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

Abstract

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. 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. Conformational diseases arise when a protein undergoes a change in size or a fluctuation in shape, which results in self-association and tissue deposition (1Carrell R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar). This process is now recognized to underlie a whole range of diseases including the amyloidoses, prion encephalopathies, glutamine repeat diseases, and Alzheimer's and Parkinson's disease (2Carrell R.W. Lomas D.A. N. Engl. J. Med. 2002; 346: 45-53Crossref PubMed Scopus (354) Google Scholar). The paradigm for the conformational diseases was provided by the serpinopathies, which result from mutations in members of the serine proteinase inhibitor or serpin superfamily. The most well characterized of these is the severe plasma deficiency that is associated with the Z allele of α1-antitrypsin (3Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar). α1-Antitrypsin is the most abundant circulating proteinase inhibitor and the archetypal member of the serpin superfamily (4Potempa J. Korzus E. Travis J. J. Biol. Chem. 1994; 269: 15957-15960Abstract Full Text PDF PubMed Google Scholar, 5Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P. Irving J. Lomas D.A. Moyer R.W. Pemberton P.A. Remold O'Donnell E. Salvesen G.S. Travis J. Whisstock J. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar). Most individuals have two M α1-antitrypsin alleles, but ∼1 in 2000 are homozygous for the Z variant. The Z mutation results from a Glu → Lys substitution at amino acid 342 (6Jeppsson J.-O. FEBS Lett. 1976; 65: 195-197Crossref PubMed Scopus (147) Google Scholar) and leads to retention of α1-antitrypsin as inclusion bodies within the hepatocyte. These inclusions predispose to neonatal hepatitis, juvenile cirrhosis, and adult hepatocellular carcinoma (7Sveger T. Acta Paediatr. Scand. 1988; 77: 847-851Crossref PubMed Scopus (198) Google Scholar, 8Eriksson S. Carlson J. Velez R. N. Engl. J. Med. 1986; 314: 736-739Crossref PubMed Scopus (471) Google Scholar, 9Sharp H.L. Bridges R.A. Krivit W. Freier E.F. J. Lab. Clin. Med. 1969; 73: 934-939PubMed Google Scholar). The resulting secretory defect accounts for the low circulating plasma level of α1-antitrypsin, which is only 15% of normal in the Z homozygote. This plasma deficiency exposes the lungs to uncontrolled proteolytic attack that in turn causes early onset panacinar emphysema particularly in Z α1-antitrypsin homozygotes who smoke (10Piitulainen E. Eriksson S. Eur. Respir. J. 1999; 13: 247-251Crossref PubMed Scopus (116) Google Scholar). The structure of α1-antitrypsin is based on a five-stranded β-sheet A and a mobile reactive center loop (11Elliott P.R. Abrahams J.-P. Lomas D.A. J. Mol. Biol. 1998; 275: 419-425Crossref PubMed Scopus (130) Google Scholar, 12Ryu S.-E. Choi H.-J. Kwon K.-S. Lee K.N. Yu M.-H. Structure. 1996; 4: 1181-1192Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 13Kim S.-J. Woo J.-R. Seo E.J. Yu M.-H. Ryu S.-E. J. Mol. Biol. 2001; 306: 109-119Crossref PubMed Scopus (53) Google Scholar). Our previous studies have shown that the Z mutation promotes opening of β-sheet A to facilitate a sequential interaction between the reactive center loop of one molecule and β-sheet A of a second, resulting in polymer formation (3Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar, 14Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 15Sivasothy P. Dafforn T.R. Gettins P.G.W. Lomas D.A. J. Biol. Chem. 2000; 275: 33663-33668Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Mahadeva R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). These polymers tangle within the rough endoplasmic reticulum of hepatocytes to form the periodic acid-Schiff-positive inclusions that are associated with liver disease (3Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar, 17Janciauskiene S. Dominaitiene R. Sternby N.H. Piitulainen E. Eriksson S. J. Biol. Chem. 2002; 277: 26540-26546Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The significance of the reactive loop-β-sheet linkage was underscored by two other α1-antitrypsin variants, Siiyama (Ser-53 → Phe) and Mmalton (Δ52 → Phe) that also resulted in hepatic inclusions and severe plasma deficiency of α1-antitrypsin. Both of these mutants spontaneously formed polymers in vivo (18Lomas D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar, 19Lomas D.A. Elliott P.R. Sidhar S.K. Foreman R.C. Finch J.T. Cox D.W.J.C.W. Carrell R.W. J. Biol. Chem. 1995; 270: 16864-16870Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Moreover, this linkage accounts for the mild plasma deficiency observed with both S (Glu-264 → Val) and I (Arg-39 → Cys) α1-antitrypsin (20Elliott P.R. Stein P.E. Bilton D. Carrell R.W. Lomas D.A. Nat. Struct. Biol. 1996; 3: 910-911Crossref PubMed Scopus (78) Google Scholar, 21Mahadeva R. Chang W.