Structural and functional characterization of sHsp from the marine cyanobacterium Picosynechococcus sp. NKBG15041c

Small heat-shock proteins (sHsps) are ATP-independent chaperones whose oligomerization and client capture are regulated by a conserved C-terminal IXI/V motif. Here, we identify and characterize an sHsp from the marine cyanobacterium Picosynechococcus sp. NKBG15041c (NKBG-sHsp), which has an acidic C-terminal extension following IXI/V. We dissect the role of its acidic C-terminal extension using truncation mutants, one with the acidic C-terminal extension deleted (NKBG-sHspΔC), and another with both the acidic C-terminal extension and the IXI/V motif deleted (NKBG-sHspΔΔC). By size-exclusion chromatography by HPLC, NKBG-sHsp and NKBG-sHspΔC existed as large oligomers at 20 °C and reversibly dissociated into smaller species at 50 °C, whereas NKBG-sHspΔΔC remained a small oligomer without a temperature response. By analytical ultracentrifugation, NKBG-sHsp and NKBG-sHspΔC predominantly appeared as dodecamers with relatively large assemblies, whereas NKBG-sHspΔΔC migrated as a hexamer. In chaperone assays, NKBG-sHsp suppressed DTT-induced lysozyme aggregation in a dose-dependent manner. NKBG-sHspΔC showed weaker protection activity and was prone to coaggregation, and NKBG-sHspΔΔC failed to protect lysozyme and co-aggregated with lysozyme. NKBG-sHsp protected glyceraldehyde-3-phosphate dehydrogenase from thermal aggregation. The protection ability of NKBG-sHspΔC was weaker. Curiously, NKBG-sHspΔΔC exhibited comparable or superior activity. These data indicate that the IXI/V motif is essential for higher-order assembly, whereas acidic C-terminal extension promotes the formation of soluble client complexes and suppresses coaggregation. NKBG-sHsp demonstrates significant chaperone activity by preventing protein aggregation, potentially enhancing the stress resistance of Picosynechococcus sp. NKBG15041c. The acidic C-terminal extension may help mitigate this risk of NKBG-sHsp congregation.