The goal of this, now amended, first renewal remains to understand Signal Recognition Particle (SRP) dependent targeting of membrane and secretory proteins to the translocation machinery of the cell, at the level of molecular structure and chemical mechanism. This process is highly conserved from bacteria throughout all kingdoms. We have determined structures of the key conserved SRP 54 kD protein (Ffh) of SRP from a thermophilic species, T. aquaticus, including the signal sequence and SRP RNA binding M domain. We determined the structures of 'N' and GTPase domains of Ffh in the apo state which has a key role in the targeting cycle, and the product-like complexes with GDP and Mg++-GDP bound. We have similarly determined the structure of the SRP receptor (SR), FtsY from T. aquaticus, in its apo and its Mg++ GMPPNP bound species. To understand the mechanisms of how these molecules interact as to direct targeting we recently crystallized, and obtained a 1.9A structure of the complex formed between Ffh and FtsY in the presence of a non-hydrolysable GTP analog. This structure beautifully showed how the complex leads to activation of both the GTPases by "substrate twinning" in which each GTP 3'OH forms a hydrogen bond to the gamma-PO4 of the other GTP. The absence of 3'OH on either of the ribose rings reduces total GTPase activity by approximately 400 fold confirming its role. Thus hydrolysis of GTP would release this linkage and dissociate the complex, as required in the SRP mediated cycle. Mutagenesis suggested by the structure provides support for roles of side chains in the active site. These include an aspartate that is key to activation within each GTPase. We seek to elucidate how binding of signal sequences to SRP, assisted by SRP RNA, is transmitted to the interface formed between SRP and SR. We also seek to harness mutagenesis and structure of the T. aquaticus proteins to define contributions to the specificity of the SRP-SR interface. We also seek to understand the only known case in biology in which the SRP does not contain RNA, in chloroplasts (cp), where a protein cpSRP43 fulfills the role of the RNA, aiming first for crystal structures of the cpSRP-cpSRP43-cpSR complex. These will instruct in the mechanism of replacing the role of RNA by protein, and therefore in the essence of the dynamic roles of SRP RNA and cpSRP43.
NIH ExPorter
NIH ExPorter