Abstract
(Part1) The 70-kDa heat shock proteins (Hsp70s) are highly conserved ATP-dependent molecular chaperones composed of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD) in a bilobate structure. Interdomain communication and nucleotide-dependent structural motions are critical for Hsp70 chaperone functions. Our understanding of these functions remains elusive due to insufficient structural information of functionally intact Hsp70s in different chaperone cycle states. We report here the crystal structures of DnaK from Geobacillus kaustophilus HTA426 bound with ADP-Mg2+-Pi at 2.37 Å and 70-kDa heat shock cognate protein from Rattus norvegicus bound with ADP-Pi at 3.5 Å. The NBD and SBD in these structures are significantly separated from each other and they may be corresponding to the ADP-bound conformation. Moreover, a Trp reporter was introduced at the potential interface region between NBD and interdomain linker of GkDnaK to probe the environmental changes. The result of fluorescence measurement further supports that the substrate binding enhanced domain disjoining behavior for Hsp70 chaperone family. (Part2) The tail-anchored (TA) proteins are a typical class of membrane proteins, which present a single transmembrane domain (TMD) located near their C-termini. By anchoring the single TMD into the phospholipid bilayer surrounding cellular organelles, the N-terminal cytosolic portion of TA proteins can be properly arranged to cytosol for particular functional purposes. Get3, Get4 and Get5 in Saccharomyces cerevisiae participate in the insertion of tail-anchored proteins into the endoplasmic reticulum membrane. We elucidated the interaction between Get4 and Get5 and investigated their interaction with Get3. Based on crystallographic studies, Get4 and Get5 formed a tight complex, suggesting that they constitute subunits of a larger complex. The Get4 structure shows an overall oblong shape and uses its C-terminal part to interact with Get5. Yeast two-hybrid experiment revealed that Get4 mediates the communication between Get3 and Get5 by interact with these two proteins through its different bisections.