Structural and functional studies on a small Heat Shock Protein from Entamoeba histolytica

Abstract

Small Heat Shock Proteins (sHSPs) are molecular chaperones that bind to unfolding proteins during cellular stress to prevent them from aggregation. Once the normal conditions are restored, sHSPs release the bound substrate proteins to appropriate cellular machinery either for degradation or for further folding to attain their functional forms. Due to this property of sHSPs to prevent the aggregation of non-native proteins which helps in maintaining proteostasis, they are implicated in several diseases involving defective protein folding. sHSPs are present is all kingdoms of life. Functional characterization of several mammalian, plant and bacterial sHSPs have been reported whereas the structural information is limited. In solution, sHSPs are known to exist as a heterogeneous mixture of a variety of oligomers in a dynamic equilibrium involving constant exchange of subunits. Crystal structures of a few of the higher oligomers as cage-like closed particles have been reported. Studies on protozoan sHSPs are limited. The roles of only a few protozoan sHSPs in infection, survival and migration in the host have been reported so far and no structural information is available. Here we present the structural and functional studies that we carried out on a sHSP (HSP18.5) from the protozoan Entamoeba histolytica. The activity of HSP18.5 has been demonstrated as it could (i) protect the restriction enzyme Nde I at high temperatures and (ii) prevent the aggregation of chemically denatured lysozyme. Fluorescence and FRET studies with bis-ANS established the binding of bis-ANS in the hydrophobic sites of HSP18.5 and its proximity to and interaction with tryptophan residues. HSP18.5 showed a pH-dependent change in its oligomeric state which varied from a tetramer to oligomers higher than 48-mers. Functional studies on tetramers, octamers and higher oligomers were carried out and compared. HSP18.5 has been crystallized and the structure has been determined to a resolution of 3.28 Å. There are four subunits in the asymmetric unit of the C2221 cell forming two dimers which interact with each other through the C-terminal I/V-X-I/V motif of each dimer with a hydrophobic groove of the other dimer. This interaction is known to be involved in the formation of dimer-dimer association in higher oligomers of sHSPs. The other C-terminus of each of the dimers forms a β-strand and is involved in two novel types of interactions with symmetry-related dimers: one is a β-sheet type of interaction with the antiparallel C-terminal strand and the other is through the I/V-X-I/V motif. However, this motif is not the same as the previous motif between the two dimers in the asymmetric unit, as HSP18.5 has the sequence V-X-V-X-I (Val159-Asn160-Val161-Glu162-Ile163) with overlapping motifs; the first interaction is with the first three residues and the second one is with the last three. Application of crystal symmetry shows a three-dimensional extended network of the molecules but no closed cage-like structure is formed. However, preliminary electron microscopy studies revealed the presence of spherical structures of various sizes and also the formation of long fibrils. Thus, the structure of HSP18.5 shows significant pH-dependent variations in its oligomeric association and reveals new type of interactions between dimers involving the C-terminus