No abstract

Polymerase δ-interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, "moonlighting" protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism and DNA replication and repair. How POLDIP2 interacts with many different proteins is unknown. Towards this goal, we present the crystal structure of POLDIP2 to 2.8 Å, which exhibited a compact two-domain β-strand-rich globular structure, confirmed by circular dichroism and small angle X-ray scattering approaches.POLDIP2 comprised canonical DUF525 and YccV domains, but with a conserved domain linker packed tightly, resulting in an "extended" YccV module.A central channel was observed, which we hypothesize could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full-length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N-terminal region tethered to the YccV-domain by an extended linker, potentially facilitating interactions with distal binding partners. Models of POLDIP2 complexed with two of its partners, PrimPol and PCNA, indicated that dynamic flexibility of the POLDIP2 N-terminus and loop regions likely mediate protein interactions.

Borrelia are microaerophilic spirochetes capable of causing multisystemic diseases such as Lyme disease and Relapsing Fever. The ubiquitous Fe/Mndependent superoxide dismutase (SOD) provides essential protection from oxidative damage by the superoxide anion. Borrelia possess a single SOD enzyme-SodA that is essential for virulence, providing protection against host-derived reactive oxygen species (ROS). Here we present a method for recombinant expression and purification of Borrelia burgdorferi SodA in E. coli. Metal exchange or insertion into the Fe/Mn-SOD is inhibited in the folded state. We therefore present a method whereby the recombinant Borrelia SodA binds to Mn under denaturing conditions and is subsequently refolded by a reduction in denaturant. SodA purified by metal affinity chromatography and size exclusion chromatography reveals a single band on SDS-PAGE. Protein folding is confirmed by circular dichroism. A coupled enzyme assay demonstrates SOD activity in the presence of Mn, but not Fe. The apparent molecular weight determined by size exclusion corresponds to a dimer of SodA; a homology model of dimeric SodA is presented revealing a surface Cys distal to the dimer interface. The method presented of acquiring a target metal under denaturing conditions may be applicable to the refolding of other metal-binding proteins.

Polymerase δ-interacting protein 2 (POLDIP2, PDIP38) is a multifaceted, ‘moonlighting’ protein, involved in binding protein partners from many different cellular processes, including mitochondrial metabolism, DNA replication and repair, and reactive oxygen species generation. POLDIP2 is found in multiple cellular compartments, potentially shuttled depending on its role. How POLDIP2 binds to and coordinates many different proteins is currently unknown. Towards this goal, we present the crystal structure of the ‘mitochondrial’ fragment of POLDIP2 to 2.8 Å. POLDIP2 exhibited a compact two-domain β-strand-rich globular structure, confirmed by circular dichroism and small angle X-ray scattering approaches. POLDIP2 comprised canonical DUF525 (ApaG) and YccV-like domains, but with the conserved domain linker packed tightly, resulting in an ‘extended’ YccV module. A central channel through POLDIP2 was observed which we hypothesise could influence structural changes potentially mediated by redox conditions, following observation of a modified cysteine residue in the channel. Unstructured regions were rebuilt by ab initio modelling to generate a model of full length POLDIP2. Molecular dynamics simulations revealed a highly dynamic N-terminal region tethered to the YccV-domain by an extended linker, potentially facilitating interactions with distal binding partners. Finally we build models of POLDIP2 interacting in complex with two of its partners in genome stability, PrimPol and PCNA. These indicate that dynamic flexibility of the POLDIP2 N-terminal and loop regions are critical to mediate protein-protein interactions.