Báo cáo khoa học: Distinguishing between calpain heterodimerization and homodimerization doc

His6-tagged PEF domains of calpains 1, 3, 9 and 13 were coexpressed with the PEF domain of the small subunit that had been tagged with an antifreeze protein.. The human small subunit lac

and homodimerization

Ravikiran Ravulapalli1, Robert L Campbell1, Sherry Y Gauthier1, Sirano Dhe-Paganon2 and Peter

L Davies1

1 Department of Biochemistry, Queen’s University, Kingston, Canada

2 Structural Genomics Consortium and the Department of Physiology, University of Toronto, Canada

Calpains are a family of intracellular cysteine

prote-ases They are Ca2+-dependent and function by

modu-lating the biological activities of target proteins

through selective cleavage [1] Genome sequencing

projects have revealed numerous calpain isoforms in

vertebrates, invertebrates, plants, microorganisms and,

recently, in kinetoplastid parasites [1–7] In the human

genome, 14 different calpain isoforms have been

identi-fied to date Several calpain isoforms are ubiquitously

expressed, whereas many demonstrate tissue-specific

expression patterns [8] Although their precise func-tions are poorly understood, calpains are implicated in many intracellular processes linked to calcium signal-ing, such as cell motility, apoptosis, and cell cycle reg-ulation, as well as cell-type-specific functions, such as cell fusion in myoblasts and long-term potentiation in neurons [9–12] Several pathologic conditions (ischemic injury, Alzheimer’s disease, limb-girdle muscular dystrophy 2A, type II diabetes mellitus, gastric cancer, etc.) have been associated with disturbances of the

Keywords

calcium; calpain; dimerization; EF-hand

protease

Correspondence

P L Davies, Department of Biochemistry,

Queen’s University, Kingston, ON K7L 3N6,

Canada

Fax: +1 613 533 2497

Tel: +1 613 533 2983

E-mail: daviesp@queensu.ca

(Received 28 August 2008, revised 13

November 2008, accepted 4 December

2008)

doi:10.1111/j.1742-4658.2008.06833.x

The two main mammalian calpains, 1 and 2, are heterodimers of a large

80 kDa and a small 28 kDa subunit that together bind multiple calcium ions during enzyme activation The main contact between the two subunits

of these intracellular cysteine proteases is through a pairing of the fifth EF-hand of their C-terminal penta-EF-hand (PEF) domains From model-ing studies and observation of crystal structures, it is not obvious why these calpains form heterodimers with the small subunit rather than homodimers of the large subunit, as suggested for calpain 3 (p94) There-fore, we have used a differential tagging system to determine which of the other PEF domain-containing calpains form heterodimers and which form homodimers His6-tagged PEF domains of calpains 1, 3, 9 and 13 were coexpressed with the PEF domain of the small subunit that had been tagged with an antifreeze protein As predicted, the PEF domain of cal-pain 1 heterodimerized and that of calcal-pain 3 formed a homodimer The PEF domain of digestive tract-specific calpain 9 heterodimerized with the small subunit, and that of calpain 13, prevalent in lung and testis, was mainly found as a homodimer with a small amount of heterodimer These results indicate whether recombinant production of a particular calpain requires coexpression of the small subunit, and whether this calpain is likely to be active in a small subunit knockout mouse Furthermore, as the endogenous inhibitor calpastatin binds to PEF domains on the large and small subunit, it is less likely that the homodimeric calpains 3 and 13 with two active sites will bind or be silenced by calpastatin

Abbreviations

AFP, antifreeze protein; PEF, penta-EF-hand.

calpain system [13–18] Therefore, elucidating the

spe-cific role of calpains in these pathologies may facilitate

treatment of these diseases

The ubiquitous and well-characterized members of

the family, calpains 1 and 2 (l-isoform and m-isoform,

respectively), are heterodimers, containing a large

80 kDa subunit (domains I–IV) and a small 28 kDa

subunit (domains V and VI) [19–21] Both enzymes

share a papain-like protease core (domains I and II)

characterized by the presence of the catalytic triad

resi-dues cysteine, histidine and asparagine Domains III

and IV are the C2-like and penta-EF-hand (PEF)

domains, respectively The PEF domain (IV) of the

large subunit pairs with the homologous PEF

domain VI of the small subunit through EF-hand 5,

thus forming a heterodimer In the absence of Ca2+,

both isoforms are catalytically inactive, and upon

binding Ca2+, the heterodimer undergoes multiple

structural changes to form the active calpain enzyme

Structural events, such as autoproteolysis, subunit

dis-sociation, intradomain⁄ interdomain rearrangement and

phospholipid binding, are suggested to be involved in

this complex regulation of activation [22–25]

