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Crystallographic and cellular characterisation of two mechanisms stabilising the native fold of alpha1-antitrypsin: implications for disease and drug design.

Gooptu B, Miranda E, Nobeli I, Mallya M, Purkiss A, Brown SC, Summers C, Phillips RL, Lomas DA, Barrett TE - J. Mol. Biol. (2009)

Bottom Line: To understand these effects, we have crystallised both mutants and solved their structures.The 2.2 A structure of Thr114Phe alpha(1)-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation.We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease.

View Article: PubMed Central - PubMed

Affiliation: School of Crystallography, Birkbeck College, University of London, London, UK. b.gooptu@mail.cryst.bbk.ac.uk

ABSTRACT
The common Z mutant (Glu342Lys) of alpha(1)-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native alpha(1)-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 A structure of Thr114Phe alpha(1)-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-beta-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z alpha(1)-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of alpha(1)-antitrypsin and increase secretion of monomeric protein in cell models of disease.

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Related in: MedlinePlus

Schema for formation of a partially loop-inserted M⁎ species from native α1-antitrypsin. Findings from the Thr114Phe and Gly117Phe α1-antitrypsin crystal structures are incorporated together with previous observations of requirements for loss of the s1C26 strand, remodeling of the F helix9,12,28 and destabilising of interactions involving shutter region residues.25 Sequential insertion of the reactive loop into the upper s4A position is depicted by two chimeras. These are derived from structures of native (1QLP) and latent (1IZ2) α1-antitrypsin, murine α1-antichymotrypsin (1YXA—demonstrating changes associated with opening of the P14 acceptor site) and thyroxine binding globulin (2CEO—demonstrating changes on expansion at the P12 insertion site). In all cases, the reactive loop modelled is that of α1-antitrypsin. The final image shows the α1-antitrypsin M⁎ model, generated as described in Materials and Methods, after energy minimisation with simulated annealing molecular dynamics. The effects of mutations that will facilitate this transition are shown in red whilst those that block it are shown in blue. Thus, in this scheme, Thr114Phe retards M⁎ formation by cushioning β-sheet A against expansion whilst Gly117Phe stabilises the upper turns of helix F and abolishes the steric requirement for their remodelling in response to partial loop insertion. Conversely, mutations may accelerate polymerisation by favouring partial loop insertion (e.g., Z) or by opening the lower s4A position directly (e.g., Siiyama).
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fig4: Schema for formation of a partially loop-inserted M⁎ species from native α1-antitrypsin. Findings from the Thr114Phe and Gly117Phe α1-antitrypsin crystal structures are incorporated together with previous observations of requirements for loss of the s1C26 strand, remodeling of the F helix9,12,28 and destabilising of interactions involving shutter region residues.25 Sequential insertion of the reactive loop into the upper s4A position is depicted by two chimeras. These are derived from structures of native (1QLP) and latent (1IZ2) α1-antitrypsin, murine α1-antichymotrypsin (1YXA—demonstrating changes associated with opening of the P14 acceptor site) and thyroxine binding globulin (2CEO—demonstrating changes on expansion at the P12 insertion site). In all cases, the reactive loop modelled is that of α1-antitrypsin. The final image shows the α1-antitrypsin M⁎ model, generated as described in Materials and Methods, after energy minimisation with simulated annealing molecular dynamics. The effects of mutations that will facilitate this transition are shown in red whilst those that block it are shown in blue. Thus, in this scheme, Thr114Phe retards M⁎ formation by cushioning β-sheet A against expansion whilst Gly117Phe stabilises the upper turns of helix F and abolishes the steric requirement for their remodelling in response to partial loop insertion. Conversely, mutations may accelerate polymerisation by favouring partial loop insertion (e.g., Z) or by opening the lower s4A position directly (e.g., Siiyama).

