Structural and functional studies of the Leishmania braziliensis mitochondrial Hsp70: Similarities and dissimilarities to human orthologues

Highlights

• LbmtHsp70 was produced soluble, folded and in the monomeric state.

• It has an elongated shape and low ATPase activity, which is stimulated by LbHep1.

• LbmtHsp70 interacts with ATP and ADP with divergent thermodynamic signatures.

• LbHep1 interacts with LbmtHsp70 with a nanomolar dissociation constant.

• LbHep1 has a multifunctional role over LbmtHsp70.

Abstract

Heat shock protein 70 kDa (Hsp70) is a conserved molecular chaperone family involved in several functions related to protein homeostasis. In eukaryotes, Hsp70 homologues are found in all cell compartments. The mitochondrial Hsp70 isoform (mtHsp70) is involved in import of mitochondrial matrix proteins as well as their folding and maturation. Moreover, mtHsp70 has the propensity to self-aggregate, and it depends on the action of the co-chaperone Hsp70-escort protein 1 (Hep1) to be produced functional. Here, we analyze the solution structure and function of mtHsp70 of Leishmania braziliensis (LbmtHsp70). This recombinant protein was obtained folded, in the monomeric state and it has an elongated shape. We observed that LbmtHsp70 suffers thermal aggregation that depends on the protein concentration and is composed of domains with different thermal stabilities. LbmtHsp70 interacted with adenosine nucleotides with a thermodynamic signature different from those reported for human orthologues and interacted, driven by both enthalpy and entropy, with L. braziliensis Hep1 (LbHep1) with a nanomolar dissociation constant. Moreover, LbHep1 stimulated the LbmtHsp70 ATPase activity. Since little is known about mitochondrial Hsp70, particularly in protozoa, we believe that our data are of interest for understanding protozoan Hsp70 machinery.

Introduction

Molecular chaperones and Heat Shock Proteins (Hsps) are essential proteins that guarantee the viability of the cell. These proteins act both in normal and stress conditions, have a plethora of functions inside and outside cells, and provide a quick and efficient cellular stress response [1], [2]. Among all the molecular chaperones, the Hsp70 family is ubiquitous and highly conserved in organisms, as it is present in all cellular compartments [3]. This protein family plays a critical role in protein metabolism. The Hsp70 family acts as a pivot, receiving and distributing unfolded, partially folded or even folded client proteins among other molecular chaperones [1], [2], [4]. Therefore, Hsp70s perform quality control functions on client proteins and newly synthesized proteins and interact indiscriminately with a large variety of folded, unfolded and/or partially folded proteins [1], [2], [5], [6], [7].

Hsp70s are modular proteins with two main domains connected by a linker: an N-terminal domain or nucleotide binding domain (NBD) having ATPase activity and a C-terminal domain or peptide-binding domain (PBD) responsible for binding client proteins [2], [5]. The functionality occurs through a bidirectional heterotrophic allosteric mechanism, in which the presence of the adenosine nucleotide on the NBD or client proteins on the PBD changes the protein conformation reciprocally [2]. There are two main conformational states for Hsp70 described in the literature: i) open state: with ATP present on the NBD, which is characterized by a high rate of release of client proteins on the PBD, and ii) closed state: in this case, after ATP hydrolysis, ADP is present on NBD. That conformation “locks” the client proteins in the PBD and Hsp70 then can perform its action on the client protein [1], [5], [8]. The exchange of ADP for ATP in the NBD causes Hsp70 conformational changes leading to the open state, thereby allowing the client protein to be released and follow its fate. The entire functional cycle of Hsp70 can be regulated by co-chaperones (reviewed in Ref. [2]).

The mitochondrial Hsp70 (also called mtHsp70, GRP75, Hsp70A9 or mortalin in mammalian cells) [9], [10], acts mainly in the mitochondrial matrix providing the strength of the driving force for the translocation of cytosolic proteins into the mitochondrial matrix [11]. In mitochondria, it still operates in the folding of nascent proteins, protein degradation, and the response to oxidative stress and it works to ensure mitochondrial homeostasis [9]. However, in mammalian cells, the role of mortalin is not restricted only to the mitochondrial matrix [12]. This protein is also found in the extra-mitochondrial medium, which has multiple functions, including folding of nascent proteins, intracellular trafficking, antigen processing, control of cell proliferation, interaction with growth factors, cell differentiation, and many other functions [9], [10], [12], [13].

Thus, due to its high importance in several cellular environments, the structural and functional studies of mtHsp70 and its interaction with other chaperones and co-chaperones are of scientific importance and may provide a greater understanding of their cellular roles. Despite being known for a long time, the heterologous expression of mtHsp70 results in an insoluble form of the recombinant protein, which precludes in vitro structural and functional studies. Thus, structural and functional information about this protein, along with its interaction with chaperones, co-chaperones and client proteins, has remained limited. In 2005, a new co-chaperone acting on mtHsp70 to aid in correct folding and maintaining it in the soluble fraction was described [14]. This small mitochondrial protein is called Hep1 (Hsp70-escort protein 1), and through its co-expression with mtHsp70/mortalin, it was possible to obtain the later recombinant protein in its monomeric and functional state [15], [16], [17], [18].

Interestingly, the Hsp70s are involved in the stress response and directly affect Leishmania survival during the adaptation of vector to host [19]. Protozoa parasites of the genus Leishmania spp cause a neglected disease called Leishmaniasis, which affects 0.9–1.3 million people annually, leading 0.2–0.3 million to death. Moreover, some Leishmaniasis symptoms, such as destruction of the mucous membranes and internal organs, make the disease lethal to the host, especially in some countries in the tropics such as Brazil, Colombia, Ecuador, Venezuela and India [20], [21]. During Leishmania protozoa digenetic life cycle, the parasite is required to adapt to a temperature range of approximately 25 °C (insect vector) to 37 °C (mammalian host) [21]. This thermal stress triggers a parasite response that directs the protein synthesis to promote adaptation to the new environment. Among the classes of proteins to be produced, Hsp70, Hsp90 and their co-chaperones have a critical role in the survival and adaptation of the parasite to the temperature variation [21], [22], [23].

Here, we bring up the structure and function of mtHsp70 of Leishmania braziliensis (LbmtHsp70), which also depends on the Hep1 co-chaperone to be produced in soluble and functional form. We already showed the importance of both human Hep1 and Leishmania braziliensis Hep1 for obtaining the recombinant human mortalin and LbmtHsp70, respectively, in a functional state [18], [24]. Summarily, our present data suggest that LbmtHsp70 was produced in a monomeric state with secondary/tertiary structures and exhibited a low ATPase activity, which attested to its functionality. Moreover, LbmtHsp70 was quite elongated, as has been shown for other Hsp70s [25], [26]. Similar to human mortalin and human cytoplasmic Hsp70-1A, LbmtHsp70 interacted with adenosine nucleotides with high affinity. However, it presented a divergent thermodynamic signature. Isothermal titration calorimetry indicated that both enthalpy and entropy drove the LbHep1 interaction with LbmtHsp70 resulting in a dissociation constant of approximately 300 nM, which indicates a high affinity between the two proteins. This result helps to explain the LbHep1 sub-stoichiometric action over LbmtHsp70 thermal-induced aggregation prevention [24]. In addition, LbHsp70 ATPase activity can be stimulated by LbHep1, suggesting that this co-chaperone has additional functions over mtHsp70. Finally, the data suggest that LbmtHsp70 shows a concentration-dependent oligomerization/aggregation process, even at temperatures of 37 °C.