PDBsum entry 3df0

Hydrolase

PDB id: 3df0

Contents

Protein chains

676 a.a.

174 a.a.

56 a.a.

Metals

_CA ×10

References listed in PDB file

Key reference

Title

Concerted multi-Pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.

Authors

T.Moldoveanu, K.Gehring, D.R.Green.

Ref.

Nature, 2008, 456, 404-408. [DOI no: 10.1038/nature07353]

PubMed id

19020622

Abstract

The Ca(2+)-dependent cysteine proteases, calpains, regulate cell migration, cell death, insulin secretion, synaptic function and muscle homeostasis. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown. Here we report the 3.0 A structure of Ca(2+)-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca(2+), illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca(2+) binding. Fully activated calpain binds ten Ca(2+) atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.

Figure 1.
Figure 1: Complex between Ca^2+-bound m-calpain and calpastatin. a, Overall structure shows regions A, B and C of calpastatin bound to DIV, DI–III and DVI of calpain, respectively. The intervening sequences of calpastatin are devoid of electron density (red dots). The central part of the inhibitory region B forms the occluding loop at the active site. The active site in the protease core DI–II is stabilized by DIII. Calpain heterodimerization is largely defined at the DIV–DVI interface. Alternate conformations at the interface between the DI–III core and the DIV–DVI heterodimer (black dots) are possible^30. b, ^15N,^1H-HSQC (heteronuclear single quantum correlation) spectrum of the complex between ^13C,^15N-labelled calpastatin (residues 128–226, Supplementary Fig. 2b) and unlabelled calpain identified flexible/disordered residues of calpastatin. Connected sample strips from the HNCA NMR experiment are inset.

Figure 4.
Figure 4: Calpain–calpastatin proteolytic system. A schematic diagram illustrating the Ca^2+-induced activation of calpain and its inhibition by calpastatin. DIII has a fundamental role in relaying the Ca^2+-induced structural changes (red dotted arrows) from the peripheral domains to the catalytically competent yet labile protease core. Concerted binding of the intrinsically unstructured protein (IUP) calpastatin to peripheral domains and the active site of calpain results in low-nanomolar inhibition.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 456, 404-408) copyright 2008.

Secondary reference #1

Title

A ca(2+) switch aligns the active site of calpain.

Authors

T.Moldoveanu, C.M.Hosfield, D.Lim, J.S.Elce, Z.Jia, P.L.Davies.

Ref.

Cell, 2002, 108, 649-660. [DOI no: 10.1016/S0092-8674(02)00659-1]

PubMed id

11893336

Figure 4.
Figure 4. Ca^2+-Induced Conformational Changes in the Active Site Region of Calpain and Proposed Activation Mechanism(A) DI and II of inactive human m calpain (Strobl et al., 2000). The ribbon presentation is colored pink, with the side chains of three critical residues (equivalent to μ R104, W298, and E333) colored orange. DI and II are rotated 5° relative to each other, and C105 and H262 are 10.5 Å apart.(B) DI of μI-II (blue) was overlapped onto DI from m calpain (pink) using the program Align (Cohen, 1997). The gold sphere indicates the Ca^2+ ion.(C) Exposure of the Ca^2+ binding site in DII (cyan) resulting from attraction of the E333 side chain by R104 from DI.(D) R104-E333 double salt bridge stereo view.(E) Overlap of DII from μI-II (cyan) onto DII from m calpain (pink) showing the loops that coordinate the second Ca^2+. Note: a discrepancy in the m calpain structure around G295 results in a discontinuity in that peptide loop.(F) Stereo view of the hydrophobic pocket formed by Ca^2+ binding to DII.(G) Ca^2+ bound μI-II, showing the arrangement of the Ca^2+ ions relative to the active site cleft. This is a 90° rotation of the view in Figure 2A.

Figure 6.
Figure 6. Regulation of Heterodimeric Calpain by Ca^2+A generic model for Ca^2+ bound calpain was constructed by substituting the Ca^2+ bound structure of DI-II into the human m calpain heterodimer (Strobl et al., 2000) while overlapping DIV and VI with the Ca^2+ bound DVI heterodimer structure (Blanchard et al., 1997). DII was positioned to optimize DIII interactions. The anchor peptide (red helix) was placed in the Ca^2+-free conformation where it interacts with DVI (gray). Two consecutive yet cooperative levels of Ca^2+ regulation are proposed, both acting on a different segment of the circularized structure. Stage 1 includes anchor release (Nakagawa et al., 2001), shown by the red dotted arrow. As well, under certain conditions small subunit dissociation (Pal et al., 2001) and the potential binding of Ca^2+ to DIII (Hosfield et al. 2001 and Tompa et al. 2001) may help free the protease region from constraints. Stage 2 is active site assembly (black dotted arrows) as seen in μI-II. It follows the onset of stage 1 but may also influence it if the tendency to realign the active site pulls against the restraint. Ca^2+ ions are colored gold (seen in X-ray structures) or red (postulated or confirmed by mutagenesis; Dutt et al., 2000). Transparent spheres in DIV and VI are Ca^2+ at EF-4 sites that are likely filled only at high CaCl[2] (>20 mM) concentrations. Calpain association with membranes (double gray lines) may also contribute to activation (as reviewed in Nakagawa et al., 2001).

