a number of drugs are currently available to treat malaria ; however , treatment is becoming complicated by drug resistance , toxicity , and high cost .
recently , drug resistance against the new effective drug , artemisinin , is also emerging [ 4 , 5 ] , and we need new effective drugs to treat malaria .
therefore , the development of other classes of effective antimalarials , especially compounds that act against novel biochemical targets , is required .
to develop such compounds , it is very important to characterize the structural and biochemical features of new drug targets . among potential new targets for antimalarial chemotherapy
proteases are druggable targets , and at present protease inhibitors are now licensed as well as in clinical development to treat different diseases for example osteoporosis , diabetes , cancer , hypertension , and infectious diseases [ 69 ] .
recent advances , including the sequencing of plasmodium genomes ( http://www.plasmodb.org/ ) and development of new tools for manipulating plasmodium genes , have improved our understanding of the cysteine proteases of parasites .
therefore , given the importance of the cysteine proteases , this paper will focus on structural - functional relationship of falcipains , major cysteine proteases of p. falciparum .   in cases of malaria ,
the parasite relies on human hemoglobin hydrolysis to supply amino acids for protein synthesis and to maintain osmotic stability [ 11 , 12 ] .
cysteine proteases are involved in hemoglobin hydrolysis and have been validated as promising drug targets [ 13 , 14 ] . in a recent report , ch'ng et al .
, using cysteine protease inhibitors , demonstrate that clan ca cysteine proteases of p. falciparum are also involved in chloroquine - mediated programmed cell death .
the best characterized plasmodium cysteine proteases are the falcipains , papain family ( clan ca ) enzymes ( figure 1 ) . among the four p. falciparum cysteine proteases , falcipain-2 and falcipain-3 appear to be the principal food vacuolar hemoglobinases [ 1618 ] .
when the falcipain-2 gene is disrupted , undegraded hemoglobin accumulates in the food vacuole , confirming that this enzyme participates in haemoglobin hydrolysis .
however , disruption of falcipain-3 could not be achieved , but the gene was readily replaced with a tagged functional copy , indicating that falcipain-3 is essential for erythrocytic parasites .
falcipain-2 and falcipain-3 share 67% sequence identity and contribute more or less equally to the digestion of hemoglobin in the food vacuole . comparing with other papain family proteases ,
specifically , this paper will survey available information on structure and function of different domains of falcipain-2 and falcipain-3 and their interaction with inhibitors .
falcipains are major cysteine protease , named due to the function of a catalytic cysteine , which mediates protein hydrolysis via nucleophilic attack on the carbonyl carbon of a susceptible peptide bond .
falcipains have two main domains , the prodomain and the mature domain . the prodomain is further divided into different small domains . at the n - terminus of the prodomain ,
the first 35 amino acids are cytosolic , followed by a 20 amino acid transmembrane domain and a 188 amino acid lumenal domain .
the inhibitory domain is present in the c - terminal part of the prodomain ( figure 2 ) .
cysteine proteases of the parasite hydrolyze hemoglobin in the acidic food vacuole [ 20 , 21 ] .
recently , it has been demonstrated that prodomain has unique motifs which are responsible for targeting the falcipains . using transfection technology and constructing different chimeras with portions of the n - terminal part of prodomain
fused to green fluorescent protein , it has been shown that the prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole .
once enzymes are in the er , using the signal from transmembrane domain , the proteins appear to traffic to the plasma membrane . at the plasma membrane falcipain-2 and falcipain-3
appear to be endocytosed in a process requiring the presence of both cytoplasmic and luminal prodomain trafficking motifs , followed by vesicular transport to the food vacuole .  
in summary , serial truncation and deletion studies showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the enzymes were essential for food vacuolar trafficking ( figure 3 ) .
like many other proteases , falcipains are synthesized as a zymogen , and the prodomain inhibits the activity of the mature enzyme . to investigate how the prodomain regulates the activity of falcipain-2 , pandey et al
. expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2 .
it has been found that a c - terminal segment ( leu  asp ) of the prodomain , including two motifs ( erfnin and gnfd ) that are also conserved in cathepsin l subfamily proteases , mediates prodomain inhibitory activity .
