Complex of human neutrophil elastase with 1/2SLPI

The 1/2SLPI and HNE complex structure was solved at 1.7 Å resolution and compared with the interaction mechanism of elafin.


Introduction
The SLPI (secretory leukocyte protease inhibitor) similar domain motif is present in several other secreted proteins called the WAP four-disulfide core (WFDC) domain family. Elafin is one of these proteins (Wiedow et al., 1990(Wiedow et al., , 1991Zani et al., 2004), a 57-residue protease inhibitor that is a potent and specific strong inhibitor of PPE and HLE (human leukocyte elastase). However, the SLPI C-terminal domain inhibits HNE but inhibits PPE weakly (Masuda et al., 1994).
The X-ray structure shows that elafin inhibits PPE in a similar way as the SLPI C-terminal domain (Tsunemi et al., 1996). How are these differences in selectivity against serine proteases between SLPI and elafin caused? Elafin and SLPI have multiple important roles both in normal homeostasis and at the site of inflammation. These include anti-protease and anti-microbial activity as well as modulation of the response to LPS stimulation. Both are members of larger families of proteins secreted predominantly at mucosal sites and are modulated under multiple pathological conditions, and control the conditioning of the innate immune system. The functions of SLPIs are still unclear (Williams et al., 2006). We report here the details of their interactions with 1/2SLPI and HNE and describe the comparison with elafin:PPE complexes (Tsunemi et al., 1996).

Materials and methods
2.1. Crystallization 1/2SLPI was designed as a half-size SLPI, which corresponds to the C-terminal domain and is expressed in E. coli. HNE was purchased from Elastin Products Company (Pacific, MO, USA). To obtain the HNE and 1/2SLPI complex, the mixed sample was dialyzed against 20 mM Na-acetate buffer (pH 5.0) using Slide-A-Lyzer (5000 Mw) to remove excess 1/2SLPI molecules. The crystallization was performed using the sitting-drop vapor-diffusion method at 293 K. Jena Bioscience Screening kits were used for the initial condition search. The crystals appeared from 2.0 M Na-formate at pH 4.5 in a few days and grew to a typical crystal size of 0.4 Â 0.2 Â 0.01 mm in one week.

Data collection and processing
Data collection was carried out under nitrogen stream at 100 K using Quantum315 at beamline BL41XU of SPring-8. Paraton was chosen as a cryoprotectant and added to the crystallization condition 40%(v/v). The crystal belongs to P4 2 2 1 2 with unit-cell constants of a = b = 106.64 Å , c = 55.12 Å . The data set was processed using Crystal Clear 1.3.5. The initial phase was determined by molecular replacement with only HNE using MOLREP (CCP4.6.01 suite).

Refinements
Refinements were carried out using REFMAC5 (CCP4.6.01 suite). After several cycles of map fitting of the HNE protein structure, we identified the electron density corresponding to 1/2SLPI. The 1/2SLPI model was constructed using QUANTA2000 (MSI) with reference to the elafin (Tsunemi et al., 1996) main-chain trace. The crystallographic data are shown in Table 1.

