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How is activated by calcium

Ahmad Khorchid and Mitsuhiko Ikura

The discovery of two unexpected Ca2+-binding sites in the structure of a minimal catalytic domain of -calpain reveals a new mechanism underlying the Ca2+-dependent activation of .

Calpains are a family of cytosolic cysteine a large subunit proteinases whose enzymatic activities depend on Ca2+. Members of the calpain C2-like EF hand family are believed to function in various dI dII dIII dIV biological processes, including integrin- mediated cell migration, cytoskeletal remodeling, cell differentiation and **** 1 2+ 12345 apoptosis . At the pathological level, over- autolysis novel Ca -binding acidic activation of calpain as well as mutations sites loop abrogating calpain activity have been EF hand implicated in muscular dystrophy, cardiac 12345 and cerebral ischemia, platelet aggrega- small subunit tion, neurodegenerative diseases, rheuma- toid arthritis, cataract formation and dV dVI b autolysis Alzheimer’s disease2. Despite numerous dIIb biochemical and structural studies3, there dIIa Fig. 1 Schematic diagram of human has been no definitive description of the m-calpain. a, Schematic representation of molecular mechanism by which Ca2+ acti- the domain structure of the large and small vates calpain enzymatic activity. subunits of m-calpain. Novel Ca2+-binding sites, as found by Davies et al.4 in their struc- In a recent issue of Cell, Davies and col- ture of a mini-calpain, are depicted by 4 leagues report the crystal structure of dIII asterisks and sites of autolysis by arrows.

© http://structbio.nature.com Group 2002 Nature Publishing an enzymatically active fragment of b, Ribbon drawing of m-calpain in the dVI 6 µ-calpain that contains two new Ca2+- dI absence of calcium binding sites within the catalytic core. This crystallographic study, together with bio- chemical data, indicates that Ca2+ binding dIV to these sites is cooperative and required for full enzymatic activity. These findings finally resolve the long-standing mystery of calpain’s Ca2+-dependent activity.

Domain architecture of ‘classic’ A previous crystal structure of the full- vation of calpain6,8. Domains dIV and calpains length human m-calpain in the Ca2+-free dVI are well-characterized Ca2+-binding The calpain family includes numerous state6 revealed the overall architecture of domains, each containing five EF-hand members found in organisms ranging this (Fig. 1b). Domain dI com- motifs. These domains are responsible from mammals to Drosophila melano- prises a single α-helix anchored in a for heterodimerization of the large and gaster and Caenorhabditis elegans, as well cavity of dVI, thereby stabilizing the cir- small subunits through a unique inter- as homologs in both yeast and bacteria3,5. cular domain arrangement of the protein. action between their fifth EF-hand Currently, at least 12 different calpains Domain dII contains the catalytic site, motifs. Domain dV of the small subunit have been identified in mammals with and can be further divided into two sub- contains a cluster of Gly residues in the ubiquitous (such as m- and µ-calpains) domains, dIIa and dIIb. These sub- N-terminal region, which is largely unre- and tissue-specific (such as p94 calpain 3 domains have been shown to contain a solved in the crystal structure. While the in skeletal muscle) expression patterns. putative similar to the one structure of the apo m-calpain explained The m- and µ-calpains are the best char- found in cysteine proteinases such as why the protein is inactive without Ca2+, acterized members in the family. Both . Domain dIII consists of eight it could not explain the mechanism of are heterodimers composed of a strands with a topology similar to the C2 activation upon Ca2+ binding. While the large 78–80 kDa catalytic subunit and a domain found in various proteins includ- structure of Ca2+-bound calpain would common small 29 kDa regulatory sub- ing protein C and phospholi- provide insights into this process, efforts unit. The large subunit comprises four pase C, which is known to interact with in this regard have been hampered by the domains (dI–dIV), while the small sub- Ca2+ and phospholipids7. Indeed, an dissociation of the small subunit and the unit has two domains (dV and dVI) acidic loop within dIII has been suggested aggregation of the large subunit under (Fig. 1a). to play a role in the Ca2+-promoted acti- crystallization conditions.