earlier work with other cathepsin l subfamily proteases suggests key roles for conserved hydrophobic amino acids ( phenylalanine and tryptophan ) as well as the erfnin and gnfd motifs in maintaining prodomain structure .
later , pandey et al . explored the roles of conserved motifs in maintaining prodomain structure by circular dichroism analysis .
secondary structure was seen in a fragment with potent inhibitory activity ( leu  asp ) , but not in two larger constructs that lacked any sequence downstream of the erfnin and gnfd motifs or in a peptide spanning the erfnin and gnfd motifs .
these results indicate that , together with erfnin and gnfd motifs , an upstream region including two conserved phe * residues is required for proper folding or maintenance of secondary structure of the prodomain ( figure 2 ) .
the 3d structures of the mature domain of falcipain-2 and falcipain-3 have been solved [ 25 , 26 ] , and the profalcipain-2 model suggests that conserved residues provide stability to the inhibitory domain ( figure 4(a ) ) .
modeling , based on the solved structure of procathepsin b and procathepsin l [ 27 , 28 ] , shows that the charged pair arg and glu of the prodomain appear to form a salt bridge ( figure 4(b ) ) .
further , glu from the gnfd motif may form a separate salt bridge with lys in the mature domain .
phe may participate in nonpolar interactions and possibly pi stacking interactions , with two tryptophan residues in the mature domain , trp and trp .
the model also suggests that the prodomain of falcipain-2 binds the mature domain in a manner similar to that of procathepsin k and l , inhibiting catalytic activity by blocking substrate access to the active site ( figure 4(a ) ) .
they encode short n - terminal extensions of the mature domain , which is unique among described papain family proteases [ 13 , 14 ] .
looking at the family of c1a cysteine protease sequences on the merops database ( http://merops.sanger.ac.uk/ ) have identified more than 40 members with an n - terminus extension of 12 amino acids at this location .
notably , 18 of these sequences were those of falcipains and homologues from other plasmodial species
to better characterize the determinants of folding for falcipain-2 , pandey and sijwali et al . expressed multiple prodomain and mature constructs of the enzyme in e. coli and assessed their abilities to refold by checking their activity [ 23 , 29 ]
this folding study showed that refolding of the mature domain could be achieved when the mature domain was covalently joined with the n - terminus extension ( figure 5 ) .
only 12 amino acids of the n - terminus extension ( refolding domain ) are necessary for refolding .
deletion of 12 amino acids from n - terminus segment of the mature domain yielded a construct incapable of correct folding , but inclusion of this segment in trans allowed folding to active falcipain-2 .
correct folding also occurred when the catalytic domain was refolded with a separate prodomain - folding domain construct but not with an isolated folding domain peptide [ 23 , 29 ] ( figure 5 ) .
these results indicated that the prodomain mediates the interaction between the mature and folding domain when they were not covalently bound .
however , it was not clear from these studies , whether the amino - terminal extension is required only for folding or whether it also plays an essential role in mediating enzyme activity . to determine whether the folding domain was required for activity , pandey et al
after refolding and purification of the mature domain by anion exchange chromatography , the purified refolded mature domain still showed activity .
together , these results suggest that the refolding domain is only required for refolding , and once refolding is accomplished , the refolding domain is not required for activity . the n - terminus extensions of different falcipain subfamily enzymes have limited ( 2045% ) sequence identity but are functionally conserved .
chimeras of the falcipain-2 catalytic domain with the refolding domain of six other plasmodial proteases folded normally and had similar kinetic parameters to those of recombinant falcipain-2 .
these results showed that refolding domain can be swapped between the plasmodial proteases that harbor the same function .
the above - mentioned experiment also indicates that all plasmodial cysteine proteases including falcipain-3 also use n - terminus extension as a refolding domain .
structural analysis further reveals that the folding domain , in fact , has a short but significant element of secondary structure .
glutamate of the mature domain forms a buried hydrogen bond with tyrosine and a salt bridge with arginine of the refolding domain .
this suggests that the small folding domain , though shorter than the standard papain family prodomain , still plays a very important role in folding by stabilizing the mature domain by a hydrogen bond and a salt bridge .