Mentions: The Thr114Phe and Gly117Phe mutations act by different mechanisms to increase the global stability of native α1-antitrypsin. Expansion of β-sheet A is a common feature in both proposed mechanisms of polymerisation.9,13 In order for α1-antitrypsin to adopt either M⁎ conformation, residue 114 must move laterally by 4.6 Å in the plane of β-sheet A. This is limited by local steric clashes in Thr114Phe α1-antitrypsin. In the case of the Gly117Phe mutation, the structural mechanism whereby the changes in the vicinity of the F helix may impede formation of a β-hairpin donor/acceptor M⁎ species is less intuitive. There is no apparent increase in stabilising interactions between the helix F–s3A linker and s5A, and the changes in packing in the interface between the F helix and β-sheet A would not of themselves be expected to impede opening of the β-sheet. Nevertheless, our data are not inconsistent with this model since they may simply reflect overall cooperativity of the final steps on the folding pathway (and initial steps on the unfolding pathway) for α1-antitrypsin. However, our findings can be mechanistically integrated with other biochemical data for the native-to-M⁎ transition posited for the single-strand linkage model in which opening of the s4A site is associated with partial intramolecular loop insertion. Various data support the following features of this model: upper s4A opening precedes lower s4A opening9,25,31,32 and is associated with partial insertion of the reactive site loop.9,33,34 Subsequent lower s4A opening around the site of P8 residue insertion is associated with breaking of a network of interactions between s4A, s5A and shutter region residues.25 This can be induced by the insertion of a cleaved reactive loop or a peptide analogue into β-sheet A from the P14–P9 sites, producing species that are highly polymerogenic.31,32,35,36 In α1-antitrypsin, modelling the insertion of the reactive site loop to the P12 position necessitates both the release of s1C and remodelling of the upper helix F—secondary structural changes known to occur during the formation of polymers from native α1-antitrypsin.12,26 However, stabilisation of the upper turns of the F helix and/or its downward displacement as seen in the structure of Gly117Phe α1-antitrypsin will reduce the propensity for its remodelling. It will, therefore, reduce M⁎ formation if such remodelling is integral to the formation of the intermediate. This process is shown as a schema in Fig. 4. The model of the single-strand donor/acceptor M⁎ is shown following energy minimisation and simulated annealing molecular dynamics (coordinates are supplied as Supplementary Data).


Crystallographic and cellular characterisation of two mechanisms stabilising the native fold of alpha1-antitrypsin: implications for disease and drug design.

Gooptu B, Miranda E, Nobeli I, Mallya M, Purkiss A, Brown SC, Summers C, Phillips RL, Lomas DA, Barrett TE - J. Mol. Biol. (2009)

Schema for formation of a partially loop-inserted M⁎ species from native α1-antitrypsin. Findings from the Thr114Phe and Gly117Phe α1-antitrypsin crystal structures are incorporated together with previous observations of requirements for loss of the s1C26 strand, remodeling of the F helix9,12,28 and destabilising of interactions involving shutter region residues.25 Sequential insertion of the reactive loop into the upper s4A position is depicted by two chimeras. These are derived from structures of native (1QLP) and latent (1IZ2) α1-antitrypsin, murine α1-antichymotrypsin (1YXA—demonstrating changes associated with opening of the P14 acceptor site) and thyroxine binding globulin (2CEO—demonstrating changes on expansion at the P12 insertion site). In all cases, the reactive loop modelled is that of α1-antitrypsin. The final image shows the α1-antitrypsin M⁎ model, generated as described in Materials and Methods, after energy minimisation with simulated annealing molecular dynamics. The effects of mutations that will facilitate this transition are shown in red whilst those that block it are shown in blue. Thus, in this scheme, Thr114Phe retards M⁎ formation by cushioning β-sheet A against expansion whilst Gly117Phe stabilises the upper turns of helix F and abolishes the steric requirement for their remodelling in response to partial loop insertion. Conversely, mutations may accelerate polymerisation by favouring partial loop insertion (e.g., Z) or by opening the lower s4A position directly (e.g., Siiyama).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2723994&req=5

