Zn2+ REMOVAL FROM HUMAN DNAJA CO-CHAPERONES AFFECTS BOTH THEIR STABILITY AND THE FORMATION OF A PARTIALLY FOLDED INTERMEDIATE STATE

Introduction: The human zinc-binding proteins DNAJA1 and DNAJA4 are HSP40 class A co-chaperones of the HSP70 chaperone, that share about 75% of identity and hence a highly conserved structural set of domains, such as a cysteine-rich domain formed by four repeats of the CXXCXGXG type motif. These motifs fold in a zinc-dependent manner allowing DNAJAs to carry two Zn2+ ions by monomer in their native states. This work investigates the importance of zinc by obtaining both DNAJA1 and DNAJA4 recombinant proteins and extracting their Zn2+ ions by using chelating agents. This approach allows for the study and comparison of the zinc bound and unbound states of the protein by powerful biophysical techniques. Materials and methods: Both proteins were expressed in E. coli and purified by Ni2+ affinity and gel filtration chromatography. EDTA and 1,10-Phenanthroline were used as chelators to remove their Zn2+ cofactors and ICP-MS was applied for zinc quantification. Circular dichroism (CD) spectropolarimetry was used to check protein folding and to isolate both DNAJA1 and DNAJA4 protein unfolding intermediates. Protein oligomeric states and hydrodynamics were studied by size exclusion chromatography combined with a multi-angle light scattering detector (SEC-MALS). Thermal stability was assessed by Differential Scanning Fluorescence (DSF). Finally, Thioflavin T (ThT) and Bis-ANS fluorescence essays were used to get more information on their isolated unfolding intermediate structures. Results and discussion: DNAJA1 and DNAJA4 were obtained with high purity and their CD spectra exhibited negative bands around 208 nm and 225 nm. Both showed at least one unfolding intermediate state in thermal denaturing essays monitored by CD at 222 nm, whose CD spectra indicate a gain of structure when compared to the native and chelating-treated forms. SEC-MALS experiments demonstrated that both proteins display a dimeric form in solution and similar hydrodynamics radius and diffusion coefficients. DSF data showed that Zn2+-removed forms are less stable than native ones, and extrinsic fluorescence essays revealed that both intermediates are capable of binding bis-ANS and ThT fluorescent probes. Conclusion: These results show that both DNAJA1 and DNAJA4 are structurally similar. Furthermore, they suggest that Zn2+ ions are important for their stability and their absence seems to contribute to their unfolding intermediate state formation and isolation.

This work was supported by the São Paulo Research Foundation (FAPESP #2017/26131-5) and by the National Council for Scientific and Technological Development (CNPq) and by CAPES Foundation.