Results and discussions
3.1. Crystal structure of HNE and 1/2SLPI complex Fig. 1(a) shows the complex structure of HNE and 1/2SLPI. In the contact region there are no covalent bonds between HNE and 1/2SLPI. The main-chain trace of HNE shows no differences from PDB (Protein Data Bank) id 1B0F (Cregge et al., 1998). 1/2SLPI consists of four anti-parallel strands (strand from A to D) and four disulfide bridges (Cys64i-Cys93i, Cys71i-Cys97i, Cys80i-Cys92i, Cys86i-Cys101i), which keep the shape of 1/2SLPI like a rigid wedge.
There are a small number of intra-hydrogen bonds and electrostatic interactions in the 1/2SLPI structure (Fig. 1b).
In these four strands, only two internal strands form a doublestranded twisted -sheet (Lys91i-Gly95i and Gly98i-Val102i) connected by five main-chain-main-chain hydrogen bonds. These two strands are joined by a -hairpin loop at Met96i-Cys97i. No mainchain-main-chain hydrogen bond is formed between the two parallel strands A and C, and one hydrogen bond is formed between strands B and D. In the region between the long loop AB and the adjacent hairpin loop CD, only one hydrogen bond is formed, connecting N of Cys71i and O of Met96i.
1/2SLPI has almost the same structure as the C-terminal domain of the full SLPI structure (W. Bode, private communication), with the root-mean-square deviation (RMSD) of the backbone atoms being 1.04 Å for residues Arg58i-Ala107i. The AB loop (between strands A and B) of 1/2SLPI binding to the active site of HNE has no serious affect on the enzyme structure. Both 1/2SLPI and elafin have four disulfide bridges at the same positions. The sequence identity is 42%; however, the P1-residue of elafin is Ala24i. The main-chain structures of the AB loop of both 1/2SLPI and elafin are similar. The AB loop of elafin should have a large flexibility, like 1/2SLPI (unpublished NMR data).
The primary interaction appears to be the P1-residue Leu72i, located in the S1 pocket, and the carbonyl oxygen accommodated in the oxyanion hole.  Table 2 Inhibition constant (K i values) of elafin (Zani et al., 2004) and 50% inhibitory concentrations (IC50) of 1/2SLPI and against some serine proteases (Masuda et al., 1994).  at P2 0 -P3 in the primary contact site as elafin:PPE. Another hydrogen bond, Tyr68i (OH)Á Á ÁArg217A (N"1), in the primary contact region is added (Fig. 2a). Met96i (CD loop) is located near Phe215 with a longer distance than the usual van der Waals contact in the secondary contact region. On the other hand, elafin has additional hydrogen bonds between the carbonyl oxygen of Leu20i (P5) and the main-chain NH of Arg217A in the primary contact region, and between the hydroxyl group of Ser48i and the carbonyl oxygen of Asp97 shown in Fig. 2(b) (Tsunemi et al., 1996). The RMSD of the backbone atoms between 1/2SLPI and elafin is 1.01 Å , calculated by superposing residues Arg59i-Val105i of 1/2SLPI onto Thr11i-Gln57i of elafin. As shown in Table 2, 1/2SLPI shows a wide range of inhibitory activity against some serine proteases. On the other hand, elafin is a potent and specific protein inhibitor of PPE and HNE.
3.2. Difference in the inhibitory mechanism for elastases between elafin and 1/2SLPI 1/2SLPI:HNE has a similar interaction mode at P2 0 -P3 in the primary contact region as elafin:PPE. Figs. 3(a) and 3(b) show the S1 pocket of 1/2SLPI:HNE and elafin:PPE, respectively. They show that elafin does not fill the S1 pocket in the elafin:PPE complex structure and that SLPI fills the S1 pocket of HNE better. Leu72i (P1) of 1/2SLPI provides a large contribution in the recognition with HNE and PPE (Eisenberg et al., 1990). Fig. 3(c) shows PPE superposing onto 1/2SLPI:HNE; the S1 pocket of PPE is similar to those of HNE. 1/2SLPI should show the same inhibitory activity against PPE on the S1 pocket, but 1/2SLPI inhibits HNE more than PPE (Table 2). To understand 1/2SLPI's selectivity for HNE, we compared the interface of HNE and PPE around P5 and found a remarkable difference in these structures (Fig. 3). In the PPE's binding interfaces, Tyr68i (P5) conflicts with a region (Ser169-Val176) of PPE (shown in Figs. 3d and 3e). Tyr68i may be flipping away to avoid the conflict and cannot interact with PPE. In the 1/2SLPI:HNE complex, 1/2SLPI requires at least six hydrogen bonds between the main chains at P2 0 -P3 for the inhibitory activity owing to Leu72i (P1) occupation in the S1 pocket. Then the additional interactions at Tyr68i (P5) are used to increase the inhibitory activity of 1/2SLPI against HNE, but the interaction of the second contact region is not so critical.
On the other hand, elafin needs a full set of hydrogen-bonding interactions (seven hydrogen bonds of the main chain in the primary contact region and one in the secondary contact region) which should be essential for the inhibitory activity because Ala24i (P1) is insufficient in occupying the S1 site. The interacting mode of elafin:PPE may be kept in the elafin:HNE complex structure. It seems that the P1 and P5 residues of 1/2SLPI are important residues causing the selectivity against HNE. Schematic representation of the hydrogen-bonding interactions between the contact regions of (a) HNE and 1/2SLPI and (b) PPE and elafin (Tsunemi et al., 1996). Inhibitor residues are designated by 'i' after the sequence number. Intermolecular hydrogen bonds are indicated by dashed lines.

Figure 3
Electrostatic surface representations of (a) the HNE complex with 1/2SLPI (ball and stick model), and (b) the PPE:elafin complex around the S1 pocket. Superposed representations of the PPE molecule onto HNE of the X-ray structure HNE:1/2SLPI. The solid surface shows HNE and the meshed surface shows PPE. Comparison of HNE and PPE (purple mesh) around (c) the S1 pocket and (d) the S5 pocket. (e) The sequence alignment between HNE and PPE around the S5 pocket. The conflict residues of PPE with Y68i are shown in the purple square.