The above figures are reproduced from the cited reference with permission from Cell Press

Secondary reference #2

Title

Calpain silencing by a reversible intrinsic mechanism.

Authors

T.Moldoveanu, C.M.Hosfield, D.Lim, Z.Jia, P.L.Davies.

Ref.

Nat Struct Biol, 2003, 10, 371-378. [DOI no: 10.1038/nsb917]

PubMed id

12665854

Figure 2.
Figure 2. Structural comparison of the calcium-bound - and m-minicalpains. a, Overlap of mI-II onto I-II using ALIGN30. Gold spheres indicate Ca^2+ ions. The protease core of mI-II (red/pink) is superimposed on I-II (transparent blue/cyan). -strands and -helices are numbered sequentially from the N terminus (N) to the C terminus (C). The side chain atoms of the catalytic triad residues are colored in red (oxygen), dark blue (nitrogen) and gray (carbon), and the bonds have the domain color. b, Stereo view of the mI-II region showing oxygen coordinations to DI Ca^2+ (eight) and DII Ca^2+ (pentagonal bipyramid), as well as the double salt bridge between Arg94 and Glu323. Side chains are structured exactly as predicted from the Ca^2+-bound I-II structure.

Figure 4.
Figure 4. Hydrophobic core collapses in mI-II but not in I-II. The hydrophobic core stabilized by helix 7 is shown for the two minicalpains. The color scheme is the same as that in Fig. 2, with Phe207/217 and Trp106/116 shown in green and pink, respectively. a, Stereo view of mI-II core. The helix-breaking Gly 203 is orange. b, Stereo view of the I-II core. The peptide 207 -213 (yellow ribbon) is structured in I-II because Ala 213 (orange) stabilizes helix 7.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd

Secondary reference #3

Title

Crystal structure of calpain reveals the structural basis for ca(2+)-Dependent protease activity and a novel mode of enzyme activation.

Authors

C.M.Hosfield, J.S.Elce, P.L.Davies, Z.Jia.

Ref.

EMBO J, 1999, 18, 6880-6889. [DOI no: 10.1093/emboj/18.24.6880]

PubMed id

10601010

Figure 3.
Figure 3 Calpain has a unique N-terminal anchor. (A) The helical anchor (residues 2 -16 are shown) makes contacts only with D-VI (colors as in Figure 1). (B) View down the helical axis highlights interactions between the residues in the anchor (magenta type) and D-VI (black type), represented as an electrostatic GRASP surface (Nicholls et al., 1991) (red, acidic; blue, basic). (C) Side view of (B) illustrates the depth of the hydrophobic pocket in D-VI, which interacts with hydrophobic residues Ala2, Gly3, Ile4, Ala5, Leu8 and Ala9 of the anchor. This anchor inhibits active site assembly by associating with the regulatory subunit, thus restricting flexibility of protease D-I. The anchor also acts as a co-chaperone in concert with D-VI, ensuring proper folding of the catalytic subunit. (B) and (C) were created with the program GRASP (Nicholls et al., 1991).

Figure 4.
Figure 4 Domain III shares similar characteristics with a C[2] domain. A typical C[2] domain exists as an anti-parallel -sandwich with several acidic residues at one end that form a binding cradle for Ca^2+. The first C[2] domain from synaptotagmin (cyan, PDB accession code 1RSY) (Sutton et al., 1995) and D-III (green) have approximately the same overall dimensions, though slightly differing topologies. Numerous acidic residues (red) result in a highly negative potential, which is partially stabilized by adjacent basic residues (blue).

The above figures are reproduced from the cited reference which is an Open Access publication published by Macmillan Publishers Ltd

Secondary reference #4

Title

The crystal structure of calcium-Free human m-Calpain suggests an electrostatic switch mechanism for activation by calcium.

Authors

S.Strobl, C.Fernandez-Catalan, M.Braun, R.Huber, H.Masumoto, K.Nakagawa, A.Irie, H.Sorimachi, G.Bourenkow, H.Bartunik, K.Suzuki, W.Bode.

Ref.

