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The Native Strains in the Hydrophobic Core and Flexible Reactive Loop of A

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The Native Strains in the Hydrophobic Core and Flexible Reactive Loop of A View metadata, citation and similar papers at core.ac.uk brought to you by CORE Researchprovided Article by Elsevier1181 - Publisher Connector The native strains in the hydrophobic core and flexible reactive loop of a serine protease inhibitor: crystal structure of an uncleaved a1-antitrypsin at 2.7 Å Seong-Eon Ryu*, Hee-Jeong Choi, Ki-Sun Kwon, Kee Nyung Lee and Myeong-Hee Yu Background: The protein a1-antitrypsin is a prototype member of the serpin Address: Protein Engineering Research Division, (serine protease inhibitor) family and is known to inhibit the activity of neutrophil Korea Research Institute of Bioscience and elastase in the lower respiratory tract. Members of this family undergo a large Biotechnology, KIST, P.O. Box 115, Yusong, Taejon 305-600, South Korea. structural rearrangement upon binding to a target protease, involving cleavage of the reactive-site loop. This loop is then inserted into the main body of the enzyme *Corresponding author. following the opening of a central b sheet, leading to stabilization of the E-mail: [email protected] structure. Random mutageneses of a1-antitrypsin identified various mutations Key words: a1-antitrypsin, conformational that stabilize the native structure and retard the insertion of the reactive-site loop. transition, loop flexibility, metastability, stabilizing Structural studies of these mutations may reveal the mechanism of the mutations conformational change. Received: 25 June 1996 Revisions requested: 19 July 1996 Results: We have determined the three-dimensional structure of an uncleaved Revisions received: 8 August 1996 a1-antitrypsin with seven such stabilizing mutations (hepta a1-antitrypsin) at 2.7 Å Accepted: 29 August 1996 resolution. From the comparison of the structure with other serpin structures, we Structure 15 October 1996, 4:1181–1192 found that hepta a1-antitrypsin is stabilized due to the release of various strains that exist in native wild type a1-antitrypsin, including unfavorable hydrophobic © Current Biology Ltd ISSN 0969-2126 interactions in the central hydrophobic core. The reactive-site loop of hepta a1-antitrypsin is an extended strand, different from that of the previously determined structure of another uncleaved a1-antitrypsin, and indicates the inherent flexibility of the loop. Conclusions: The present structural study suggests that the uncleaved a1-antitrypsin has many folding defects which can be improved by mutations. These folding defects seem to be utilized in a coordinated fashion in the regulation of the conformational switch of a1-antitrypsin. Some of the defects, represented by the Phe51 region and possibly the Met374 and the Thr59 regions, are part of the sheet-opening mechanism. Introduction and ovalbumin (a non-inhibitory serpin) the loop forms a Proteins in the serpin (serine protease inhibitor) family distorted or regular a helix, respectively [5,10]. In have a common structural motif of about 370–400 amino antithrombin the loop has an extended conformation and acids comprising two central b sheets surrounded by a the starting region of the loop is partially inserted into small b sheet and several helices [1,2]. The functions of sheet A [11,12]. It is proposed that the helical conformation serpin proteins range from blood coagulation and fibrinoly- is the native form, and the extended conformation is the sis to tumor suppression [3]. Many of these functions are inhibitory form which is used for the target protease mediated by the protease inhibitor activity of serpins. Two binding [10]. However, the role of the mobility and confor- characteristic features of the inhibitory serpins are the mation of the loop in the serpin function is yet to be char- metastability of the native state [4] and the mobility of the acterized in detail. The stability of the inhibitory serpins is reactive loop [4,5]. The inhibitory serpins have a unique greatly increased upon the cleavage and insertion of the loop of about 20 residues that binds tightly to the active loop [4]. The difference in stability between the native and site of the target proteases to inhibit the proteolytic activ- loop-inserted states has an important role in the regulation ity. This reactive loop is cleaved at a specific site by a of the inhibitory-serpin function [2,8,13–15]. The non- target protease [6] and the cleaved loop is incorporated into inhibitory serpins including ovalbumin and angiotensino- b-sheet A, leading to a large rearrangement of the strands gen, do not show the structural transition [15,16]. in sheet A with concomitant stabilization of the structure [5,7–9]. Two kinds of loop conformations were found in The protein a1-antitrypsin, which is a member of the serpin different uncleaved serpin structures. In antichymotrypsin family, inhibits the activity of neutrophil elastase in the 1182 Structure 1996, Vol 4 No 10 lower respiratory