this is analogous to a similar interaction in falcipain-2 , where tryosine of the refolding domain of falcipain-3 provides hydrogen bonding anchor to glutamate of the mature domain of falcipain-3 .
these results indicate that the folding domains of falcipain-2 and falcipain-3 stabilize the mature domain in a similar fashion .
falcipain-2 and 3 efficiently hydrolyze human hemoglobin in the acidic food vacuole of parasite [ 17 , 18 ] .
it has recently been suggested that hemoglobin hydrolysis is not a highly ordered process but rather proceeds with rapid cleavage by falcipains at multiple sites .
falcipain subfamily proteases contain an unusual motif near the c - terminus , which is present between the highly conserved active site histidine and asparagine residues ( figure 2 ) .
a motif of identical size ( 14 aa ) is found in all studied proteases of this subfamily of falcipain , although sequence identity is modest . in case of falcipain-2 and falcipain-3 , motifs
smaller motifs are present in the sequences encoding p. falciparum serine repeat antigens ( 10 aa ) and dipeptidyl peptidase
i ( 8 aa ) , and larger ( 20 aa ) motifs are present in two other putative p. falciparum dipeptidyl peptidase genes [ 14 , 33 ] .
to evaluate the function of the unusual c - terminal motif in falcipains , a mutant enzyme was made by deleting that motif lacking 10 amino acids from falcipain-2 , and the biochemical properties of wild and mutant enzymes were compared .
native page , biacore , and gel filtration studies indicated that the motif mediates specific interactions with hemoglobin .
in fact , falcipain-2 has relatively higher affinity for methemoglobin ( kd is 0.8  m ) than hemoglobin ( kda is 3.3  m ) .
it had been demonstrated that several factors contribute to the formation of methemoglobin during malarial infection , including acidic ph of plasmodial food vacuole , oxidative damage in rbc [ 35 , 36 ] , which causes an increase in methemoglobin content to up to 2042% in the plasmodial food vacuole .
thus , the higher affinity of falcipain-2 for methemoglobin looks like an adaptation to the specific conditions in the infected rbc .
the enzyme without this motif showed negligible activity against hemoglobin or globin . further , a peptide encoding the motif blocked hemoglobin hydrolysis but not the hydrolysis of casein , indicating that the motif specifically interacts with hemoglobin .
thus , this study suggests that the motif mediates the most biologically relevant activity of falcipain , the hydrolysis of hemoglobin during trophozoite stage .
falcipains may also have other functions in erythrocytic malaria parasites , and it would be interesting to explore the role of the motif in hydrolysis of other proteins of potential biological relevance . in the case of other members of the falcipain
subfamily , it is likely that the encoded motifs share the same functions as the falcipain motif , because a number of members of this subfamily have been also shown to be hemoglobinases .
the sequence conservation among the motifs of falcipain subfamily proteases is not high , but , as in the case of the n - terminal extension that mediates protein folding , the presence of these unusual motifs in all related proteases probably indicates a conserved function . for genes encoding more distantly related enzymes , including falcipain-1 , dipeptidyl peptidases , and serine repeat antigens , c - terminal motifs vary greatly in sequence and size , and specific functions of predicted motifs are undefined .
data indicates that falcipain-2 captures hemoglobin via its c - terminal motif before subsequently cleaving the substrate at multiple sites .
further , the structure of falcipain indicates a protruding configuration for the motif , surrounded by a predominant negative charge , and it may be that charged residues are crucial for interaction with hemoglobin .
it is proposed that hemoglobin , which has many charged surface residues , first binds at the motif through charge - charge interaction and brings hemoglobin closer to the active site before hydrolysis .
the function of the motif in mediating catalysis despite its separation from the active site somewhat resembles that of the hemopexin domain of matrix metalloproteases .
the hemopexin domain is required for both the efficient cleavage of collagen   and the formation of complexes with both inhibitors and proteases to mediate biological activities .
thus , as is the case for the falcipain-2 motif , the hemopexin domain serves to facilitate biologically relevant protein - substrate and protein - inhibitor interactions .
