Crystal Structures And Functional Studies Of The Pyrococcus Furiosus RecJ-like Nuclease And Using Xenon For Phase Determination Of Protein Trichosanthin Structure

Euryarchaea Pyrococcus furiosus RecJ-like protein (PF2055, PfuRecJ) has beenidentified as a nuclease belonging to the DHH phosphatase family, which degradesingle-stranded DNA from5’end, as well as ssRNA from3’end. In the first part of mythesis work, we successfully solved the novel crystal structure of PfuRecJ by usingSe-SAD method. Furthermore, it is the first time that we define an extra structuraldomain (domain M) located between the N terminal catalytical domain (domain I) andthe C terminal substrate-specificity domain (domain II). Protein sequence BLASTresults show that the middle domain exists in euryarchaeal protein RecJ as well as itseukaryotic homolog Cdc45, but not in bacteria RecJ protein. Although the exactfunction of this domain is still unknown, it is supposed to be involved in theformation of archaeal chromosomal replicative helicase complex Cdc45/RecJ-MCM-GINS as found in eukaryotic cells.Following the first solved PfuRecJ structure, a series of structures of PfuRecJcomplexed with various divalent metal ions (Mn2+, Mg2+and Zn2+) and singlenucleotides (5’-CMP and5’-dCMP) were determined. The residues involved in metalion binding and substrate catalysis were identified. In general, divalent metal ionshave their preferred coordination numbers in the complex with proteins. The affinityof metal ion for a given set of ligands and therefore the relative stability of complexesformed follows the Irving-Williams series order: Mg2+＜Mn2+＜Fe2+＜Co2+＜Ni2+＜Cu2+＞Zn2+. As it is shown in structures of PfuRecJ-metal ion complexes, there arethree Zn2+identified in the metal ion binding site, but only one Mn2+or Mg2+.Moreover, it is found that the occupied position of the third Zn2+is highly overlappedwith the5’-phosphate group of single nucleotide that mimic the substrate bound. Thisindicates the binding of third zinc ion will block the substrate accessing to thecatalytic site of PfuRecJ. Based on the structural results, we propose the mechanismhow different divalent metal ions impacted on the nuclease activity of PfuRecJ. The noble gas, xenon (Xe) can be used as a “heavy” atom for determining phasesin protein crystallography. In the second part of my thesis work, the Xe gas waspressurized into the crystals of recombinant protein trichosanthin (TCS) under thepressures ranging from1to3MPa. Using single anomalous scattering diffractionmethod, we demonstrate that an interpretable electron density map can be obtained forprotein trichosanthin from a single xenon derivative. Moreover, for the first time, weidentified a pre-existing hydrophobic cavity just under the protein surface oftrichosanthin. Combined with the mica surface induced epitaxial crystal growthproperties of TCS, it seems that TCS would be developed into a powerful andgenerally applicable fusion tag in protein crystallography to solve the “unknown”protein structure.