Structural and molecular dynamics simulation studies support Symplekin's protein scaffolding role and a novel fold in the TraI relaxase-helicase C-terminus is essential for conjugative DNA transfer

The majority of eukaryotic pre-mRNAs are processed by 3'-end cleavage and polyadenylation, although in metazoa the replication-dependant histone mRNAs are subject only to 3'-end cleavage and not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the ∼1,160-residue protein Symplekin. Secondary structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain was investigated to begin to elucidate the role Symplekin plays in mRNA maturation. The crystal structure the D. melanogaster Symplekin HEAT domain was determined to 2.4 A resolution using SAD phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 29 amino acid loop between the fourth and fifth repeat (loop 8) that is both unique and conserved in Symplekin sequences. Molecular dynamics simulations of this domain show that loop 8 dampens the overall motion of the HEAT domain, therefore providing a stable surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts, and the Symplekin HEAT region structurally aligns with several established scaffolding proteins. Taken together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.;The TraI relaxase-helicase is the central catalytic component of the multi-protein relaxosome complex responsible for conjugative DNA transfer (CDT) between bacterial cells. CDT is a primary mechanism for the lateral propagation of microbial genetic material, including the spread of antibiotic resistance genes. The 2.4 A resolution crystal structure of the C-terminal domain of the multifunctional E. coli F plasmid TraI protein (TraI-CT) is presented, and specific structural regions essential for CDT are identified. The crystal structure reveals a novel fold composed of a 39-residue N-terminal alpha-helical extension connected by a proline-rich loop to a compact alpha/beta core domain. Both the globular nature of the alpha/beta-domain and the presence and rigidity of the proline-rich loop are required for DNA transfer and single-stranded DNA binding. Taken together, these data establish the specific structural features of this non-catalytic domain that are essential to DNA conjugation.