy. However, for some proteins an energy barrier prevents them from adopting the lowest free energy state, thus trapping them in a metastable state. Some typical examples are the membrane fusion proteins of some viruses, α-lytic proteases, arrestin and serpins. Serpins are a large and widely distributed family of serine protease inhibitors which include plasma protease inhibitors such as α1-antitrypsin, α1-antichymotrypsin, antithrombin III, plasminogen activator inhibitor-I, C1 inhibitor, as well as non-inhibitory members such as ovalbumin and angiotensinogen (1). Inhibitory serpins share a common tertiary structure consisted of three β sheets (A, B, C) and 8 to 9 helices, with the extended reactive center loop exposed at one end of the molecule for protease binding (Fig. 1a) (2). Upon cleavage by a protease, the amino terminal portion of the cleaved reactive center loop (RCL) of the serpin inserts into the central beta sheet A with the protease covalently linked to it (3). This conformational transition from stressed (S) (also referred to as “native”) form to relaxed (R) form results in a dramatic increase in thermal stability of the molecule (4). Unfavorable interactions in the native form such as side-chain overpacking, buried polar groups and cavities present in serpin molecule have been identified as the structural basis for native metastability (5-8) and such metastability thus turns out to be absolutely crucial for efficient protease inhibitor activity. The metastability of serpins and their ability to undergo controlled conformational changes also rendered these molecule convert to a latent inactive state (3). In the latent form, the RCL of the serpin molecule inserts into β-sheet A, therefore cannot react with the target protease. This also makes them vulnerable to misfolding and polymerization (9). Figure 1 (A) Structure of the metastable form of α1AT. β-sheet A is shown in black and other functionally significant regions are indicated with arrows. (B) Close up view of the structure of G117F (21). Phe117 is shown in red and surrounding residues ... Extensive mutagenesis studies have probed the role of local and global stability in serpins (10-14). Single stabilizing mutations near the RCL and in β-sheet A result in decreased activity, indicating that local instability in these regions is important for function (10-12). On the other hand, it has been shown that combinations of multiple stabilizing mutations can compromise activity regardless of location, provided that the degree of stabilization exceeds a threshold of ∼13 kcal/mole (14). From these studies it appears that both local and global instability contribute to serpin function. One drawback to these studies is that, while they determined the effect of mutations on the stability of α1AT, they did not investigate their effect on conformational flexibility, which is also expected to play a role in inhibitory function. Recently, the local distribution of both stability and conformational flexibility in α1AT was investigated using hydrogen/deuterium exchange and mass spectrometry (15-16). Conformational flexibility was found to vary widely between different regions, for example, the top of the F-helix is extremely labile while most of β-sheets A and B are rigid (15). In contrast, stability was much more uniformly distributed. The entire molecule undergoes a concerted transition to a molten globule state at 0.8 M GdnHCl, while the ΔG of unfolding for all regions clustered around a value of 5 kcal/ mole (16). In the present study we have employed HXMS to study the local dynamics and local/global unfolding in a mutant of α1AT in which glycine 117 has been substituted with phenylalanine. Gly 117 is on β-sheet 2A and the Cα of the residue is surrounded by Phe-119, Tyr-160, and val-185 on the outer face of β-sheet A (Fig. 1b). This site forms a cavity that can accommodate larger side chains without appreciably altering the main structure. Substitution of Gly by the larger hydrophobic residue Phe fills a cavity between helix F and β-sheet A, and this substitution was shown to both increase the thermodynamic stability of α1AT and to reduce its inhibitory activity toward elastase (12). As the rate of association with elastase was unchanged, it was assumed that activity loss resulted from reduced efficiency of RCL translocation and insertion. Using this mutant, we tried to further elucidate the respective roles of conformational flexibility and stability in serpin function.