Five of the human calpains (calpains 5, 6, 7, 10 and

15) have significantly different domain compositions

from those of the conventional calpain large subunit,

suggestive of distinct functions [25–29] In particular,

they lack a PEF domain with which to dimerize, and

are presumed to be monomers The other members of

the calpain family (calpains 3, 8, 9, 11, 12 and 13) do

have a PEF domain (domain IV) Considering their

similarity in domain arrangement to the classic

cal-pains 1 and 2, these isoforms have the potential to

form heterodimers with the small subunit However,

recent biophysical studies on the recombinant PEF

domain of calpain 3 showed that it forms a very stable

homodimer [30] Molecular modeling demonstrated

that this interaction could be the basis for

homodimer-ization of the whole enzyme A 180 kDa protein was

formed by recombinant expression of inactive

cal-pain 3 in the absence of the small subunit, which is

consistent with homodimerization [31] The situation

with native calpain 3 (p94) is unclear, because the

enzyme is unstable and rapidly autoproteolysed during

purification, but the small subunit does not seem to

copurify with the 94 kDa large subunit Thus, it

can-not be assumed that the presence of a C-terminal PEF

domain in other calpain isoforms will lead to

heterodi-merization with the small subunit One of the reasons

why it is important to establish which calpains form

heterodimers is that calpastatin, the natural inhibitor

of calpains 1 and 2 [32], binds to sites on the PEF

domains of both the large and small subunits [33,34]

In the presence of Ca2+, subdomains A and C of calpastatin tightly associate with PEF domain IV of the large subunit and domain VI of the small subunit, respectively This binding ensures a high local concen-tration of subdomain B that binds and blocks the active site cleft of the enzyme In the absence of the small subunit, calpastatin would lose one of its binding sites and might not associate tightly enough with the large subunit to inhibit it More to the point, a homodimer of the large subunit would have two active sites at opposite ends of the molecule, and these certainly could not both be inhibited by one calpastatin inhibitory domain In this context,

we sought to examine all known PEF domains from human calpain isoforms, including calpain 3, to establish whether they exist as heterodimers or homodimers

In order to screen these PEF domains, a coexpres-sion system with differential tags on the recombinant proteins was established The human small subunit lacking the glycine-rich domain (21 kDa) was tagged with type III antifreeze protein (AFP) (7 kDa) [35] in its place on the N-terminus, whereas the recombinant domain IVs of other calpain isoforms had a His6-tag

on the N-terminus (Fig 1) This approach gave us the opportunity to exploit two distinct purification methods, ice affinity purification [36] and Ni2+ –nitri-lotriacetate–agarose chromatography, to characterize these recombinant proteins

Results

Multiple constructs representing the domain IV region

of human calpain isoforms 1, 3, 8, 9, 11, 12 and 13 were designed in an effort to improve the likelihood of expressing these recombinant isoforms Recombinant calpains 1, 3, 9 and 13 domain IV constructs produced high yields when expressed alone or when coexpressed with human small subunit (Table 1) Constructs of cal-pains 8, 11 and 12 failed to express Further trials to stabilize their expression by coexpression with human small subunit did not influence the yield

Establishing the validity of the screening method using calpain 1 domain IV

To test the functionality of the N-terminally AFP-tagged small subunit in forming a natural heterodimer [20,21], we coexpressed it with inactive rat calpain 2 (C105S-m-80 kDa) large subunit and with human cal-pain 1 domain IV The rat large subunit was chosen for this purpose because the human ortholog is poorly expressed in Escherichia coli and the residues involved

in heterodimer formation are highly conserved in the

two mammals As expected, both calpain 2 large

sub-unit and the isolated domain, calpain 1 domain IV

(21 kDa), formed heterodimers with recombinant

type III AFP-tagged human small subunit (28 kDa)

This was established by Ni2+–nitrilotriacetate–agarose

column purification, where both the coexpressed

con-structs were detected in the imidazole-eluted fractions

(Fig 2A, lane 4; Fig 2C, lane 3) In Fig 2A lane 4,

the relative staining of large (80 kDa) and small

(21 kDa) subunits is consistent with their 1 : 1

stoichi-ometry When an immunoblot of the gel, shown in

Fig 2A, was probed with antibody against AFP, the

only protein band detected was 28 kDa AFP-tagged

small subunit (Fig 2B, lane 1) Similarly, when a

duplicate immunoblot was probed with antibody against His-tag, the only protein band detected was

80 kDa His-tagged large subunit (Fig 2B, lane 2) One immediate advantage of the type III AFP-tagged small subunit construct is the increase in its molecular mass from 21 to 28 kDa, which readily distinguishes it from domain IV constructs Thus, in lane 3 of Fig 2C, the upper 28 kDa band of the small subunit is well separated from the lower, more abun-dant His6-tagged calpain 1 domain IV Although the presence of AFP-tagged small subunit in the affinity-purified His6-tagged calpain 1 domain IV shows that the two different PEF domains form heterodimers, the relative staining of these two bands suggests that calpain 1 domain IV is present in excess

Fig 1 Three possible scenarios derived from coexpression of recombinant PEF fusion proteins (A) Homodimer model of His6-tagged PEF domain (B) Homodimer model of type III AFP-tagged PEF domain (calpain small subunit domain VI) (C) Heterodimer model of fusion protein containing type III AFP-tagged (blue) small subunit (cyan) forming a dimer with His6-tagged (light brown) PEF domain (orange) Rat small subunit (1AJ5) was used for modeling All structures were drawn with PYMOL [51].

