MINIREVIEW SERIES The structural basis of calpain behavior Yves Benyamin UMR5539, EPHE-CNRS-UM2, cc107, Universite ´ de Montpellier II, France Calpains are intracellular Ca 2+ -regulated cysteine pro- teases which mediate regulatory cleavage of specific substrates. They cover a broad range of physiological functions including proteolysis of molecules involved in cytoskeletal organization, the cell cycle, signal trans- duction, apoptosis, and protein renewal during growth and tissue regeneration. Originally found in mamma- lian skeletal muscle then in numerous organisms inclu- ding protists and plants, their expression is ubiquitous [1,2]. Among the 14 members of the calpain gene family with different expression patterns in tissue develop- ment, two ubiquitous isoforms, microcalpain (l-cal- pain or calpain 1) and milli-calpain (m-calpain or calpain 2), have been the focus of three decades of intensive characterization. In vitro analysis has shown that the Ca 2+ concentration required for optimal activity is 5–50 lm for calpain 1 and 0.2–1 mm for cal- pain 2. A large range of substrates and a common inhibitor (calpastatin), also found in the nucleus, were identified. Knowledge of the 3D structure of Ca 2+ -free calpain 2 and a chimeric l,m-calpain has provided mechanistic concepts for understanding their allosteric regulation [3–6]. Briefly (Fig. 1), ubiquitous calpains have an 80-kDa catalytic subunit (backbone representation) and a 28-kDa regulatory subunit (space-filled representation), which functions as a chaperone to stabilize the 80-kDa structure. The critical importance of the 28-kDa sub- unit, which is common to the two calpains, was shown by the fact that transgenic mice with a knock-out gene die at an early stage of embryonic development. This subunit contains a mobile hydrophobic segment (dashed purple segment) and a penta EF-hand domain (DV and DVI, respectively). In the 80-kDa subunit, the N-terminal segment (DI) interacts with DVI which anchors DII (catalytic domain) to the 28-kDa subunit. DII (DIIa and DIIb) interacts strongly with DIII via electrostatic bonds (+ ⁄ –). Each DII subdomain includes a part of the catalytic triad (red stars). This Ca 2+ -free configuration corresponds to a structurally inactive conformation of calpain 2. DIII is related to the spatial organization of the C2 domain and binds Ca 2+ ions and phospholipids. DIV, structurally similar to DVI, also contains five sets of EF-hand motifs. Note that the fifth set of EF-hand motifs of each domain are unable to bind calcium and serve to bridge the two subunits (crossed yellow squares). Upon Ca 2+ binding (yellow squares), salt bridges that keep the catalytic domain (DII) in an open con- formation are disrupted (transducer signals) and the Fig. 1. Calpain 2 (accession number: 1DFO) representation using the Cn3D software from NCBI. The probable position of DV [4] is added to the picture. The different calpain domains of the catalytic subunit circled in black (DI), pink (DIIa), blue (DIIb), brown (DIII), green (DIV) are indicated. Other details are in the text. doi: 10.1111/j.1742-4658.2006.05353.x FEBS Journal 273 (2006) 3413–3414 ª 2006 The Author Journal compilation ª 2006 FEBS 3413 two parts, DIIa and DIIb, come closer together, with concomitant release of constraints between the domains (large arrows). This active conformation of DII successively initiates proteolysis activity, intermo- lecular cleavage in DI then DV with possible subunit dissociation, substrate proteolysis, and, finally, loss of calpain activity upon increased autolysis of the 80-kDa subunit, all in a few minutes. At physiological [Ca 2+ ], calpastatin blocks calpains via several contacts (blue squares), which tie up the mobile structure to form a long-lived complex. Several questions are raised by these molecular stud- ies. What is the correlation between the in vitro and in vivo Ca 2+ requirements for calpain activity? How do the two isoforms target their specific substrates in vivo? Can we obtain a clearer picture of calpain mobility between calpastatin complexes and proteolysis sites, including the translocation to membrane phos- pholipids and the nucleus? We also need to identify the cell’s safeguards which prevent inappropriate clea- vage during transient increases and oscillations of [Ca 2+ ], in particular those involving specific phos- phorylations of calpastatin, calpains and targets. The following Minireviews develop three themes on the role of calpain 1 and 2 in cytoskeletal anchorage and sarcomere stability, on the specific behaviour of calpain 3 (p94) in muscle cells and on apoptosis mech- anisms in neuritic cells. References 1 Goll DE, Thompson VF, Li H, Wei W & Cong J (2003) The calpain system. Physiol Rev 83, 731–801. 2 Zatz M & Starling A (2005) Calpains and disease. N Engl J Med 352, 2413–2423. 3 Hosfield CM, Elce JS, Davies PL & Jia Z (1999) Crystal structure of calpain reveals the structural basis for Ca 2+ - dependent protease activity and a novel mode of enzyme activation. EMBO J 18, 6880–6889. 4 Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, Sorimachi H, Bou- renkow G, Bartunik H, et al. (2000) The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc Natl Acad Sci USA 97, 588–592. 5 Pal GP, De Veyra T, Elce JS & Jia Z (2003) Crystal structure of a micro-like calpain reveals a partially acti- vated conformation with low Ca 2+ requirement. Struc- ture (Camb) 11, 1521–1526. 6 Bozoky Z, Alexa A, Tompa P & Friedrich P (2005) Multiple interactions of the ‘transducer’ govern its function in calpain activation by Ca 2+ . Biochem J 388, 741–744. Yves Benyamin is currently Professor of Cellular Biochemistry at the Ecole Pratique des Hautes Etudes in Paris. His doctoral work on the evolution of muscle kinases was conducted at the Colle ` ge de France in Paris. In 1977 he joined the National Center of Scientific Research (CNRS) in Montpellier where he started his group on cyto- skeleton protein interactions. Since 1997 he has been studying the role of calpains in cell behavior at the Uni- versity of Montpellier. The structural basis of calpain behavior Y. Benyamin 3414 FEBS Journal 273 (2006) 3413–3414 ª 2006 The Author Journal compilation ª 2006 FEBS . 1997 he has been studying the role of calpains in cell behavior at the Uni- versity of Montpellier. The structural basis of calpain behavior Y. Benyamin 3414. representation using the Cn3D software from NCBI. The probable position of DV [4] is added to the picture. The different calpain domains of the catalytic subunit