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Authors' Summary:
Structure of a Site-2 Protease Family Intramembrane Metalloprotease

Liang Feng et al.

An unusual signaling mechanism involves the cleavage of a transmembrane protein within the lipid membrane bilayer. Cleavage is accomplished by a membrane-embedded protease enzyme (1), so this process is called regulated intramembrane proteolysis (RIP) and is used by organisms from bacteria to humans. The first RIP system to be molecularly characterized was the cleavage of the membrane-anchored transcription factor SREBP (sterol regulatory element–binding protein) by a metalloprotease known as site-2 protease (S2P) (2). This cleavage releases a transcription factor, which translocates into the nucleus of the cell and activates genes involved in synthesis and uptake of cholesterol and fatty acids. How S2P cleaves a protein embedded within the lipid bilayer has been enigmatic. Cleavage of a protein requires water molecules. How does water gain access to the buried active site of S2P? Is the metal ion cofactor that is required for catalysis exposed to the lipid environment? If not, how can a substrate protein get into the active site? Finally, what does an S2P protease look like, and how does the structure support its function? Our study of the S2P protein provides clues for the answers to all these questions.

X-ray crystallography is a powerful approach for elucidation of the detailed three-dimensional structure of macromolecules. A prerequisite is generation of crystals that are sufficiently ordered and large enough to produce useful x-ray diffraction data—a daunting challenge for membrane proteins. We were able to crystallize the S2P protein from the archaebacterial species Methanocaldococcus jannaschii by including the detergent decyl-β-D-maltopyranoside. To improve the ability of these crystals to diffract x-rays, we incorporated two additional detergents into the crystallization buffer. The structure was determined with multi-wavelength anomalous dispersion.

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Closed and open conformations of an S2P metalloprotease, which cleaves its protein substrates within the cell membrane. Substrate peptide is proposed to gain access to the catalytic zinc atom (red sphere) only in the open conformation.

Credit: Chris Bickel/Science

S2P has six transmembrane segments, TM1 through TM6 (see the figure). The catalytic zinc atom is located ~14 Å from the lipid membrane surface. Zinc is coordinated by three amino acids, His⁵⁴ and His⁵⁸ in TM2, and Asp¹⁴⁸ in TM3, which are highly conserved in all S2P proteins. TM2 and TM4 are stabilized by TM3, and together, these three segments constitute a core domain of S2P (green). Amino acid sequences for TM2–4 are similar in S2P proteins from other species, which suggests that they have a similar structure and a conserved active-site conformation. In the crystals, two molecules of S2P are contained in one asymmetric unit, the minimal element that can be built into an entire crystal. The two molecules exist in different conformations (see the figure). Although the conformations of the core domain are identical, the other TM segments are quite different; TM1 and TM6 are 10 to 12 Å farther apart in one S2P molecule than in the other. The conformational difference has a direct consequence: The active site is accessible only in the S2P molecule in which TM1 and TM6 are farther apart. Hence, these two conformations likely represent the open and closed states of S2P. In the open state, the cleft between TM1 and TM6 can accommodate a peptide in an extended conformation. Thus, we propose that, to be properly positioned for cleavage, the peptide gains access to the active site of S2P through the lateral movement of TM1 and TM6. In the closed state, water molecules can get to the zinc in the active site through a hydrophilic channel that opens to the cytoplasmic side of the lipid membrane.

Although our structure suggests how the substrate peptide may get to the active site, further insight must await biochemical experiments. Given the flexibility of TM1, TM5, and TM6, can substrate enter the active site between TM1 and TM2 or between TM6 and the core domain? Although these possibilities cannot be ruled out, they are not supported by available sequence or structural information. Nonetheless, crystals of S2P were generated in the presence of detergents, rather than membrane lipid. Consequently, the influence of detergents on the structure remains to be characterized.

Despite these caveats, the structure of S2P serves as a framework for understanding the function of intramembrane metalloproteases. The sequence conservation among S2P family members suggests that the active site is in a similar position throughout the family. In contrast, substrate proteins are cleaved at different positions along their putative transmembrane helices. This suggests that, before cleavage, SP2 must recognize a specific sequence in the substrate to appropriately position the cleavage site. Such recognition does not necessarily occur within the lipid bilayer, as is the case for human S2P and its homolog in Bacillus subtilis.

In addition to the S2P family, there are three additional families of intramembrane proteases: serine protease rhomboid, aspartate protease presenilin, and signal peptide peptidase. The mechanisms of water entry and substrate access appear to be similar between S2P and rhomboid, the only other intramembrane protease for which structural information is available. It remains to be seen whether such mechanisms also apply to the aspartate proteases.

Summary References

1. M. S. Wolfe, R. Kopan, Science 305, 1119 (2004).
2. M. S. Brown, J. Ye, R. B. Rawson, J. L. Goldstein, Cell 100, 391 (2000).

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