Table 1 Screening results of domain IV constructs from calpains 1, 3, 8, 9, 11, 12, and 13 Column 1: calpains used for screening Col-umn 2: number of constructs designed and cloned ColCol-umn 3: number of constructs expressed ColCol-umn 4: yields of constructs when expressed alone in the absence of human small subunit Column 5: results from coexpression of the domain IV construct with human small subunit Column 6: results obtained by biophysical analysis of these constructs NA, data not available; +++, very high expression; ++, high expression; +, low expression.

++

++

a Predominant form found as homodimer.

Calpain 3 domain IV is a homodimer

Calpain 3 domain IV is suggested to favor

homodi-merization, even though small subunit-containing

calpains are produced in muscle cells Earlier studies

showed that recombinant calpain 3 domain IV, when

expressed in isolation, formed a homodimer [30] In

further support of this argument, we show below that

His6-tagged recombinant calpain 3 domain IV

coex-pressed with type III AFP-tagged human small subunit

(28 kDa) exclusively forms a homodimer Upon

purifi-cation by Ni2+–nitrilotriacetate–agarose

chromato-graphy, the 28 kDa subunit was not detected in the

imidazole-eluted fraction along with calpain 3 domain

IV (Fig 3A, lane 4) The 28 kDa subunit was present

in the fractions that did not bind to the Ni2+

–nitrilo-triacetate–agarose column (Fig 3A, lane 2) Indeed, it

was the most abundant protein in the flow-through

fraction from that column

Calpain 9 domain IV forms a heterodimer with

the small subunit

The recombinant calpain 9 domain IV construct has

200 amino acids, including its His6 N-terminal tag It

has a theoretical pI of 5.71 and a calculated molecular

mass of 23 130 Da The amino acid sequence is 43% identical with domain IV of calpain 1, and 40% identi-cal with the small subunit (28 kDa) When identi-calpain 9 domain IV was coexpressed with the 28 kDa small subunit fusion protein, it formed a heterodimer Both subunits were detected in the imidazole-eluted fraction (Fig 3B, lane 3) Their stoichiometry was close to

1 : 1 To confirm the identity of the two subunits, the gel was immunoblotted and probed with the two anti-bodies used in Fig 2B The antibody against AFP detected the upper band as a 28 kDa AFP-tagged small subunit (Fig 3C, lane 1) Similarly, the antibody against His-tag reacted with the N-terminally His6-tagged calpain 9 domain IV (Fig 3C, lane 2)

In the converse approach using ice affinity purifica-tion, His6-tagged calpain 9 domain IV was included in the ice because of its heterodimerization with the AFP-tagged small subunit (Fig 4, lane 2) Here, the amount

of the His6-tagged calpain 9 domain IV in the ice frac-tion was slightly lower than would be predicted from the expected 1 : 1 stoichiometry with the small subunit

as seen in the liquid fraction (Fig 4, lane 3) This seems to be due to a small amount of subunit dissocia-tion that occurs as the ice grows over and pushes past the adsorbed AFP-tagged subunit The shearing forces

of the ice are apparently sufficient to disrupt

Fig 2 SDS ⁄ PAGE and immunoblot analysis of differentially tagged calpain 1 and 2 heterodimers (A) Lane 1: molecular mass standards indi-cated at the side of the gel Lanes 2, 3 and 4: flow-through, wash and eluate samples, respectively, from the Ni 2+ –nitrilotriacetate–agarose column chromatography of 80 kDa subunit (C105S-m-80 kDa) (triangle) coexpressed with 28 kDa AFP-tagged small subunit (dot) (B) Lanes 1 and 2: immunoblots of lane 4 from (A) probed with antibody against AFP and antibody against His-tag, respectively (C) Lanes 1, 2 and 3: flow-through, wash and eluate samples from the Ni 2+ –nitrilotriacetate–agarosecolumn chromatography of calpain 1 domain IV (C1DIV) (square) coexpressed with 28 kDa AFP-tagged small subunit (dot) Both coexpressed constructs are predominantly detected in fractions eluted with imidazole.

nary structure in a portion of the dimers, but do not

break covalent bonds between the AFP moiety and a

fusion partner [36] A similar partial dissociation of

subunits was seen during ice affinity purification of full

length l-calpain heterodimerized to the AFP-tagged

subunit (results not shown) The control experiment in

this series showed that His6-tagged calpain 9 domain

IV, when expressed alone, was not included in the ice

but remained in the liquid fraction (Fig 4, lanes 4 and

5, respectively)

Calpain 13 domain IV

The recombinant calpain 13 domain IV construct

con-tains 174 amino acids, including the His6 N-terminal

tag It has a theoretical pI of 6.75 and a calculated

molecular mass of 19 901 Da Unlike other calpain

PEF domains, it has low sequence identity with

domai-n IV of calpaidomai-n 1 (28%) adomai-nd the small subudomai-nit (29%)

