Small Heat Shock Proteins from Bacteria and a Bacteriophage

Abstract

Proteins can unfold during heat and other types of stress and tend to aggregate with subsequent loss of function. This event is prevented by small Heat Shock Proteins (sHSPs) acting as molecular chaperones. sHSPs do not bind to native proteins but interact with unfolding proteins during stress conditions and transfer them to the other heat shock proteins for proper folding once normalcy is restored. sHSPs thus maintain cellular proteostasis and unlike the other heat shock proteins of higher molecular weight, sHSPs function in an ATP-independent manner. sHSPs are present in all kingdoms of life with many organisms having multiple sHSPs with little overall sequence identity. The molecular weight of sHSPs varies between 12 to 40 kDa. The structure of -crystallin domain present in sHSPs is conserved and is flanked by flexible N- and C- terminal regions with roles mainly in substrate binding and oligomerization, respectively. sHSPs often form large oligomeric assemblies with the dimeric unit as the basic building block. sHSPs exist in a dynamic equilibrium by constant exchange of subunits between oligomers of various sizes. They have a broad substrate specificity, however, there is a dearth of knowledge on the exact mechanism of substrate binding and the necessity for the formation and exact functioning of different oligomers. The available structural information on sHSPs is limited as the three-dimensional structures of only a few higher order oligomers have been determined to date. To obtain insights into the functions and structures of sHSPs and the correlation between them and as a part of an ongoing project on sHSPs, we have carried out biophysical, biochemical and structural investigations on sHSPs of three bacteria: Bacillus cereus, Staphylococcus aureus and a cyanobacterium Synechococcus sp. WH7803; and a bacteriophage of Synechococcus sp. WH7803, called Synechococcus phage S-ShM2, which are presented in this thesis. The full length and a few deletion constructs of these four sHSPs were generated. The chaperone activity of all the constructs was investigated using two denatured substrates: NdeI and lysozyme. The size and the content of the oligomers were determined by SEC-MALS and DLS. The importance of the C-terminal I-X-I motif in higher oligomer formation and the role of the N-terminus in the chaperone activity and oligomerization of sHSPs were examined. Though crystallization of all the constructs was attempted, crystals of only the cyanophage sHSP (SM2), which diffracted to a low resolution of 7 Å could be obtained. SM2 crystallized as a 24-mer with 432 symmetry similar to the sHSP of Methanococcus jannaschii and one of the forms of AgsA of Salmonella typhimurium. The dimer is similar to that found in sHSPs of non-metazoans. Negative stain EM images showed that the protein is heterogeneous in nature with a major population of large cage-like assemblies and a minor population of smaller cages. 3D structures of both of them have been determined by Cryo-EM 3D image reconstruction at a resolution of ~ 8 Å. The larger particles are 60-mers with 52 symmetry and the smaller ones are 48-mers with 432 symmetry. The crystallographic and EM studies show that SM2 is capable of forming various types of oligomers, which vary in size and symmetry. This is the first structural report of a viral or a phage sHSP. This is the largest sHSP particle (60-mer) of all reported structures and the 52 symmetry is observed for the first time in sHSPs. Despite the variations, certain common features are observed in the three particles. Hexameric sub-assemblies made up of three dimers with 32 symmetry are present in all the three oligomers. Though the structures were determined at low resolutions, it is clear that the C-terminal I-X-I motif interacts with the neighboring molecules in a way similar to that observed in other sHSP structures. The flexibility of the C-terminal region combined with slight differences in the arrangement of dimers in the hexamer enabled the variation in oligomerization. Our studies confirmed the highly polydisperse and heterogenic nature of sHSPs, however, we could study the structures of three types of particles in isolation which revealed new features related to the oligomerization and symmetry of sHSPs.