fig4: Schema for formation of a partially loop-inserted M⁎ species from native α1-antitrypsin. Findings from the Thr114Phe and Gly117Phe α1-antitrypsin crystal structures are incorporated together with previous observations of requirements for loss of the s1C26 strand, remodeling of the F helix9,12,28 and destabilising of interactions involving shutter region residues.25 Sequential insertion of the reactive loop into the upper s4A position is depicted by two chimeras. These are derived from structures of native (1QLP) and latent (1IZ2) α1-antitrypsin, murine α1-antichymotrypsin (1YXA—demonstrating changes associated with opening of the P14 acceptor site) and thyroxine binding globulin (2CEO—demonstrating changes on expansion at the P12 insertion site). In all cases, the reactive loop modelled is that of α1-antitrypsin. The final image shows the α1-antitrypsin M⁎ model, generated as described in Materials and Methods, after energy minimisation with simulated annealing molecular dynamics. The effects of mutations that will facilitate this transition are shown in red whilst those that block it are shown in blue. Thus, in this scheme, Thr114Phe retards M⁎ formation by cushioning β-sheet A against expansion whilst Gly117Phe stabilises the upper turns of helix F and abolishes the steric requirement for their remodelling in response to partial loop insertion. Conversely, mutations may accelerate polymerisation by favouring partial loop insertion (e.g., Z) or by opening the lower s4A position directly (e.g., Siiyama).
Mentions: The Thr114Phe and Gly117Phe mutations act by different mechanisms to increase the global stability of native α1-antitrypsin. Expansion of β-sheet A is a common feature in both proposed mechanisms of polymerisation.9,13 In order for α1-antitrypsin to adopt either M⁎ conformation, residue 114 must move laterally by 4.6 Å in the plane of β-sheet A. This is limited by local steric clashes in Thr114Phe α1-antitrypsin. In the case of the Gly117Phe mutation, the structural mechanism whereby the changes in the vicinity of the F helix may impede formation of a β-hairpin donor/acceptor M⁎ species is less intuitive. There is no apparent increase in stabilising interactions between the helix F–s3A linker and s5A, and the changes in packing in the interface between the F helix and β-sheet A would not of themselves be expected to impede opening of the β-sheet. Nevertheless, our data are not inconsistent with this model since they may simply reflect overall cooperativity of the final steps on the folding pathway (and initial steps on the unfolding pathway) for α1-antitrypsin. However, our findings can be mechanistically integrated with other biochemical data for the native-to-M⁎ transition posited for the single-strand linkage model in which opening of the s4A site is associated with partial intramolecular loop insertion. Various data support the following features of this model: upper s4A opening precedes lower s4A opening9,25,31,32 and is associated with partial insertion of the reactive site loop.9,33,34 Subsequent lower s4A opening around the site of P8 residue insertion is associated with breaking of a network of interactions between s4A, s5A and shutter region residues.25 This can be induced by the insertion of a cleaved reactive loop or a peptide analogue into β-sheet A from the P14–P9 sites, producing species that are highly polymerogenic.31,32,35,36 In α1-antitrypsin, modelling the insertion of the reactive site loop to the P12 position necessitates both the release of s1C and remodelling of the upper helix F—secondary structural changes known to occur during the formation of polymers from native α1-antitrypsin.12,26 However, stabilisation of the upper turns of the F helix and/or its downward displacement as seen in the structure of Gly117Phe α1-antitrypsin will reduce the propensity for its remodelling. It will, therefore, reduce M⁎ formation if such remodelling is integral to the formation of the intermediate. This process is shown as a schema in Fig. 4. The model of the single-strand donor/acceptor M⁎ is shown following energy minimisation and simulated annealing molecular dynamics (coordinates are supplied as Supplementary Data).

Bottom Line: To understand these effects, we have crystallised both mutants and solved their structures.The 2.2 A structure of Thr114Phe alpha(1)-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation.We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease.

View Article: PubMed Central - PubMed

Affiliation: School of Crystallography, Birkbeck College, University of London, London, UK. b.gooptu@mail.cryst.bbk.ac.uk

ABSTRACT
The common Z mutant (Glu342Lys) of alpha(1)-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native alpha(1)-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 A structure of Thr114Phe alpha(1)-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-beta-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z alpha(1)-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of alpha(1)-antitrypsin and increase secretion of monomeric protein in cell models of disease.

Show MeSH
Related in: MedlinePlus