When the recombinant calpain 13 domain IV construct

was coexpressed with type III AFP-tagged human

small subunit (28 kDa), calpain 13 domain IV was

pre-dominantly seen in the eluant The 28 kDa small

sub-unit was mainly observed in the flow-through,

although a faint band was seen in the wash and eluant

(Fig 5) On the basis of these SDS⁄ PAGE results, a small amount of heterodimer is produced but cal-pain 13 domain IV is predominantly a homodimer

Discussion

The PEF domain was first described in calpain [37– 39], and has since been found in other proteins such as ALG-2, grancalcin, sorcin and peflin [40,41] It is char-acterized by having a fifth EF-hand available to pair with that of another PEF domain to form heterodi-mers or homodiheterodi-mers More than half of the human calpain isoforms (1, 2, 3, 8, 9, 11, 12 and 13) have a PEF domain Of these, the ubiquitous well-studied cal-pains 1 and 2 are known to form heterodimers with the small subunit PEF domains However, previous investigations on calpain 3 suggest that PEF domain-containing calpain isoforms need not necessarily form

a heterodimer, like calpains 1 and 2 In this study, we set out to determine what kind of dimers the different calpain isoforms make

Modeling studies using shape complementarity as a tool to measure the likelihood of forming a hetero-dimer or homohetero-dimer were performed using calpain 2, the previously generated model of calpain 3 domain IV

Fig 3 SDS ⁄ PAGE and immunoblot analysis of calpain 3 domain IV (C3DIV) and calpain 9 domain IV (C9DIV) samples coexpressed with small subunit (A) Lane 1: molecular mass standards indicated at the side of the gel Lanes 2, 3 and 4: flow-through, wash and eluate sam-ples, respectively, from the Ni2+–nitrilotriacetate–agarose column chromatography of His-tagged (C3DIV) (triangle) coexpressed with 28 kDa AFP-tagged small subunit (dot) Only the C3DIV domain is detected in the eluant (B) Lanes 1–3: flow-through, eluate and wash samples from the Ni 2+ –nitrilotriacetate–agarose column of His-tagged C9DIV (square) coexpressed with 28 kDa AFP-tagged small subunit (dot) Both the human small subunit and C9DIV are present in the eluant (C) Lanes 1 and 2: immunoblots of lane 3 from (B) probed with antibody against AFP and antibody against His-tag, respectively.

[30], and the small subunit structures as a guide In

addition, models were generated for artificial structures

of the calpain 3 domain IV–small subunit heterodimer

and of the calpain 2 domain IV homodimer Shape

complementarity values differed only slightly between

the different dimers In order of best to worst, the

complementarity values were calpain 3 domain IV

homodimer (0.751), small subunit homodimer (0.751),

calpain 3 domain IV–small subunit heterodimer

(0.734), calpain 2 domain IV–small subunit

hetero-dimer (0.734) and calpain 2 domain IV homohetero-dimer

(0.715) These values are not significantly different

from each other, and therefore do not appear to

pro-vide a method for distinguishing correct from incorrect

dimers Comparison of the buried surface areas for the

various complexes also shows little variation, with the

calpain 2 domain IV homodimer displaying the

small-est surface area (average value of 1182 A˚2) as

com-pared to the others (average values ranging from 1311

to 1391 A˚2) As tight packing of residues involved in the dimerization interfaces might not be the only factor influencing dimer formation, we used experimen-tation to distinguish which isoforms form heterodimers

or homodimers

The recombinant small subunit domain VI has a molecular mass of 21 264 Da, and forms a homo-dimer when expressed alone [42] Its molecular mass

is close in value to those of isolated calpain PEF domains (domain IV), making it hard to distinguish whether they formed homodimers or heterodimers when coexpressed In order to overcome this uncer-tainty, we devised a differential tag approach whereby all the calpain PEF domains contain a His6 N-termi-nal tag and the small subunit has an N-termiN-termi-nal type III AFP tag (7 kDa), allowing us to distinguish these two domains by size Like the rat small subunit, the recombinant 28 kDa human small subunit fusion protein formed a homodimer when expressed alone (results not shown)

Fig 4 Ice affinity purification of type III AFP-tagged small subunit

and calpain 9 domain IV (C9DIV) Lane 1: molecular mass standards

indicated at the side of the gel Lanes 2 and 3: equal volumes of

the ice and liquid fractions obtained from the distribution of

coex-pressed 28 kDa AFP-tagged small subunit (dot) with His-tagged

C9DIV (square) Lanes 4 and 5: equal volumes of the ice and liquid

fractions obtained from the distribution of His-tagged C9DIV

(square) in the absence of 28 kDa AFP-tagged small subunit.

Fig 5 SDS ⁄ PAGE analysis of calpain 13 domain IV (C13DIV) sam-ples from the Ni 2+ –nitrilotriacetate–agarose column Lane 1: molec-ular mass standards indicated at the side of the gel Lanes 2, 3 and 4: flow-through, wash and eluate fractions from the column, respectively The 28 kDa subunit and C13DIV proteins are indicated

by dot and square symbols, respectively.