Proc Natl Acad Sci U S A, 2000, 97, 588-592. [DOI no: 10.1073/pnas.97.2.588]

PubMed id

10639123

Figure 1.
Fig. 1. Ribbon structure of human m-calpain in the absence of calcium, shown in reference orientation. The 80-kDa L-chain starts in the molecular center (green, dI), folds into the surface of the dIIa subdomain (gold, I II linker), forms the papain-like left-side part of the catalytic domain dII (gold, dIIa) and the right-side barrel-like subdomain dIIb (red), descends through the open II III loop (red), builds domain dIII (blue), runs down (magenta, III IV), and forms the right-side calmodulin-like domain dIV (yellow). The 30-kDa S-chain becomes visible from Thr95S onwards (magenta, dV) before forming the left-side calmodulin domain dVI (orange). The catalytic residues Cys105L, His262L, and Asn286L together with Trp106L, Pro287L, and Trp288L (top) are shown with all non-hydrogen atoms. The figure was made with SETOR (34).

Figure 3.
Fig. 3. Superposition of the m-calpain catalytic domain and papain. The papain-like part of the catalytic domain (gold, dIIa) and the barrel-like subdomain dIIb (red) are superimposed with papain (18) (blue) after optimal fit of the left-side papain half to the helical subdomain dIIa. The active site residues Cys105L, His262L, and Asn286L, and Pro287L, Trp288L, and Trp106L are shown in full structure. This "standard view" of papain-like cysteine proteinases (18) is obtained from Fig. 1 by a 90° rotation around a horizontal axis. The figure was made with SETOR (34).

Secondary reference #5

Title

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.

Authors

B.Todd, D.Moore, C.C.Deivanayagam, G.D.Lin, D.Chattopadhyay, M.Maki, K.K.Wang, S.V.Narayana.

Ref.

J Mol Biol, 2003, 328, 131-146. [DOI no: 10.1016/S0022-2836(03)00274-2]

PubMed id

12684003

Figure 2.
Figure 2. Ribbon representation of the domain VI crystal structure. (a) Stereographic Ribbon diagrams of the domain VI monomer present in the asymmetric unit. The bound calcium atoms are represented as silver colored spheres. Helices are labeled according to their EF-hand numbering ranging from EF1 to EF5, respectively. The bound DIC19 peptide in helical conformation is represented in yellow and the "mysterious peptide" appeared in the same location as observed in ALG-2 crystal structure[52.] is presented in purple. (b) Ribbon representation of the DVI dimer, depicting interactions through the crystallographic 2-fold axis.

Figure 5.
Figure 5. Stereo view of the surface plots of the hydrophobic inhibitor binding sites. (a) The DIC19 binding region in calcium-bound DVI. DIC19, represented as a helical segment, yellow in color, clearly displays its amphipathic nature with its bulky hydrophobic side-chains buried deep into DVI and polar residues exposed to the solvent (side-chains removed for clarity). (b) Bulky hydrophobic ring of the inhibitor PD150606 and Phe610 of DIC19 occupy the same region of DVI. However, the hydrophobic region that accommodates these inhibitor molecules seems to be flexible enough, varying in size to accommodate different sized inhibitors. c) View of the inhibitor binding regions in the calcium-free DVI structure. DIC19 is positioned in the same place, as in previous Figures, indicating the narrowness of the hydrophobic region.

The above figures are reproduced from the cited reference with permission from Elsevier

Secondary reference #6

Title

Crystal structures of calpain-E64 and -Leupeptin inhibitor complexes reveal mobile loops gating the active site.

Authors

T.Moldoveanu, R.L.Campbell, D.Cuerrier, P.L.Davies.

Ref.

J Mol Biol, 2004, 343, 1313-1326. [DOI no: 10.1016/j.jmb.2004.09.016]

PubMed id

15491615

Figure 4.
Figure 4. Close-up of µI-II-inhibitor interactions. Stereoview of the interactions between leupeptin (green) and E64 (magenta) with calpain µI-II. Residues within domains I and II are colored blue and cyan, respectively. The residues found within 4 Å of leupeptin and E64 are colored by atom type (carbon, yellow; oxygen, red; nitrogen, blue; and sulfur, orange). Hydrogen-bonding interactions are indicated by dotted blue lines. (A) and (B) Surface and stick representations of the calpain µI-II-leupeptin complex. (C) and (D) Stick and surface representations of the calpain µI-II-E64 complex.

Figure 7.
Figure 7. Comparison of calpain's active site with that of papain and cathepsins K and B. (A) Calpain µI-II-leupeptin complex. (B) Papain-leupeptin complex (PDB code 1POP). (C)-(F) Close-up views of active site cleft (rotated about 90° from (A) and (B)). (C) Calpain µI-II-leupeptin. (D) Papain-leupeptin. (E) Cathepsin K-E64 (PDB code 1ATK). (F) Cathepsin B-benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone (PDB code 1THE45).

The above figures are reproduced from the cited reference with permission from Elsevier