however , the hemopexin domain is approximately 20 times larger than the falcipain motif and , unlike the motif , is separated from the catalytic domain by a hinge region .
therefore , it appears that falcipains and matrix metalloproteases use similar means of biological control , but the specific mechanisms by which the proteases mediate interactions with substrates and inhibitors are different .
another papain family protease , cathepsin k , forms a pentameric complex with chondroitin sulfate to facilitate hydrolysis of collagen . having analogy to falcipain-2-hemoglobin interaction , structural requirements for complex formation between cathepsin k and chondroitin sulfate
it is interesting to raise a question , why do falcipains contain a unique motif that mediates the hydrolysis of hemoglobin ?
this mechanism might not have evolved simply to facilitate hemoglobin hydrolysis , because other papain family enzymes without c - terminus motifs still can hydrolyze hemoglobin .
it looks like that the utilization of a specific motif at this region to mediate enzyme - substrate interaction is an unusual but conserved feature of plasmodial cysteine proteases .
it may be possible that an evolutionary bottleneck occurred in ancestral plasmodial cysteine proteases , and those enzymes might not be that efficient in hemoglobin hydrolysis .
in addition , plasmodial cysteine proteases might have evolved with introduction of specific motif for efficient hemoglobin hydrolysis .
the interaction of a major hemoglobinase , falcipain-2 , with most biologically relevant substrate , hemoglobin , could be an interesting starting point for the development of an effective drug .
the structure of falcipain-2 hemoglobin binding domain will guide the design of inhibitors that should interfere with hemoglobin binding .
there are two major classes of cysteine protease inhibitor , small inhibitors like leupeptin , vinyl sulfones , e64 , and another class known as macromolecular inhibitor .
these endogenous cysteine protease inhibitors have been described in a number of eukaryotic systems . here
, we will discuss three major cysteine protease inhibitors , cystatin , chagasin , and falstatin .
excluding the two unique motifs discussed earlier , the rest of the falcipains are structurally similar to homologous proteases in the papain family .
cystatin was used as a falcipain inhibitor with the expectation that protein - protein interaction over large surfaces would enhance crystal quality and thus facilitate structural determination .
cystatins inhibit a wide range of papain family cysteine proteases with high affinity making them ideal candidates for cocrystallization with falcipain .
falcipain and chicken egg white cystatin formed 1  :  1 complex ( figure 6(a ) ) .
the inhibitory constants ( ki ) of cystatin for falcipain-2 and falcipain-3 are 6.5  nm and 100  nm , respectively .
it is interesting that cystatin is a more potent inhibitor of falcipain-2 than falcipain-3 , which suggests that cystatin regulates both the falcipains with different rates .
the falcipain - cystatin complex shares many features with two known cysteine protease - cystatin structures : cathepsin h - stefin a and papain - stefin b ( the stefins are a subclass within the cystatin superfamily [ 41 , 42 ] ) . these cystatins bind to target proteases in similar orientations , resulting in extensive interactions with the target proteases ( figure 6(b ) ) .
cystatin largely interacts with falcipain-2 at prime sites where substrate - binding pockets are relatively shallow and less defined , but falcipains also have binding specificity at nonprimed sites .
although , the binding preference is not as pronounced as that found at the s2 pocket ( figure 6(b ) ) .
cystatin is only able to access the solvent exposed periphery of the nonprime site of falcipain-2 and majority of its binding occurs at the prime end of the active site . unlike most other cathepsin l - like cysteine proteases ,
crystal structures indicate that this preference may be due to ile in falcipains , because it creates a small protrusion in the otherwise flat base of the s2 pocket , which restricts aromatic side - chain binding   ( figure 6(b ) ) .
the falcipain  cystatin complex can be exploited to design potent inhibitors of the malaria parasite .
a recent study indicates that a peptide based on cystatin binding residues blocked the activity of falcipain-2 and led to accumulation of undegraded hemoglobin in the food vacuole .
chagasin is a cysteine protease inhibitor that was first identified in trypanosoma cruzi as the physiological regulator of cruzain , the major cysteine protease [ 45 , 46 ] .
cruzain is also a papain - like ( clan ca ) cysteine protease that is expressed in all stages of t. cruzi life cycle .
it is a key virulence factor of t. cruzi , the infectious agent responsible for the leading cause of heart disease in latin america , called the chagas disease .
chagasin is associated with cruzain during its trafficking to specific compartments of the parasite cell , and accumulated evidence suggests that the primary role of chagasin is in posttranslational regulation of protease activity .