Calpain 1, 3, 9 and 13 PEF domains were

success-fully cloned and coexpressed as soluble recombinant

products However, numerous attempts to express

cal-pain 8, 11 and 12 PEF domain constructs in E coli

were unsuccessful, and thus the dimerization potential

of these PEF domains could not be analyzed As the

wild-type calpains 1 and 2 are both known to form

heterodimers, we used calpain 2 large subunit and

cal-pain 1 domain IV as controls in our experiments Even

in the absence of its adjacent domains, calpain 1

domain IV formed a heterodimer with the small

sub-unit, rather than a homodimer It should be noted that

this construct lacks the N-terminal anchor peptide,

which, on the basis of the structure of calpain 2

[19,21], should make additional heterodimerization

contacts between the large and small subunits

Recombinant calpain 3 domain IV was previously

shown to form a homodimer when expressed alone [30]

In this study, it was coexpressed with small subunit

(28 kDa) but still formed a homodimer, further

support-ing the argument that calpain 3 is a natural homodimer

Calpain 9 has been previously suggested to form a

hete-rodimer when coexpressed with small subunit in the

baculovirus expression system [43] Coexpression of

recombinant proteins calpain 9 domain IV and small

subunit fusion product (28 kDa) led these proteins to

associate as a heterodimer, in agreement with these

pre-vious studies As with calpain 1, the absence of the other

domains in the large subunit did not alter the propensity

of calpain 9 domain IV to heterodimerize When

expressed alone, calpain 9 domain IV formed an

oligo-mer, unlike other PEF domains (results not shown)

Calpain 13 is a tissue-specific calpain expressed

predom-inantly in testis and lung Its physiological role is not

well understood, and its dimerization state is unknown

[8] Calpain 13 domain IV appeared as a predominant

homodimer when coexpressed with small subunit fusion

protein (28 kDa), although there were small amounts of

heterodimer present in the eluate from the Ni2+

–nitri-lotriacetate–agarose column

Most of the PEF domains in calpain isoforms share

a high degree of sequence identity; however, it is not

clear why they prefer one form of dimerization over

the other Further analysis of these constructs by

determining their structure through crystallography

may help us to gain more insight into the preference

for homodimerization versus heterodimerization

Meanwhile, on the basis of these results, we predict

that calpain 9 can be bound and silenced by

calpasta-tin Silencing of calpains 3 and 13 would require the

simultaneous binding of two calpastatin inhibitory

domains Although this is a theoretical possibility,

especially as calpastatin has four inhibitory domains

and is an intrinsically unstructured protein, the absence of a small subunit in these two calpains would deprive calpastatin of one of its three calpain-binding sequences The loss of this binding site would signifi-cantly weaken the overall binding interaction

Experimental procedures

High-throughput cloning

The cDNA fragments encoding the domain IV regions of cal-pains 1, 9, 11, 12 and 13 were obtained by PCR amplification

of full-length cDNA templates of human calpains 1, 9, 11, 12 and 13 obtained from the Mammalian Gene Collection, using Expand high-fidelity DNA polymerase (Roche, India-napolis, IN, USA) Human calpain 8 domain IV was obtained by PCR amplification of reverse transcripts (RT-PCR) of total RNA from human stomach (Stratagene,

La Jolla, CA, USA), using an RT-Thermoscript kit (Invitro-gen, Carlsbad, CA, USA) and Expand high-fidelity DNA polymerase Human calpain 3 domain IV was obtained as previously described [30] Multiple constructs were designed for each of these domains The amplified fragments encoding domain IV regions of calpains 1, 8, 9, 11, 12 and 13 were inserted using the infusion ligation independent cloning system (BD Biosciences, Mountainview, CA, USA) into a modified pET28-LIC expression vector (EMD-Novagen, Gibbstown, NJ, USA) using a 96-well format high-through-put approach [44], downstream of the nucleotide sequence encoding MGSSHHHHHHSSGLVPRLGS This 20 amino acid sequence contains a hexahistidine tag (His6-tag) and a thrombin cleavage site

Type III AFP-tagged human small subunit

The cDNA fragment encoding domain VI of the human small subunit was obtained by PCR amplification of reverse transcripts (RT-PCR) of total RNA from human stomach (Stratagene, La Jolla, CA, USA), using an RT-Thermo-script kit (Invitrogen, Carlsbad, CA, USA) and Expand high-fidelity DNA polymerase (Roche, Indianapolis, IN, USA) The amplified product was cloned into the modified pET vector (pAC-pET) as previously described [45] The type III AFP sequence was previously prepared by gene synthesis [46] It was cloned into the pAC-pET vector 5¢ of the truncated 21 kDa subunit sequence At the protein level, the two domains are joined by a linker of three alanine residues