-S.W. Dafforn T. Oakley D.J. Foreman R.C. Calvin J. Wight D. Lomas D.A. J. Clin. Invest. 1999; 103: 999-1006Crossref PubMed Scopus (166) Google Scholar). Further support for polymer formation as the mechanism responsible for the retention of mutant α1-antitrypsin within hepatocytes came from studies utilizing the Xenopus oocyte expression system. Point mutations that attenuated polymerization of Z α1-antitrypsin in vitro (14Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 22Kim J. Lee K.N. Yi G.-S. Yu M.-H. J. Biol. Chem. 1995; 270: 8597-8601Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) increased the secretion of Z α1-antitrypsin in vivo (23Sidhar S.K. Lomas D.A. Carrell R.W. Foreman R.C. J. Biol. Chem. 1995; 270: 8393-8396Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Our understanding of the mechanism underlying polymerization has allowed the design of strategies to prevent polymer formation (3Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (877) Google Scholar, 16Mahadeva R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). To date, however, these have been based on peptides that bind to β-sheet A and as a consequence inactivate α1-antitrypsin as a proteinase inhibitor. A more useful strategy would be to identify cavities in α1-antitrypsin that can bind peptides, or their mimetics, and block polymerization without a loss of inhibitory activity. Our high resolution crystal structure of α1-antitrypsin revealed a large hydrophobic cavity bounded by strand 2 of β-sheet A and helices D and E (Fig. 1). The cavity is present in monomeric α1-antitrypsin but is obliterated during polymerization (24Elliott P.R. Pei X.Y. Dafforn T.R. Lomas D.A. Protein Sci. 2000; 9: 1274-1281Crossref PubMed Scopus (169) Google Scholar). This cavity could provide an ideal target for drug design to prevent polymer formation and the associated liver disease (11Elliott P.R. Abrahams J.-P. Lomas D.A. J. Mol. Biol. 1998; 275: 419-425Crossref PubMed Scopus (130) Google Scholar, 13Kim S.-J. Woo J.-R. Seo E.J. Yu M.-H. Ryu S.-E. J. Mol. Biol. 2001; 306: 109-119Crossref PubMed Scopus (53) Google Scholar, 24Elliott P.R. Pei X.Y. Dafforn T.R. Lomas D.A. Protein Sci. 2000; 9: 1274-1281Crossref PubMed Scopus (169) Google Scholar). We have used site-directed mutagenesis to explore the role of this surface cavity in the conformational of α1-antitrypsin in vitro and in and of α1-antitrypsin → was used as the of the to the protein a inhibitor of has the and of polymerization as α1-antitrypsin P. Dafforn T.R. Gettins P.G.W. Lomas D.A. J. Biol. Chem. 2000; 275: 33663-33668Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, E. Gettins P.G.W. Sci. S. 1997; 4: Scopus Google Scholar). The α1-antitrypsin mutants → Thr-114 → Gly-117 → Leu-100 → → Leu-100 → → and Thr-114 → → were by site-directed and the were by The α1-antitrypsin were the and and expression was with α1-antitrypsin was from the E. by and (11Elliott P.R. Abrahams J.-P. Lomas D.A. J. Mol. Biol. 1998; 275: 419-425Crossref PubMed Scopus (130) Google Scholar). this was for of the cysteine cavity the α1-antitrypsin cavity mutants were also in the that an were from the of E. by and as Carrell R.W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The were and was by of and were characterized by and activity was by of site Scopus Google Scholar) with of α1-antitrypsin site of in a of with The for at and the proteolytic activity was by the addition of the to a of (18Lomas D.A. Finch J.T. Seyama K. Nukiwa T. Carrell R.W. J. Biol. Chem. 1993; 268: 15333-15335Abstract Full Text PDF PubMed Google Scholar). The change in the A was site were by proteolytic activity the of α1-antitrypsin and to the K. J. Travis J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were formed by of the or α1-antitrypsin with α1-antitrypsin variant at in at for to at