1 binding with falcipain-2 ( figure 7 ) . the protease - binding loops ( bc , de , and fg ) in chagasin form a well - aligned wedge that fills the active site groove of falcipain-2 to obstruct substrate binding ( figure 7 ) .
the bc loop is one of the three signature motifs that contribute to the inhibition of the cysteine protease .
this is confirmed by a synthetic peptide corresponding to the bc loop of the chagasin like protein from e. histolytica , which specifically blocked the activity of cysteine proteases .
it is noteworthy that thr 31 in the bc loop of chagasin binds to the catalytic cys at the falcipain-2 active site by water - mediated hydrogen bonds ( figure 7 ) .
it is likely that the highly conserved thr serves as a key functional residue among chagasin - like inhibitors .
another interesting feature of the falcipain-2-chagasin interaction is the highly mobile de loop like in e64 , a strong irreversible inhibitor of cysteine protease , which occupies the nonprime site . in summary ,
the bc loop , mobile de loop , and the rpw / f motif in the fg loop are the key elements for binding with falcipain-2 .
disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer , rheumatoid arthritis , osteoporosis , and emphysema . in case of malaria ,
falciparum parasites express falstatin , a potent inhibitor of falcipains and many other cysteine proteases .
the stage - specificity of falstatin expression and the effects of anti - falstatin antibodies on parasite development suggest that this inhibitor facilitates a process that also requires proteolytic activity , the invasion of erythrocytes by p. falciparum merozoites .
falstatin is a competitive and reversible inhibitor of falcipains , as demonstrated by increasing calculated km values but similar vmax values with increasing concentrations of falstatin . to evaluate the mechanism of inhibition of cysteine proteases by falstatin , pandey et al .
tested the ability of active site - inhibited falcipain-2 to compete with active falcipain-2 for binding with falstatin .
in contrast to results with the prodomain , the inhibitory effect of falstatin was not affected by the presence of active site inhibited falcipain-2 .
thus , the binding of falstatin to falcipain-2 appears to be via interaction with the enzyme active site .
leupeptin , e-64 , and vinyl sulfones are major cysteine protease inhibitors that bind to the active site [ 26 , 48 ] .
the active sites of both enzymes are located in a cleft between the structurally distinct domain of the papain - like fold .
the structures of falcipain-2 and falcipain-3 have been determined in complex with these small inhibitors .
e-64 , and leupeptin interact with residues in the s1 , s2 , and s3 subsites of falcipain-2 and falcipain-3 , corresponding to the p1 , and p2 , and p3 position of the inhibitors ( figure 8) .
the conserved catalytic residues of falcipain-2 and falcipain-3 ( gln , cys , his , asn , resp . )
inhibitors display binding modes with their partner enzymes similar to those found in other papain family enzymes .
the inhibitors bind to the main chain of falcipain-2 and falcipain-3 by glycine ( gly in falcipain-2 and gly in falcipain-3 ) residue that is highly conserved in the s3 subsite of clan ca cysteine proteases ( figure 9 ) . in cocrystallization , these residues form hydrogen bonds with the o and n atoms of the inhibitor backbone . in the case of the falcipain-2 complex with e-64 ,
enzyme active sites gln , ser , cys , asn , and his are involved in the formation of additional hydrogen bonds with e-64 . in the falcipain-3-leupeptin complex ,
gln , cys , and asn also act as hydrogen bonding partners to the inhibitor .
the enzyme - inhibitor complex stabilize by a series of possible hydrophobic interaction using nonpolar region of gly , tyr , gly , leu , ser , leu , asn , and ala in falcipain-2 , and tyro , gly , tyr , ile , and ser in case of falcipain-3 .