Protein expression and purification by

Ni2+–nitrilotriacetate–agarose

The pET28-LIC vectors encoding the domain IV regions were transformed along with the pAC-pET plasmid

containing the small subunit fusion construct into E coli

BL21(DE3) cells (Novagen) by electroporation The

trans-formed cells were grown in 1 L of LB medium under

kana-mycin and ampicillin selection The cells were grown to a

D600 nmof 0.8–1.0 at 37C Protein expression was induced

at 16C using 0.4 mm isopropyl thio-b-d-galactoside for

16 h The cells were collected by centrifugation, resuspended

in lysis buffer [25 mm Tris⁄ HCl, pH 7.6, 5 mm EDTA, 5%

(v⁄ v) glycerol, 10 mm 2-mercaptoethanol, and 0.1 mm

phen-ylmethanesulfonyl fluoride], and lysed by sonication The

resulting lysate was clarified by centrifugation at 27 000 g

for 45 min The supernatant obtained was incubated with

5 mL Ni2+–nitrilotriacetate–agarose resin (Qiagen,

Chats-worth, CA, USA) for 30 min at 4C with constant stirring

The Ni2+–nitrilotriacetate–agarose resin was later

trans-ferred to a column and washed with N-buffer (50 mm

Tris⁄ HCl, pH 7.6, 100 mm NaCl, 5 mm imidazole, and

0.01% sodium azide) His6-tagged proteins were eluted with

the lysis buffer containing 250 mm imidazole The samples

collected were later analyzed by SDS⁄ PAGE The inactive

recombinant rat calpain 2 large subunit (C105S-m-80 kDa)

was also coexpressed with the AFP-tagged small subunit

and purified as described previously [45]

Ice affinity purification

Ice affinity purification [36] was explored as a way of

isolat-ing and identifyisolat-ing products containisolat-ing the type III AFP

fusion In this method, the AFP fusion protein was

adsorbed from solution (50 mL) into growing

polycrystal-line ice frozen onto a cooled brass cold finger The growth

of the ice was controlled by circulating cold ethylene glycol

solution through the hollow cold finger After a thin layer

of ice ( 1 mm) had initially formed on the cold finger, it

was immersed in the AFP-containing solution prechilled to

1C in an insulated beaker The solution was gently mixed

using a stir bar, and the temperature of the cold finger was

gradually reduced at a linear rate ()0.5 to )2.5 C over

36 h), using a temperature-programmable water bath

(Ne-slab), until approximately half to two-thirds of the volume

was incorporated into the ice hemisphere The ice

hemi-sphere was then removed from the liquid and allowed to

melt for 10 min to remove any protein that was

nonspe-cifically bound to the surface of the ice The ice hemisphere

was melted to release the AFP Samples (2 and 5 lL) from

both melted ice (ice fraction) and leftover liquid (liquid

fraction) were analyzed by SDS⁄ PAGE [47]

Modeling studies

Shape complementarity of various dimer structures and

models was calculated using the program sc from the ccp4

program suite [48] Crystal structures of the rat small

sub-unit homodimer (Protein Data Bank code: 1dvi) and of the

human calpain 2 heterodimer (Protein Data Bank code:

1kfu) were used as references Homology models of the cal-pain 3 domain IV homodimer, the heterodimer of calcal-pain 3 domain IV with the small subunit and of a calpain 2 domain IV homodimer were generated using the program modeller9v3 [49] The best of 100 models were then used

in an energy minimization and molecular dynamics proto-col using the program gromacs 3.3 [50] The protein was solvated, energy minimized using the steepest descents pro-tocol, and subjected to position-restrained molecular dynamics to relax the solvent This was followed by a 2 ns molecular dynamics simulation Structures were extracted from the trajectory every 20 ps, and the surface comple-mentarity at the dimer interface was calculated with the program sc from the ccp4 program suite [48] The average

scvalue from these 100 structures is reported For compar-ison, the same molecular dynamics protocol was used on the crystal structures of the rat small subunit homodimer (Protein Data Bank code: 1dvi) and of the human calpain 2 heterodimer (Protein Data Bank code: 1kfu)

Immunoblotting

Immunoblotting was performed using 10% Tris⁄ Tricine SDS⁄ PAGE gels transferred onto poly(vinylidene difluoride) membranes Polyclonal antibodies against the His-tag and against type III AFP were raised in rabbits The secondary antibody was anti-(rabbit IgG) conjugated to horseradish peroxidase (Promega, Madison, WI, USA), which was detected by ECL (Perkin-Elmer, Fremont, CA, USA)

Acknowledgements

This research was funded by a grant to P L Davies from the Canadian Institutes for Health Research

P L Davies holds a Canada Research Chair in Pro-tein Engineering The Structural Genomics Consortium

is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Insti-tute, GlaxoSmithKline, Karolinska InstiInsti-tute, the Knut and Alice Wallenberg Foundation, the Ontario Inno-vation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Sys-tems, the Swedish Foundation for Strategic Research, and the Wellcome Trust

