Caspase-7 (PDB ID:4FEA) from Homo sapiens
Created by: Stefanie Muller
Caspase-7 (PBD ID: 4FEA) is a member of the cysteine protease family, an important group of proteins involved in apoptosis (Feldman 2012). Caspase-7 belongs to a subgroup of this family known as the caspases. The name comes from “cysteine aspartate proteases”; caspases cleave substrates “after specific aspartate residues” (Hardy 2009). Caspases are involved in two different apoptotic pathways: mitochondrial-mediated and death receptor-mediated. Transmembrane death receptors initiate the death receptor-mediated apoptotic process (Brentnall 2013). In mitochondrial-mediated apoptosis , cytochrome c from the mitochondria activates a pathway that eventually leads to caspase-9 (the initiator) activating caspase-3 and caspase-7 (the executioners) (Image: Riedel 2004, Feldman 2012). Before activation, caspases-3 and -7 are found in inactive zymogen (procaspase) forms. When bound to an inhibitor, caspase-7 adopts a conformation similar to the procaspase form. Activated caspases-3 and -7 cleave over 500 proteins and DNA (Hardy 2009). Cleavage of substrates leads to substrate degradation or to degradation of other cellular components. Caspases-3 and -7 “play primary but nonredundant roles as the executioners of apoptosis” (7). Caspase-7 is required for ROS (reactive oxygen species) production and cell detachment from the extracellular matrix; its specific role is not yet understood (Brentnall 2013). Malfunction of the mitochondrial-mediated apoptotic pathway can lead to autoimmune diseases and cancer (through over-activation), as well as neurodegenerative disorders and strokes (through under-activation). A potential direction of research aimed in treating diseases caused by over-activation is in inhibiting caspases. Inhibition can also lead to decreased inflammation (Feldman 2012). One potential problem of inhibiting caspases is that it is difficult to find a drug that can target the active site, which in all caspases is quite specific, preferring “an electrophilic carbonyl and an aspartyl functionality” (10). However, several allosteric inhibitors have been found that bind to the dimer interface, which results in an alteration of the active site (Feldman 2012).
Caspase-7 is found as a dimer in the cytoplasm, and is a globular protein (Feldman 2012, Holm 2010). Its molecular weight is 56081.7 Da, and its theoretical isoelectric point is 5.83 (Gasteiger 2005). These data were found using the ExPASy ProtParam server, which analyzes the protein sequence for physio-chemical parameters. Caspase-7 has a structure that is 22% helical, containing 7 helices of 55 residues. The structure is 17% beta sheets, containing 8 sheets of 44 residues (Kabsch 1983). However, many of the functionally important residues, such as the catalytic cysteine of the substrate binding site (Cys 186), are located on flexible loops (Feldman 2012).
In order to activate caspase-7, procaspase-7 must be cleaved in two places; the second cleavage generates a large and small subunit (Hardy 2009). “The substrate-binding site is composed of four loops; L2, L3, and L4 are contributed from one-half of the caspase dimer, and L2′ is contributed from the other half of the caspase dimer” (Hardy 2009). Any inhibitor that interferes with the dimerization of caspase-7 “could be useful in caspase-based therapeutics” (Hardy 2009). The loops appear to be very active because their x-ray structure is only observable when bound to a substrate (Hardy 2009). Feldman et al characterized an allosteric site on caspase-7 using four reversibly inhibitory compounds, A, B, C, and D. These compounds are able to inhibit all caspases. Compound A was used to characterize the crystallized structure of caspase-7. The four compounds are structurally similar; all are “pyridinyl, copper-containing molecules with a multi-ring structure”. Compound A binds to caspase-7 between two beta sheets at the dimer interface, and creates a conformational change that involves a steric hindrance between Compound A and “Arg 187 of L2 and Thr 225, Val 226 and Pro 227 of L3”. The resulting structure bears similarity to the inactive procaspase-7 form. The residues Compound A interacts with include “residues Tyr 223 and Cys 290 from one subunit and Glu 216, Phe 221, Tyr 223, Val 292 and Met 294 from the neighboring subunits" (Feldman 2012).
Hardy and Wells used thiol-containing irreversible inhibitors DICA [2-(2,4-Dichlorophenoxy)-N-(2-mercapto-ethyl)-acetamide] and FICA [5-Fluoro-1H-indole-2-carboxylic acid (2-mercapto-ethyl)-amide] to inhibit caspase-7; the resulting structure is also similar to procaspase-7 (Feldman 2012, Hardy 2009). DICA and FICA are inhibitory only to caspases-3 and -7 because these inhibitors form a disulfide bond with Cys 290 in the dimer interface of the caspases. Cys 290 is conserved in caspases -3 and -7, and not in other caspases (Feldman 2012). DICA and FICA work by causing Arg-187 to move into the active site using a hinge-like movement around Gly 188. “The L2′ loop folds down to cover the allosteric inhibitor [DICA or FICA] and assumes a zymogen-like conformation” (Hardy 2009).
Comparing monmeric structure using the DALI server, caspase-7 shares many similarities with procaspase-7 (PDB: 4JR1) and caspase-3 (PDB: 3DEJ). The Z-score with procaspase-7 is 25.8; the Z-score for caspase-3 is 24.5 (Holm 2010). The Z-score is a measure of structural similarity, and these results indicate high similarity between the two proteins. Running a BLAST search, which finds the degree of sequence homology in primary structure, caspase-7 has an E-value of 7x10-176 when compared to human procaspase-7 bound to Ac-devd-cmk (PDB: 4JR1), and an E-value of 5x10-176 when compared to caspase-3 bound to specific unnatural amino acid peptides (PDB: 4JJ8) (Altschul 1990). In addition to tertiary structure similarity found on the DALI server, the results of the BLAST search indicate a high level of primary structure similarity between caspase-7 and caspase-3 and caspase-7 and procaspase-7. Caspase-3 was compared with caspase-7 because these two caspases are similar in function; both are executioner caspases, and have a higher structural similarity with each other when compared to other caspases (Feldman 2012, Hardy 2009). Procaspase-7 bound to Ac-devd-cmk does have low levels of activity. This is due to the fact only the L2 loop is unable to fold into the active caspase-7 structure due to steric constrains. Hardy and Wells concluded that the purpose of activating procaspase-7 through cleavage is to allow the L2 loop (which is part of the substrate binding site) to fold into the proper looped structure (Hardy 2009). This cleavage at L2 is also responsible for forming the two subunits of each dimer (Thomsen 2013).
Structural differences between caspase-7, caspase-3, and procaspase-7 reveal functional differences important in the evolution of the apoptotic pathway. Hardy and Wells found that while procaspase-7 is more active than procaspase-3, after maturation, caspase-3 is more active than caspase-7. Perhaps as it matures, procaspase-7 cleaves certain substrates and “could control the threshold for apoptotic activation in cells” (Hardy 2009). The researchers proposed that procaspase-7 helps mediate a cell’s entry into the apoptotic pathway, while procaspase-3 maturation represents the committed step of this pathway. In addition, caspase-7 is more easily ubiquitinated for degradation due to the presence of a binding motif for apoptotic inhibitors that is not present on caspase-3 (Hardy 2009). Future research aimed at inhibiting apoptosis could examine different allosteric inhibitors of either caspase-7 or procaspase-7.