the active site cysteine of falcipain-2 forms a covalent , irreversible hemithioketal with the e-64 epoxy carbon ( figure 9 ) .
the active site cysteine of falcipain-3 forms a covalent , reversible hemithioacetal with the asymmetric carbonyl carbon of leupeptin ( figure 10 ) . the interaction pattern of e-64 and leupeptin with all papain family enzymes is conserved .
the carboxyl group of e-64 and carbonyl group of leupeptin occupy the oxyanion hole formed by the conserved catalytic residues . falcipain-2 and falcipain-3 have a clear preference for substrates / inhibitors that contains a leucine at p2 position , and both leupeptin and e64 contain a leu at p2 .
surprisingly , falcipain-3 has been shown to be much less efficient at hydrolyzing peptide substrates and more difficult to inhibit with small peptidyl - based inhibitors compared with falcipain-2 [ 16 , 51 ] .
the structure that forms the active site is highly conserved between falcipain-2 and falcipain-3 , and there is no notable movement in the peptide backbone or loop region .
however , superimposition of falcipain-2 and falcipain-3 structures highlights two important substitutions in the s2 subsite .
this position of the s2 subsite is known to be a key determiner of the specificity ( figures 9 and 10 ) . in comparison to asp in falcipain-2 ,
the additional side chain carbon in glu of falcipain-3 , this increased the size as well as flexibility at this position . in case of falcipain-3leupeptin complex , the larger and more flexible residue ( glu ) at the bottom of s2 subsite and bulkier residue ( try ) create a narrow wall of the s2 subsite , as compared to the falcipain  e-64 complex ( figure 10 ) .
the overall structures of falcipain-3leupeptin and falcipain-2e-64 complexes show a similar mode of binding and inhibition compared with macromolecule inhibitors like chagasin and cystatin .
like e64 and leupeptin , vinyl sulphones ( mu - leu - hph - vsph ) have been shown to be effective inhibitors of a number of papain - family - like cysteine proteases .
mu - leu - hph - vsph is a potent irreversible inhibitor of falcipain-2 and falcipain-3 .
the cocrystallization of falcipain-3 with mu - leu - hph - vsph indicates that inhibitor binds the respective s1 and s3 subsites to form an irreversible , covalent bond with the sulfur of the active site cysteine thiol in enzyme   ( figure 11 ) .
given the hydrophobic nature of the p1 and p2 substituents in mu - leu - hph - vsph , the active site of falcipain-3 is lined with a number of residues that are able to make nonpolar contacts with their respective inhibitor   ( figure 11 ) . as seen in the crystal structures of falcipain-3 with the above inhibitor , the residue at the bottom of the s2 subsite ( glu ) points out of the pocket to avoid a potentially unfavorable interaction with the bulky phe residue at the p2 position of inhibitor .
cysteine proteases of the malaria parasite may be targeted for inhibition by vinyl sulphones . indeed , vinyl sulphones - based cysteine protease inhibitors are already being used in preclinical trials for the chagas disease .
the understanding of the cysteine proteases of malaria parasites has increased markedly in recent years . since cysteine proteases that play an important role in the parasite life cycle by degrading erythrocyte proteins , most notably hemoglobin , are attractive targets for antimalarial chemotherapy . falcipain-2 and falcipain-3 are the best characterized cysteine proteases of malaria parasite , the structure and function of different domains and their interaction with small and macromolecular inhibitors are studied
. the structure - function study of falcipains and interaction with inhibitors will provide detail insights to develop rational design of inhibitor against falcipains .
structure - guided approaches should have great role in the design of potent and highly selective inhibitor .
efforts to optimize current inhibitors based on the structure - function of falcipains are currently underway .
 evidence indicates that cysteine proteases play essential role in malaria parasites ; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria . 
 studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis . 
 the best characterized plasmodium cysteine proteases are falcipains , which are papain family enzymes . 
 falcipain-2 and falcipain-3 are major hemoglobinases of p. falciparum . 
 structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain . 
 overall , the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria . 
 drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies . 
1. Introduction
2. Domains of Falcipains
3. Structural Basis of Inhibition of Falcipains
4. Summary and Conclusion