References

1 Goll DE, Thompson VF, Li H, Wei W & Cong J (2003) The calpain system Physiol Rev 83, 731–801

2 Pinter M, Stierandova A & Friedrich P (1992) Purifica-tion and characterizaPurifica-tion of a Ca(2+)-activated thiol

protease from Drosophila melanogaster Biochemistry 31,

8201–8206

3 Barnes TM & Hodgkin J (1996) The tra-3 sex

determi-nation gene of Caenorhabditis elegans encodes a

mem-ber of the calpain regulatory protease family EMBO J

15, 4477–4484

4 Huang X, Czerwinski E & Mellgren RL (2003)

Purifica-tion and properties of the Dictyostelium calpain-like

protein, Cpl Biochemistry 42, 1789–1795

5 Margis R & Margis-Pinheiro M (2003) Phytocalpains:

orthologous calcium-dependent cysteine proteinases

Trends Plant Sci 8, 58–62

6 Dear N, Matena K, Vingron M & Boehm T (1997)

A new subfamily of vertebrate calpains lacking a

calmodulin-like domain: implications for calpain

regulation and evolution Genomics 45, 175–184

7 Ersfeld K, Barraclough H & Gull K (2005)

Evolution-ary relationships and protein domain architecture in an

expanded calpain superfamily in kinetoplastid parasites

J Mol Evol 61, 742–757

8 Farkas A, Tompa P & Friedrich P (2003) Revisiting

ubiquity and tissue specificity of human calpains Biol

Chem 384, 945–949

9 Glading A, Lauffenburger DA & Wells A (2002)

Cutting to the chase: calpain proteases in cell motility

Trends Cell Biol 12, 46–54

10 Neumar RW, Xu YA, Gada H, Guttmann RP & Siman

R (2003) Cross-talk between calpain and caspase

prote-olytic systems during neuronal apoptosis J Biol Chem

278, 14162–14167

11 Janossy J, Ubezio P, Apati A, Magocsi M, Tompa P &

Friedrich P (2004) Calpain as a multi-site regulator of

cell cycle Biochem Pharmacol 67, 1513–1521

12 Suzuki T, Okumura-Noji K, Ogura A, Tanaka R,

Nakamura K & Kudo Y (1992) Calpain may produce

a Ca(2+)-independent form of kinase C in long-term

potentiation Biochem Biophys Res Commun 189, 1515–

1520

13 Cox NJ, Hayes MG, Roe CA, Tsuchiya T & Bell GI

(2004) Linkage of calpain 10 to type 2 diabetes:

the biological rationale Diabetes 53(Suppl 1),

S19–S25

14 Kuwako K, Nishimura I, Uetsuki T, Saido TC &

Yoshikawa K (2002) Activation of calpain in cultured

neurons overexpressing Alzheimer amyloid precursor

protein Brain Res Mol Brain Res 107, 166–175

15 Enns D, Karmazyn M, Mair J, Lercher A, Kountchev J

& Belcastro A (2002) Calpain, calpastatin activities and

ratios during myocardial ischemia–reperfusion Mol Cell

Biochem 241, 29–35

16 Richard I, Broux O, Allamand V, Fougerousse F,

Chiannilkulchai N, Bourg N, Brenguier L, Devaud C,

Pasturaud P, Roudaut C et al (1995) Mutations in the

proteolytic enzyme calpain 3 cause limb-girdle muscular

dystrophy type 2A Cell 81, 27–40

17 Yoshikawa Y, Mukai H, Hino F, Asada K & Kato I (2000) Isolation of two novel genes, down-regulated in gastric cancer Jpn J Cancer Res 91, 459–463

18 Wang KK & Yuen PW (1994) Calpain inhibition: an overview of its therapeutic potential Trends Pharmacol Sci 15, 412–419

19 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

20 Hosfield CM, Ye Q, Arthur JS, Hegadorn C, Croall

DE, Elce JS & Jia Z (1999) Crystallization and X-ray crystallographic analysis of m-calpain, a Ca2+-depen-dent protease Acta Crystallogr D Biol Crystallogr 55, 1484–1486

21 Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K, Irie A, Sorimachi H, Bourenkow 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

22 Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z

& Davies PL (2002) A Ca(2+) switch aligns the active site of calpain Cell 108, 649–660

23 Reverter D, Strobl S, Fernandez-Catalan C, Sorimachi

H, Suzuki K & Bode W (2001) Structural basis for possible calcium-induced activation mechanisms of calpains Biol Chem 382, 753–766

24 Tompa P, Emori Y, Sorimachi H, Suzuki K & Fried-rich P (2001) Domain III of calpain is a Ca2+-regu-lated phospholipid-binding domain Biochem Biophys Res Commun 280, 1333–1339

25 Suzuki K, Hata S, Kawabata Y & Sorimachi H (2004) Structure, activation, and biology of calpain Diabetes 53(Suppl 1), S12–S18

26 Waghray A, Wang DS, McKinsey D, Hayes RL & Wang KK (2004) Molecular cloning and characteriza-tion of rat and human calpain-5 Biochem Biophys Res Commun 324, 46–51

27 Tonami K, Kurihara Y, Aburatani H, Uchijima Y, Asano T & Kurihara H (2007) Calpain 6 is involved in microtubule stabilization and cytoskeletal organization Mol Cell Biol 27, 2548–2561

