Cysteine Proteases

The Cysteine Proteases - IV

Apo-m-Calpain (1kfu)	Zoom in on Catalytic Center

Apo-m-Calpain	Zoom in on Catalytic Center

The enzyme consists of a large variable subunit and a small constant subunit (lower right)
- These are held together by lipophilic forces involving the blue helix that extends down into the rear of the small unit; this is the sequence that is cleaved
- Variable means that the sequence varies between m- and m, whereas the small subunit is the same in both
The catalytic site consists of Cys105, His262, and Asn286 (all in CPK colors)
- Note that in m-calpain the Cys is in a different domain than the His and Asn, more than 10 A away
- Thus the catalytic site is not formed in the apoenzyme
- However, in m-calpain, the Cys (here mutated to a Ser) is much closer
- This may partly explain the greater ease of activation of the m-enzyme

Upon binding of Ca⁺⁺, the small subunit dissociates. A couple of recent crystal structures of domains I and II (the upper part in the figure above) also help to explain the initiation of activity.

Apo-m-Calpain, Domains I and II

The autolytically cleaved sequence is colored in red.

The binding of two Ca⁺⁺ in domains I and II activates m-calpain, whereas binding Ca⁺⁺ similarly in m-calpain produces only slight activity.

Domains I and II of m-Calpain	Closeup of Catalytic Center

Domains I and II of m-Calpain	Closeup of Catalytic Center

Binding of Ca⁺⁺ in domains I and II was not previously suspected; rather, at least five Ca⁺⁺ were found binding to a calmodulin-like domain IV

The positioning of the catalytic residues in both the m and m-protein clearly is appropriate for reaction

The continued unreactivity of the m-calpain is explained by the position of the side chain of W106:

It blocks access to the catalytic site in m- but not in m

The structures in which the Ca⁺⁺ is bound are like the binding segments in other calcium binding proteins, for example calmodulin:

Human Calmodulin (1cll)

Recall that this helix-loop-helix motif, with the helices roughly perpendicular is called an EF-Hand.
- They typically have 10-12, 12, and 10-12 residues in the helix, loop, and helix
- The calcium is bound by residues in the loop, which contribute backbone O and carboxylates
- Most commonly, loop residues 1, 3, 5, 7, 9, and 12 are coordinated to calcium
Here is an example of the calcium binding sites of calpain

EF-Hand in Calpain (residues 572-600)

Sequence Alignment, Calpain and Calmodulin EF Hands

Exactly how additional Ca⁺⁺ activates the m-calpain is not yet known, but it probably is related to interactions between the other Ca-binding domains and the catalytic domains.
Elce and co-workers [Biochem. J., 2000, 367, 263] found that mutations of EF-Hand residues had limited effect on the binding of calcium by the two proteins. They concluded:
- Cooperativity in binding of calcium was affected by differences in the first 40-50 residues at the N-terminal end
- Variations in the EF-Hand near the C-terminal end also affected cooperativity
Unfortunately, they were not very specific about which difference, or how cooperativity was altered.
This page last modified 2:01 PM on Tuesday April 7th, 2009.
Webmaster, Department of Chemistry, University of Maine, Orono, ME 04469

EF-Hand in Calpain (residues 572-600)

Sequence Alignment, Calpain and Calmodulin EF Hands