were assessed on a R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). were by or The melting temperature and were for α1-antitrypsin mutant as (14Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) of of and formation was assessed by of the α1-antitrypsin at in at The were by and the protein was by of of the monomeric protein was by The for polymer formation was from the of the loss in of from the Xenopus The cavity mutants Leu-100 → Thr-114 → and Gly-117 → Phe were the either M or Z α1-antitrypsin by site-directed and the were as The in vitro and of α1-antitrypsin secretion from the Xenopus oocyte were as (23Sidhar S.K. Lomas D.A. Carrell R.W. Foreman R.C. J. Biol. Chem. 1995; 270: 8393-8396Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). of and cysteine cavity mutants were with a of either used or at both and for to at to the The was by the addition of of and the protein was from on a in of α1-antitrypsin were for site-directed mutagenesis in order to explore the role of the cavity in polymer formation. Leu-100 on and Thr-114 on have that the hydrophobic Gly-117 is at the of the cavity on (Fig. large or cysteine that could be with at these was to the cavity and the effect of a molecule inhibitor. The one cysteine at in α1-antitrypsin was by a serine to that only the cavity cysteine was for The → mutation has effect on the inhibitory activity or polymerization of α1-antitrypsin P. Dafforn T.R. Gettins P.G.W. Lomas D.A. J. Biol. Chem. 2000; 275: 33663-33668Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Chang W.-S.W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Whisstock 1998; PubMed Scopus Google Scholar). of Leu-100 → → Thr-114 → → and Gly-117 → Phe α1-antitrypsin were from the of the E. (11Elliott P.R. Abrahams J.-P. Lomas D.A. J. Mol. Biol. 1998; 275: 419-425Crossref PubMed Scopus (130) Google Scholar). this on and the α1-antitrypsin cavity mutants Leu-100 → → and Thr-114 → → were to bind to the Leu-100 → → Thr-114 → → Leu-100 → Thr-114 → Gly-117 → and Leu-100 → → Phe α1-antitrypsin were an expression the at the amino were in E. and to by and were with α1-antitrypsin an the as a on and as a on from Leu-100 → → Phe α1-antitrypsin which a the mutants an that was to α1-antitrypsin on a with the of Gly-117 → which at a increased the of the was to that of α1-antitrypsin that the mutations a in the structure of the molecule of α1-antitrypsin was as an inhibitor of the cavity mutants inhibitory Leu-100 → → Phe and Leu-100 → → α1-antitrypsin were reduced to and The formed when in a site with (Fig. inhibitory melting and of polymer formation of and mutant → → → → → → → → → the cysteine with in at for 2 The inhibitory activity was and melting temperature was from at formation was assessed by α1-antitrypsin variant at and for The was from the loss of of the monomeric protein The results are the of → → → → the cysteine with in at for 2 The inhibitory activity was and melting temperature was from at formation was assessed by α1-antitrypsin variant at and for The was from the loss of of the monomeric protein The results are the of This the cysteine with in at for 2 The inhibitory activity was and melting temperature was from at formation was assessed by α1-antitrypsin variant at and for The was from the loss of of the monomeric protein The results are the of in a of and melting temperature of and mutant α1-antitrypsin was by The increased the melting temperature of the α1-antitrypsin by when with the from E. that the the mutations strand the of the Gly-117 → Phe α1-antitrypsin a melting temperature that was more the D mutations to their Leu-100 → Phe the melting temperature by a cysteine at the significantly the when with the the addition of a within the cavity at → → Phe) resulted in an in the effect of Leu-100 → Phe mutation was also assessed by or cavity mutants of α1-antitrypsin between and at of for and the by Leu-100 → → and Leu-100 → Phe α1-antitrypsin to that of Gly-117 → Phe α1-antitrypsin was the most as monomeric at for which was α1-antitrypsin. Thr-114 → → Thr-114 → and Leu-100 → → Phe α1-antitrypsin the in melting were in the of and mutant α1-antitrypsin when assessed by and on of was assessed at and for to as these to polymer formation of α1-antitrypsin that could be by The of polymer formation was from the loss of of the monomeric protein α1-antitrypsin within when at (Fig. Leu-100 → Phe α1-antitrypsin accelerated