Structural And Functional Researches On RNase D Homologous Proteins In Agrobacterium Tumefaciens

RNase D(RND)is a DEDD exonuclease.It has been reported responsible for the 3' maturation of several structured RNAs.Although RNase Ds exist in bacteria and eukaryote widely,current research mainly focused on the RNase D proteins from Escherichia coli(Ec_RND).Ec_RND contains three conserved domains:one N-terminal DEDD domain and two C-terminal HRDC domains.The DEDD domain is responsible for catalysis and the HRDC domains are involved in the substrate binding.However,among the huge number of the RNase D homologues,the C-terminal HRDC domains are not well conserved as the DEDD domain.Some RNase D homologues only have one HRDC domain and part of these homologues even lack the both HRDC domains,becoming single domain proteins.At the same time,there are more than one RNase D homologues in the genome of some bacteria.Until now,the research is scarce on these shortened RNase D proteins.It remains obscure that how these shortened RNase D proteins function without the substrate binding HRDC domains.Our research focused on the RNase D homologues in Agrobacterium tumefaciens.We identified two RNase D homologues Atul151(denoted At RND)and Atu4108(denoted At_NrnC)with blastp sever.At RND is a typical RNase D protein,which has all the three domains and has 30%similarity with the full-length Ec_RND.At_NrnC only has the DEDD domain and shares 31%similarity with the N-terminal 184 residues of Ec RND.At the same time,the similarity between the DEDD domain of At RND and At_NrnC is 32%,one not high value.We cloned and purified the At_RND and At_NrnC proteins and determined the three dimensional structure of these two proteins with X-ray diffraction method after obtaining corresponding protein crystals.At the same time,the enzymatic activities of At RND and At_NrnC towards different nucleic acid were checked and compared with previous reported Ec RND and Oligoribonuclease(Orn).Through the biochemical and structural analysis,we obtained the following results:I)At_RND contains three domains.The overall structure of At_RND is similar with that of Ec_RND.Although the relative position of the three domains in At_RND does not perfectly overlap with that in Ec_RND,the triangle architecture remains similar with that in Ec_RND.In our opinion,the conformational difference arises from the instability of the relative positions of the three domains.The connection element between these domains of both At_RND and Ec_RND is unstructured loops and no strong interactions between these domains are observed.Both above observations support our hypothesis.2)Through sequence alignment,we noticed that the last HRDC domain of At_RND is about 20 amino acids longer than that of Ec_RND.These additional 20 amino acids form two(3 strands.In crystal lattice,these two ? strands interact with the ? strands from the adjacent svmmetry At_RND molecules,forming inter-molecule ?-sheet.However,such kind of interaction formed by additional ? strands does not occur in the crystal structure of the Ec_RND.We proposed that the interaction between the adjacent symmetry HRDC domains could help crystal packing.but in solution,At_RND proteins are mainly in monomer form and this is the active state.3)Like Ec_RND,At_RND can degrade both single-stranded RNA(ssRNA)and double-stranded RNA(dsRNA)efficiently,but almost has no activity towards single-stranded DNA(ssDNA),and double-stranded DNA(dsDNA).4)At_NrnC is a nuclease containing only a single DEDD domain.Its three-dimensional structure is similar to the DEDD domain of both the At_RND and Ec_RND.However.in the asymmetry unit of the At_NrnC.the two At_NrnC molecules binding together with a huge interaction interface,thus forming high level oligomeric state.We noticed the At NrnC dimer is much different from other structure-determined DEDD nuclease in that the active pockets of the two protomer are in the same side of the dimer.5)We obtained the apo form and two kinds metal binding forms of At_NrnC structures separately.Through comparison of these three structures,we noticed the dynamic state of the active pocket:the catalytic Tyr 153 and an adjacen lopp will flip out without substrate binding after metal coordinating,thus making space for the entry of the substrate;at the same time,the distance between the two metals can change dramatically before the catalysis.6)Further,we noticed that the structure of the At_NrnC are similar to other nuclease families,such as Orn and RNase T,which also belong to the DEDD nuclease super family.It was reported that these nucleases can form homodimer,thus obtaining substrate binding surface from each other monomers and ensure the processivity of the degrading process.After analysis of the crystal structure of the At_NrmC,we hypothesize that the At_NrnC may form an octamer to overcome the lack of the substrate binding surface.The results of size-exclusion chromatography(SEC),analytical ultracentrifugation(AUC)and molecular dynamics simulation all support our hypothesis that the At_NrnC octamer is stable in solution.7)We performed the nuclease assays of At_NrnC on different substrates.Unexpectedly,these assays revealed that At_NrnC possesses remarkably different substrate specificity with Ec RND and At RND.It can hydrolyze ssRNA,ssDNA and dsDNA with high efficiency but almost has no activity on dsRNA.Furthermore,At_NrnC can hydrolyze long dsDNA with high efficiency,which is quite interesting that At_NrnC was identified as a NanoRNase before our experiment.8)We hypothesize that the forming of an octamer is necessary for the nuclease activity:eight At NrnC molecules forms two layers,constituting a central hollow architecture;the substrate has to enter the central tunnel and then reaches the active pocket;the dimensional of the central tunnel is relative stable for the strong interactions which hold the octamer state.According to our hypothesis,the central tunnel just right allows the entry of dsDNA.Due to the larger diameter of dsRNA,it cannot enter the tunnel and thus cannot be degraded by At NrnC.To further support our hypothesis,we mutated some alkaline lysine residues into acidic residues,which we proposed are involved in helping the entry of the nucleic acid.While keeping the stable octamer state,these mutated proteins completely lost the enzymatic activity.