tract [17,18]. The deficiency of a extends to the region of the P20 (20th residue in the functional a1-antitrypsin due to a variety of hereditary N-terminal direction from the protease cleavage site, P1) mutations leads to emphysema and the aggregation of even though some of the hydrogen bonds are not tight mutant a1-antitrypsin in liver cells is often associated with (Fig. 3). This is in contrast to the gap between strand 5 liver diseases such as infant hepatitis [19,20]. The and strand 3 of sheet A in the same region shown in the mutations stabilizing the uncleaved a1-antitrypsin were uncleaved antithrombin [11,12] and ovalbumin structures found by random mutageneses [21,22]. These mutations do [5]. In the uncleaved antithrombin, the gap is filled with not affect the inhibitory activity of a1-antitrypsin. One of the preinserted loop residues (P14–P17). In ovalbumin, these stabilizing mutation (Phe51→Leu) can suppress the the gap, which is relatively small, is occupied with water folding defect of the Z-type variant [23], presumably by molecules. Hepta a1-antitrypsin does not show the loop inhibiting the loop-sheet polymerization [24]. Recently, a preinsertion observed in the uncleaved antithrombin. low-resolution structure of an uncleaved a1-antitrypsin was This contradicts the proposal based on limited proteolytic determined at 3.45Å [25]. digestion [26] and peptide annealing experiments [27]. A possible method by which stabilization of sheet A occurs We report here the three-dimensional (3D) structure of an in the absence of the preinsertion will be discussed in the uncleaved a1-antitrypsin with seven such stabilizing muta- following sections. tions (hepta a1-antitrypsin) at 2.7Å resolution. The struc- ture of hepta a1-antitrypsin shows differences in loop Before superposition with the cleaved a1-antitrypsin, conformation from the previously determined uncleaved using Ca positions, we divided hepta a1-antitrypsin into a1-antitrypsin structure and suggests an inherent plasticity two fragments (proposed earlier [9]) to test whether each of the reactive loop. There is no preinsertion of the reac- fragment moves as a unit during the structural transition. tive-site loop into b-sheet A, and strands 3 and 5 of the Fragment 1 contained helices A, B, C, G, H and I, sheets sheet have a better hydrogen-bonding network than other B and C and strands 5 and 6 of sheet A (229 residues), and uncleaved serpin structures [5,11,12]. A structural compar- the smaller of the two fragments (fragment 2) comprised ison of hepta a1-antitrypsin with other serpin proteins pro- helix F and strands 1, 2 and 3 of sheet A (63 residues). vides an explanation for the mechanism of the stabilizing The flexible joint regions (helices D and E) and the reac- effect of each mutation site and insights into the function tive-site loop were excluded in the superposition. The of the serpin proteins. root mean square (rms) deviation for the superposition when both fragments were included (292 residues) was Results and discussion 1.80Å. When each fragment was superposed separately, The crystal structure of hepta a1-antitrypsin was deter- the rms deviation was reduced to 1.46Å and 1.28Å for mined by molecular replacement using the structure of fragments 1 and 2, respectively. Figure 4a shows that cleaved a1-antitrypsin (PDB code 7api) as a search model. residues of fragment 2 of hepta a1-antitrypsin are dis- The rotation and translation searches were performed with placed in one direction from those of the cleaved a1-antit- the initial low resolution data at 4.0Å. Later, data at higher rypsin when the two proteins were superposed using resolutions of up to 2.7Å were obtained and used for the residues of fragment 1. The observation that individual refinement. The refinement resulted in a model with a fragments superpose more closely than the overall protein crystallographic R value of 19.5% and excellent geome- and the concerted displacement of residues in fragment 2 tries for 3597 protein atoms of the residues 21–394. The by the superposition of fragment 1 support the proposal model agrees well with electron-density maps in most that fragment 2 slides relative to fragment 1 during the regions of the protein including the reactive-site-loop conformational transition [9]. Ovalbumin and the region and the sheet-A region which shows a large uncleaved antithrombin can be superposed with hepta rearrangement of the b strands with respect to the struc- a1-antitrypsin with rms deviations of 1.97Å and 1.98Å ture of the cleaved a1-antitrypsin (Fig. 1). respectively with both fragments included (Fig. 4b). The relative orientation of the reactive-site loop to the main Structure description body of hepta a1-antitrypsin is similar to that of ovalbumin The structure of hepta a1-antitrypsin has a similar topol- even though there are differences in local conformation. ogy to the structures of uncleaved antithrombin [11,12] and the uncleaved antichymotrypsin [10], but with several In the crystal used for the structure determination, hepta differences.
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