polymer formation in with melting temperature (Fig. Leu-100 with a cysteine (Fig. or at (Fig. within the cavity this effect as these mutants at a to α1-antitrypsin the mutations on to of polymer formation as would be from their melting (Fig. The most mutant Gly-117 → Phe α1-antitrypsin polymer formation, as polymers were only at for between and and to the reactive center loop of was used to the of β-sheet were formed by α1-antitrypsin with a of at for were on α1-antitrypsin and the cavity mutants formed a with the with a (Fig. Gly-117 → Phe α1-antitrypsin formed a with the at a α1-antitrypsin and formation was significantly retarded by Leu-100 → Phe α1-antitrypsin (Fig. The other cavity mutations of the to β-sheet Leu-100 → Phe α1-antitrypsin was able to form a with the to of the reactive loop of α1-antitrypsin, the at formation for and cavity mutants of formation → → → → → → → → → the cysteine cavity mutants with in at for 2 The results are the of → → → → the cysteine cavity mutants with in at for 2 The results are the of This the cysteine cavity mutants with in at for 2 The results are the of in a of from the Xenopus of the mutants were assessed on the polymerization of the Z variant of α1-antitrypsin. This mutant is to be as a and the mutants were assessed for their effect on the secretion of Z α1-antitrypsin in of the protein was from the with of Z α1-antitrypsin with Gly-117 → Phe and Leu-100 → Phe effect on the secretion of Z α1-antitrypsin and Thr-114 → Phe more the secretion of Z α1-antitrypsin to with Z The results are the of of accessibility of the cavity was by the cysteine Leu-100 → → and Thr-114 → → α1-antitrypsin with a of fluorophores The were in the at either or for to of Leu-100 → → α1-antitrypsin with at resulted in and when for and of Thr-114 → → α1-antitrypsin with at resulted in and when for and These results that of the cavity cysteine with was within were assessed for their to the cysteine in an to the The addition of the and the temperature both increased the of protein with but of other and as by on This could be by the to and the to 2 at this of Leu-100 → → α1-antitrypsin and of Thr-114 → → α1-antitrypsin were with only a of the protein with was to the effect of these on the of of cysteine cavity mutants of α1-antitrypsin with → → → → and and in a Our high resolution crystal structure of M α1-antitrypsin a hydrophobic cavity bounded by and that was present in the monomeric structure but to in size by during polymer formation (24Elliott P.R. Pei X.Y. Dafforn T.R. Lomas D.A. Protein Sci. 2000; 9: 1274-1281Crossref PubMed Scopus (169) Google Scholar). this cavity a target for rational drug design to prevent polymerization and ameliorate the associated disease. order to explore the role of the cavity in polymer formation, the were for site-directed mutagenesis (Fig. large at Leu-100 on and Thr-114 and Gly-117 on is to the A has been to the effect of these mutations on polymer formation in order to the hydrophobic cavity would be a target for rational drug the cavity mutants a normal were as proteinase and formed in activity when assessed of but the that the mutations to a change in the structure of α1-antitrypsin. of the mutations melting temperature and significantly the at which α1-antitrypsin formed particularly the introduction of at either or These observations are in with previous studies (14Dafforn T.R. Mahadeva R. Elliott P.R. Sivasothy P. Lomas D.A. J. Biol. Chem. 1999; 274: 9548-9555Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Moreover, the addition of a at the of Leu-100 → Phe α1-antitrypsin polymerization to that of the the mutations increased of α1-antitrypsin which is in with their effect on These provide that the cavity with mutants on α1-antitrypsin and polymer formation without inhibitory in results from the sequential of the reactive center loop of one molecule β-sheet A of P. Dafforn T.R. Gettins P.G.W. Lomas D.A. J. Biol. Chem. 2000; 275: 33663-33668Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Point mutations that polymerization are to β-sheet A to facilitate of an reactive center loop P.E. Carrell R.W. Nat. Struct. Biol. 1995; PubMed Scopus Google Scholar). The accessibility of β-sheet A was assessed in the mutants by the at which an reactive loop to form a S. E. R. Eur. J. PubMed Scopus Google Scholar). Gly-117 → Phe α1-antitrypsin formed a with the at a This was as the mutant