Abstract
Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones preventing the irreversible denaturation of proteins. While in Escherichia coli two sHSPs IbpA and IbpB work in strong cooperation, the sole Mollicute with free-living ability Acholeplasma laidlawii carries a single gene encoding the sHSP protein AlIbpA. In vitro, independently of the temperature, AlIbpA forms a heterogeneous mixture of approximately 24-mer globules, fibrils and huge protein aggregates. The removal of either 12 or 25 N-terminal amino acids led to the formation of fibrils and enhanced the protein ability to prevent the temperature-induced aggregation of insulin, assuming the fibrillar form as an active protein. In turn, the deletion of the C-terminus or substitution of C-terminal LEL motif by SEP decreased the temperature stability of AlIbpA and eliminated its chaperone function completely, although the protein remained predominantly in a globular state. This suggests that the C-terminal LEL motif is necessary for the chaperon-like activity of AlIbpA and fibril formation. Double N- and C-terminal truncations abolished both the chaperone-like activity and huge oligomer formation. Since the globular form of sHSPs is considered as their inactive form, our data suggest that the N-terminus of AlIbpA is responsible for the huge globule (low-active form) formation and behaves as an intramolecular inhibitor of the fibrils (active form) formation and substrates binding. Taken together these data demonstrate non-trivial properties of AlIbpA, in which the competitive action of N- and C-termini governs the equilibrium between either fibrillar or globular structures representing a possible molecular mechanism of the AlIbpA activity regulation.