The Schistosoma mansoni tegumental allergen protein, SmTAL1: Binding to an IQ-motif from a voltage-gated ion channel and effects of praziquantel

Highlights

• SmTAL1 binds four calcium ions per dimer.

• There are cooperative interactions between binding sites.

• SmTAL1 binds praziquantel non-cooperatively.

• SmTAL1 interacts with an IQ-motif from SmCa_v1B.

• Praziquantel interferes with this interaction in a complex manner.

Abstract

SmTAL1 is a calcium binding protein from the parasitic worm, Schistosoma mansoni. Structurally it is comprised of two domains – an N-terminal EF-hand domain and a C-terminal dynein light chain (DLC)-like domain. The protein has previously been shown to interact with the anti-schistosomal drug, praziquantel (PZQ). Here, we demonstrated that both EF-hands in the N-terminal domain are functional calcium ion binding sites. The second EF-hand appears to be more important in dictating affinity and mediating the conformational changes which occur on calcium ion binding. There is positive cooperativity between the four calcium ion binding sites in the dimeric form of SmTAL1. Both the EF-hand domain and the DLC-domain dimerise independently suggesting that both play a role in forming the SmTAL1 dimer. SmTAL1 binds non-cooperatively to PZQ and cooperatively to an IQ-motif from SmCa_v1B, a voltage-gated calcium channel. PZQ tends to strengthen this interaction, although the relationship is complex. These data suggest the hypothesis that SmTAL1 regulates at least one voltage-gated calcium channel and PZQ interferes with this process. This may be important in the molecular mechanism of this drug. It also suggests that compounds which bind SmTAL1, such as six from the Medicines for Malaria Box identified in this work, may represent possible leads for the discovery of novel antagonists.

Introduction

In trematodes and some other helminths, there is a family of unusual calcium binding proteins. These proteins are characterised by having an N-terminal domain with two EF-hand motifs and a C-terminal dynein light chain (DLC) like domain [1,2]. Thus, the proteins have an N-terminal domain which resembles the globular heads of calmodulin. This is joined by a flexible linker to a C-terminal domain which has very high structural similarity to members of the DLC family (Fig. 1) [[3], [4], [5]]. Proteins from this family have been identified and characterised in a number of species including Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Fasciola hepatica, Fasciola gigantica, Opisthorchis viverrini and Clonorchis sinensis [[6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. In general, these proteins bind to calcium ions, although there are some exceptions, for example S. mansoni tegumental allergen-like (TAL) protein 3 (SmTAL3 or Sm20.8) [10,16]. The 13 S. mansoni TAL proteins are probably the best characterised group of proteins from this family. Several of these TAL proteins have been implicated in eliciting IgE-mediated immune responses in the host [11,21]. The proteins have different tissue and life cycle stage expression profiles and it is clear from the available data that they also vary in terms of their ion and drug binding properties [10,11,16]. There is limited data on the in vivo roles of these proteins. One early report demonstrated that SmTAL3 and a dynein light chain form part of a larger complex suggesting that one function may be to regulate microtubule motors [22].

We have previously demonstrated that, despite similarities in sequence and predicted structure, that members of this protein family have different biochemical properties. For example, of the four characterised family members from F. hepatica, one dimerises in response to the presence of calcium ions (FhCaBP4), one is converted from a dimer to a monomer by calcium ions (FhCaBP3) and the oligomeric states of the two are unaffected by this ion (FhCaBP1 and FhCaBP2) [[12], [13], [14], [15]]. In the case of the 13 family members from S. mansoni, two do not appear to bind calcium ions at all (SmTAL3 and SmTAL5) [10,16]. FhCaBP2 binds calcium (and other divalent ions) primarily through its second EF-hand motif [13]. However, there is evidence from sequence analysis of the EF-hands to suggest that the some family members from S. mansoni (SmTAL7, SmTAL9, SmTAL10 and SmTAL13) bind calcium at primarily at the first EF-hand motif [16].

SmTAL1 has an increased resistance to thermal denaturation and increased resistance to proteolytic digestion in the presence of praziquantel (PZQ), providing evidence for an interaction with this drug which stabilises the structure of the protein [10]. SmTAL4, SmTAL5 and SmTAL8 are all destablised by PZQ suggesting that the drug may bind to partially unfolded forms of these proteins, promoting lower overall stability [16]. These interactions with SmTAL proteins are particularly interesting since PZQ is the drug of choice for treating S. mansoni infections. The drug is cheap, effective and has few significant side effects [23]. However, its mechanism of action is unknown although it is generally accepted that it disrupts calcium homeostasis, potentially through the antagonism of voltage-gated calcium channels [[23], [24], [25], [26]]. The existence of an in vitro interaction between the drug and SmTAL1 (and other family members) does not, of course, prove that these proteins are pharmacologically relevant targets of PZQ. Nevertheless their documented biochemical activity as calcium binding proteins makes it tempting to suggest that they may be implicated in the molecular mechanism of action of this drug. Although few, if any, cases of bone fide resistance to PZQ have been reported, it seems likely that resistance will eventually emerge, just as it has to most major classes of antibacterial drug [[27], [28], [29], [30]]. Here we present a detailed, biochemical study of SmTAL1 focusing on its ion and drug binding activities, and probing its ability to interact with IQ-motifs from voltage-gated calcium channels.