significantly retarded polymer formation. Moreover, the most Leu-100 → Phe α1-antitrypsin, formed a with the at one of the and between polymer formation and at which a mutant of α1-antitrypsin reactive loop peptides has also been observed for the Z and Mmalton deficiency D.A. Elliott P.R. Sidhar S.K. Foreman R.C. Finch J.T. Cox D.W.J.C.W. Carrell R.W. J. Biol. Chem. 1995; 270: 16864-16870Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). the for Z α1-antitrypsin has R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The Z mutation at that of strand of β-sheet A and the of the reactive center β-sheet A to of reactive This the of β-sheet A and of an reactive loop the of β-sheet A and this the Z variant to either an loop to form polymers or a that is to the of the reactive loop R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The effect of the Leu-100 → Phe mutation is to be to that of Z α1-antitrypsin. Leu-100 → Phe of the reactive loop by the opening of the of β-sheet The of the A to for the accelerated of Z α1-antitrypsin, the which is be to the more of the Leu-100 → Phe mutation in β-sheet the Gly-117 → Phe mutation the of β-sheet A to effect on polymer formation, which the of the for of an reactive loop cavities to the of α1-antitrypsin that is for inhibitory Sci. S. 1997; PubMed Scopus Google Scholar). of these cavities is associated with a loss of inhibitory activity Lee Yu M.-H. Sci. S. 2000; PubMed Scopus Google Scholar, Seo E.J. Yu M.-H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J.-P. Lee Yu M.-H. Protein Sci. 2001; PubMed Scopus Google Scholar, E.J. Yu M.-H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Our results that surface cavities can the and inhibitory activity. We have shown that the Z variant of α1-antitrypsin a from the protein R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). the Z mutation the structure of the hydrophobic pocket that has been as a target for drug This be assessed in as Z α1-antitrypsin is to be as a protein in E. To this the effect of the mutants on Z α1-antitrypsin was characterized in vivo the Xenopus oocyte expression system. This the in which hepatocytes mutants of α1-antitrypsin D.A. Elliott P.R. Sidhar S.K. Foreman R.C. Finch J.T. Cox D.W.J.C.W. Carrell R.W. J. Biol. Chem. 1995; 270: 16864-16870Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, S.K. Lomas D.A. Carrell R.W. Foreman R.C. J. Biol. Chem. 1995; 270: 8393-8396Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, R.C. FEBS Lett. PubMed Scopus Google Scholar). The importance of these studies is by the Gly-117 → Phe which the polymerization of M α1-antitrypsin in vitro without the secretion of Z α1-antitrypsin in the Thr-114 → Phe which also the polymerization of M α1-antitrypsin in significantly increased the secretion of Z α1-antitrypsin from the Xenopus oocyte expression system. the cavity is to have a in the Z α1-antitrypsin in the or has a structure when Leu-100 → → Thr-114 → → α1-antitrypsin to the that the cysteine were within the The accessibility of the cavity to was assessed by to Leu-100 → → and Thr-114 → → α1-antitrypsin. Both of these mutants a range of fluorophores that the cavity was to external agents. The fluorophores effect on polymer formation, which that more of have their cavities a is to that the conformational change in the hydrophobic cavity bounded by and is in the polymerization of α1-antitrypsin. polymer formation is an and have been including that protein Sci. S. 2000; PubMed Scopus Google Scholar, D.J. Lomas D.A. J. Respir. Mol. Biol. 2001; PubMed Scopus Google Scholar) and the of peptides that bind to β-sheet A R. Dafforn T.R. Carrell R.W. Lomas D.A. J. Biol. Chem. 2002; 277: 6771-6774Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). and have also been used in other conformational as Alzheimer's and disease J. J. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, R.A. Nat. Med. 1998; 4: PubMed Scopus Google Scholar, H.L. Nat. 1999; PubMed Scopus Google Scholar). the is is as α1-antitrypsin to β-sheet results in the of α1-antitrypsin. The surface cavity bounded by and is ideal for rational drug design as is to external that can block polymerization without an loss of inhibitory activity. of α1-antitrypsin polymerization within hepatocytes prevent the liver disease associated with Z α1-antitrypsin. Moreover, an in the of circulating α1-antitrypsin a for the associated