28 Yorikawa C, Takaya E, Osako Y, Tanaka R, Terasawa

Y, Hamakubo T, Mochizuki Y, Iwanari H, Kodama T, Maeda T et al (2008) Human calpain 7⁄ PalBH associ-ates with a subset of ESCRT-III-related proteins in its N-terminal region and partly localizes to endocytic membrane compartments J Biochem 143, 731–745

29 Dong B & Liu R (2008) Characterization of endoge-nous and recombinant human calpain-10 Biochimie 90, 1362–1371

30 Ravulapalli R, Diaz BG, Campbell RL & Davies PL (2005) Homodimerization of calpain 3 penta-EF-hand domain Biochem J 388, 585–591

31 Kinbara K, Ishiura S, Tomioka S, Sorimachi H, Jeong

SY, Amano S, Kawasaki H, Kolmerer B, Kimura S,

Labeit S et al (1998) Purification of native p94, a

mus-cle-specific calpain, and characterization of its autolysis

Biochem J 335, 589–596

32 Wendt A, Thompson VF & Goll DE (2004) Interaction

of calpastatin with calpain: a review Biol Chem 385,

465–472

33 Todd B, Moore D, Deivanayagam CC, Lin GD,

Chattopadhyay D, Maki M, Wang KK & Narayana

SV (2003) A structural model for the inhibition of

calpain by calpastatin: crystal structures of the native

domain VI of calpain and its complexes with calpastatin

peptide and a small molecule inhibitor J Mol Biol 328,

131–146

34 Hanna RA, Campbell RL & Davies PL (2008)

Cal-cium-bound structure of calpain and its mechanism of

inhibition by calpastatin Nature 456, 409–412

35 Jia Z, DeLuca CI, Chao H & Davies PL (1996)

Struc-tural basis for the binding of a globular antifreeze

protein to ice Nature 384, 285–288

36 Kuiper MJ, Lankin C, Gauthier SY, Walker VK &

Davies PL (2003) Purification of antifreeze proteins by

adsorption to ice Biochem Biophys Res Commun 300,

645–648

37 Lin GD, Chattopadhyay D, Maki M, Wang KK,

Carson M, Jin L, Yuen PW, Takano E, Hatanaka M,

DeLucas LJ et al (1997) Crystal structure of calcium

bound domain VI of calpain at 1.9 A resolution and its

role in enzyme assembly, regulation, and inhibitor

bind-ing Nat Struct Biol 4, 539–547

38 Blanchard H, Grochulski P, Li Y, Arthur JS, Davies

PL, Elce JS & Cygler M (1997) Structure of a calpain

Ca(2+)-binding domain reveals a novel EF-hand and

Ca(2+)-induced conformational changes Nat Struct

Biol 4, 532–538

39 Kretsinger RH (1997) EF-hands embrace Nat Struct

Biol 4, 514–516

40 Maki M, Kitaura Y, Satoh H, Ohkouchi S & Shibata

H (2002) Structures, functions and molecular evolution

of the penta-EF-hand Ca2+-binding proteins Biochim

Biophys Acta 1600, 51–60

41 Jia J, Han Q, Borregaard N, Lollike K & Cygler M (2000) Crystal structure of human grancalcin, a member

of the penta-EF-hand protein family J Mol Biol 300, 1271–1281

42 Blanchard H, Li Y, Cygler M, Kay CM, Simon J, Arthur C, Davies PL & Elce JS (1996) Ca(2+)-binding domain VI of rat calpain is a homodimer in solution: hydrodynamic, crystallization and preliminary X-ray diffraction studies Protein Sci 5, 535–537

43 Lee HJ, Tomioka S, Kinbara K, Masumoto H, Jeong

SY, Sorimachi H, Ishiura S & Suzuki K (1999) Charac-terization of a human digestive tract-specific calpain, nCL-4, expressed in the baculovirus system Arch Biochem Biophys 362, 22–31

44 Benoit RM, Wilhelm RN, Scherer-Becker D &

Ostermeier C (2006) An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids Protein Expr Purif 45, 66–71

45 Elce JS, Hegadorn C, Gauthier S, Vince JW & Davies

PL (1995) Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography Protein Eng 8, 843–848

46 Chao H, Davies PL, Sykes BD & Sonnichsen FD (1993) Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein Protein Sci 2, 1411–1428

47 Marshall CB, Tomczak MM, Gauthier SY, Kuiper MJ, Lankin C, Walker VK & Davies PL (2004) Partitioning

of fish and insect antifreeze proteins into ice suggests they bind with comparable affinity Biochemistry 43, 148–154

48 Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D Biol Crystallogr 50, 760–763

49 Sali A & Blundell TL (1993) Comparative protein mod-elling by satisfaction of spatial restraints J Mol Biol

234, 779–815

50 Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE & Berendsen HJ (2005) GROMACS: fast, flexible, and free J Comput Chem 26, 1701–1718

51 DeLano WL (2003) The PyMOL Molecular Graphics System DeLano Scientific, San Carlos, CA