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a Fig. 2 Ribbon drawings of the protease domain (dII) of calpain in a, the absence6 and b, the W288 N286 presence4 of calcium. Catalytic triad residues are colored in red, and the Trp in

o green. ~10 A H262 C105

Ca2+-bound and Ca2+-free calpain are rel- atively small (r.m.s. deviation of 1.6 Å for dIIa and 3.6 Å for dIIb). A major change occurs within the two loop regions involv- ing Gln 96–Cys 108 in dIIa and Ca2+-free dII m-calpain Ile 254–Val 269 in dIIb. In the presence of (inactive) Ca2+, residues in the loop region Ile 254–Val 269 come together to form a b β-sheet that provides supporting van der Ca++ Waals contacts to the active site Trp 298. Ca++ W298 The net effect of this rearrangement is that N296 the Trp 298 side chain, which acts as a

o wedge between domains IIa and IIb in the ~3 A H272 S115 apo state, moves away from the active site cleft allowing for the proper formation of the catalytic triad (Fig. 2). In addition to domain II, the full-length calpains contain two other domains that bind Ca2+ — that is, the EF-hands in domains IV and VI. How does Ca2+- Ca2+-bound dII -calpain (I-II) binding to all three domains orchestrate (active) the activation of calpains? Davies and col- leagues4 suggest a two-step mechanism for activation. The first step is the release of Mini-calpain Most of the Ca2+-coordinating residues constraints imposed by the circular The recent success by Davies and col- found in the µI-II structure, however, were arrangement of the domains. This would

© http://structbio.nature.com Group 2002 Nature Publishing leagues4 depended upon the construction not predicted earlier. Bode and col- involve subtle conformational changes in of a recombinant mini-calpain that com- leagues3,6 had also suggested that the ‘acidic dIV and dVI upon Ca2+ binding, leading prises the catalytic domain IIa-IIb loop’ in dIII (Fig. 1a) binds Ca2+ and that to the abrogation of the interaction (referred to as µI-II). This mini-calpain is binding of Ca2+ to this site may result in a between the N-terminal α-helix of dI and catalytically active only upon Ca2+- of dIIa and dIIb via the second EF-hand motif of dVI9 and loading, and tryptophan fluorescence an ‘electrostatic switch’ mechanism. perhaps promoting conformational analysis demonstrated that Ca2+-binding changes in dIII8 and dissociation of the is positively cooperative involving multi- Mechanism of Ca2+-activation small subunit from the large subunit. The ple sites. Not surprisingly, the active site of Ca2+- second step in the activation is the realign- The crystal structure of the mini- bound µ-calpain markedly resembles the ment of the active site cleft caused by the calpain revealed two novel Ca2+-binding active site seen in other cysteine of Ca2+ to dIIa and sites. The first site, located in dIIa, involves such as papain and cathepsins. The loca- dIIb. Such a two-step mechanism adds an residues Asp 106, Val 99 and Gly 101 on tion and spacing between the critical extra layer of control over calpain func- the loop preceding α-helix 3 (that contains active site residues — Cys 115, His 272, tion. This makes sense physiologically the catalytic Cys 115) and residue Glu 185, Asn 296, Gln 109 and Trp 298 — overlap considering the ubiquitous expression of which is positioned on the loop leading to well with the corresponding residues in calpains and their role as biomodulators the N-terminus of the core α-helix 5. The papain (a root mean square (r.m.s.) devia- of a wide variety of cytoskeletal, mem- second site is in dIIb, and Ca2+-binding is tion of 0.66 Å for all side chain atoms). brane and cytosolic proteins1,10. coordinated by four acidic residues and a Most remarkably, the Oγ atom of the methionine: Glu 302 and Asp 309 in the active site Ser 105 is in very close Remaining questions loop containing Trp 298, another residue proximity (3.7 Å) to the immidazole Nδ of The residues involved in the newly charac- in the active site, and residues Asp 331, His 262, a central histidine in the catalytic terized Ca2+-binding sites in dII are highly Glu 333 and Met 329 in the loop between triad of µ-calpain (Fig. 2b). By contrast, conserved in calpains from Drosophila to α-helices 8 and 9. the distance between these residues in humans. However, members in the cal- Based on the inactive Ca2+-free m- Ca2+-free calpain is 10.5 Å (Fig. 2a). pain family have very different Ca2+ sensi- calpain structure, Bode and colleagues3,6 What are the backbone conformational tivities. For example, µ-calpain is active in had previously predicted the involvement changes that trigger these side chain the Ca2+ concentration range of 5–50 µM, of Asp 321 in Ca2+-binding to m-calpain. rearrangements required for the activa- where as m-calpain is active in the Indeed, the corresponding residue in tion of µI-II, which are also likely for 200–1,000 µM range; the activity of the µ-calpain, Asp 331, is part of the second native µ-calpain? Interestingly, the differ- tissue-specific human calpain 3 is Ca2+- Ca2+-binding site in the structure of µI-II. ences in backbone conformation between independent. A possible explanation for

