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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 call-cal-pain 1 and milli-c

M I N I R E V I E W S E R I E S

The structural basis of calpain behavior

Yves Benyamin

UMR5539, EPHE-CNRS-UM2, cc107, Universite´ de Montpellier II, France

Calpains are intracellular Ca2+-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 cal(l-cal-pain 1) and milli-cal(l-cal-pain (m-cal(l-cal-pain or

calpain 2), have been the focus of three decades of

intensive characterization In vitro analysis has shown

that the Ca2+ 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 Ca2+-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

Ca2+-free configuration corresponds to a structurally

inactive conformation of calpain 2 DIII is related to

the spatial organization of the C2 domain and binds

Ca2+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 Ca2+ 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

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 [Ca2+],

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 Ca2+ 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

[Ca2+], 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 Ca2+ -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 Ca2+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 Ca2+ 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.