240 nature structural biology ¥ volume 9 number 4 ¥ april 2002 news and views

these differences is the high sequence C2-like dIII of classic calpains binds Ca2+ Canada M5G 2M9. Correspondence should diversity in the acidic loop of dIII among and that its affinity to Ca2+ is enhanced by be addressed to M.I. email: mikura@ the calpains. µ-calpain has three more the presence of di- and triphosphoinosi- oci.utoronto.ca

acidic residues in the dIII loop relative to tide-containing liposomes. Furthermore, 1. Suzuki, K. & Sorimachi, H. FEBS Lett. 433, 1–4 (1998). m-calpain; human calpain 3 has no acidic Ca2+-binding to calpain promotes several 2 .Huang, Y. & Wang, K.K.W. Trends Mol. Med. 7, 355–362 (2001). residue in this loop. As the central processes: (i) translocation of the 3. Reverter, D., Sorimachi, H. & Bode, W. Trends domain, dIII may play a major role in the to the plasma membrane15 (ii) autolysis in Cardiovasc. Med. 11, 222–229 (2001). 2+ 6,8 4. Moldoveanu, T. et al. Cell, 108, 649–660 (2002). Ca -mediated activation of calpain . It is both the large and small calpain sub- 5. Sorimachi, H. & Suzuki, K. J. Biochem. 129, 653–664 conceivable that changes in dIII affect the units16,17 (Fig. 1a), and (iii) dissociation of (2001). 6. Strobl, S. et al. Proc. Natl. Acad. Sci. USA 97, catalytic and structural integrity of the the two subunits into truncated frag- 588–592 (2000). catalytic dII, as well as that of the other ments1. Together these processes con- 7. Rizo, J. & Südhof, T.C. J. Biol. Chem. 273, 2+ 15879–15882 (1998). Ca -binding motifs in calpain. Further tribute to the biological activity of calpain 8. Hosfield, C.M., Elce, J.S., Davies, P.L. & Jia, Z. EMBO studies are needed to understand the vari- in the cell, adding a complexity to the J. 24, 6880–6889 (1999). 2+ 2+ 9. Nakagawa, K., Masumoto, H., Sorimachi, H. & ability of calpain’s Ca -sensitivity. mechanism underlying the Ca -depen- Suzuki, K. J. Biochem. 130, 605–611 (2001). Another major unresolved question con- dent activation of calpains. The next chal- 10. Glading, A., Lauffenburger, D.A. & Wells, A. Trends 2+ Cell Biol. 12, 46–54 (2002). cerns the intracellular Ca level, which is lenge for calpain structural biology would 11. Takano, E., Ma, H., Yang, H.Q., Maki, M. & generally 1 µM at most, even in stimulated be the determination of a membrane- Hatanaka, M. FEBS Lett. 362, 93–97 (1995). 2+ 12. Benetti, R. et al. EMBO J. 20, 2702–2714 (2001). cells, and never reaches the high concentra- bound, fully Ca -activated enzyme. 13. Saido, T.C., Shibata, M., Takenawa, T., Murofusi, H. tion range at which the calpains become & Suzuki, K. J. Biol. Chem. 267, 24585–24590 (1992). active. Thus, it may be that other biological Ahmad Khorchid and Mitsuhiko Ikura 14. Tompa, P., Emori, Y., Sorimachi, H., Suzuki, K. & molecules such as protein inhibitors (cal- are in the Division of Molecular and Friedric, P. Biochem. Biophys. Res. Comm. 280, 11 12 1333–1339 (2001). pastatin and Gas-2 ) as well as phospho- Structural Biology, Ontario Cancer 15. Molinari, M., Anagli, J. & Carafoli, E. J. Biol. Chem. lipids13 are required to modulate the Institute, and the Department of Medical 269, 27992–27995 (1994). 2+ 16. Michetti, M. et al. FEBS Lett. 392, 11–15 (1996). Ca -sensitivity of calpains. Recently, Biophysics, University of Toronto, 610 17. Baki, A., Tompa, P., Alexa, A., Molnar, O. & Tompa et al.14 reported that the isolated University Avenue, Toronto, Ontario, Friedrich, P. Biochem. J. 318, 897–901 (1996).

Single-handed recognition of a sorting

© http://structbio.nature.com Group 2002 Nature Publishing traffic motif by the GGA proteins Tom Kirchhausen

Selective transport of cargo between membrane-bound organelles is vital for the well-being of cells. The crystal structure of a short peptide signal from the cytoplasmic tail of the mannose-6-phosphate receptor bound to the VHS domain of GGA proteins gives hints to how sorting works.

In most cells, it takes an hour or less to cytosolic proteins, which function as tors (MPRs), transmembrane proteins recycle lipids and many transmembrane adaptors to link the cargo and the coat that are essential for normal lysosomal proteins between various cellular mem- machinery responsible for membrane function in mammalian cells. For exam- branes. Vesicles and tubulo-vesicular deformation and budding1. ple, these receptors recognize the man- carriers shuttle these components, but Cellular processes that depend on accu- nose-6-phosphate groups on lysosomal remarkably, the compositions of the vari- rate membrane traffic range from endo- and are involved in transport- ous membrane compartments remain cytosis of , nutrients and viruses ing these from the TGN to endo- distinct (Fig. 1). This feat depends in part to protease secretion, antigen presenta- somes. After making such a delivery, the on the controlled formation of the vesi- tion and membrane recycling during receptors cycle back to the TGN for cles and tubulo-vesicular structures that neurotransmission. The bi-directional another round of traffic. bud from the donor membrane, their traffic along the secretory and endocytic How is this trafficking pattern main- movement inside the cell, and their tar- pathways intersect at the interface tained? Enter the GGAs (Golgi-localized geting and fusion with the acceptor between the trans Golgi network (TGN, a γ-ear-containing ARF binding pro- membrane. Much like the zip code tubulo-vesicular network abutting the teins)2–5. Unknown until barely two years addressing system for letters and pack- distal side of the characteristic Golgi ago, GGAs are ubiquitous cytosolic pro- ages, sorting at the beginning of the membrane stacks) and the endosomal/ teins of 613–721 amino acids that cycle process ensures correct selection of cargo lysosomal compartment (a tubulo-vesicu- between the cytosol and the TGN and link molecules for transport and delivery. lar network that is more dispersed clathrin to membrane-bound ARF–GTP. Sorting of transmembrane proteins throughout the cells). One class of cargo The three mammalian GGAs — GGA1, involves recognition of short peptide sig- proteins in this bi-directional traffic com- GGA2 and GGA3 — are responsible for nals in their cytoplasmic tails by special prises the mannose-6-phosphate recep- the accurate trafficking of MPRs and

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