An Overview of Dps: Dual Acting Nanovehicles in Prokaryotes with DNA Binding and Ferroxidation Properties.

DNA binding proteins under starvation (Dps) are proteins belonging to the ferritin family with the capacity for DNA binding, in addition to iron storage and ferroxidation. Present only in the prokaryotes, these multifaceted proteins have been assigned with a number of roles, from pathogenesis to nucleoid condensation and protection. They have a significant role in protecting the cells from free radical assaults, indirectly by sequestration of iron and by directly binding to the DNA. Due to their symmetry, stability and biomineralization capacity, these proteins have ever increasing potential applications in biotechnology and drug delivery. This chapter tries to bring together all these aspects of Dps in the view of current understanding and older perspectives by studies of our group as well as other experts in the field.

A histidine aspartate ionic lock gates the iron passage in miniferritins from Mycobacterium smegmatis.

Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 Å for the various mutants to compare structural alterations vis à vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

Dps Functions as a Key Player in Bacterial Iron Homeostasis.

Iron plays a vital role in the maintenance of life, being central to various cellular processes, from respiration to gene regulation. It is essential for iron to be stored in a nontoxic and readily available form. DNA binding proteins under starvation (Dps) belong to the ferritin family of iron storage proteins and are adept at storing iron in their hollow protein shells. Existing solely in prokaryotes, these proteins have the additional functions of DNA binding and protection from oxidative stress. Iron storage proteins play a functional role in storage, release, and transfer of iron and therefore are central to the optimal functioning of iron homeostasis. Here we review the multifarious properties of Dps through relevant biochemical and structural studies with a focus on iron storage and ferroxidation. We also examine the role of Dps as a possible candidate as an iron donor to iron-sulfur (Fe-S) clusters, which are ubiquitous to many biological processes.

Flexible aspartates propel iron to the ferroxidation sites along pathways stabilized by a conserved arginine in Dps proteins from Mycobacterium smegmatis.

DNA-binding proteins under starvation (Dps) are dodecameric nano-compartments for iron oxidation and storage in bacterial cells. These proteins have roughly spherical structures with a hollow interior where iron is stored. Through mutational analysis of a conserved arginine residue in the second Dps protein from Mycobacterium smegmatis, we have identified residues which stabilize the interfaces between the iron entry and ferroxidation sites. Also, we have used X-ray crystallography to determine the structures of co-crystals of iron and Dps in varying proportions and compare the changes in these ligand-bound forms with respect to the apo-protein. The iron-loaded proteins of low, medium and high iron-bound forms were found to exhibit aspartate residues with alternate conformations, some of which could be directly linked to the sites of ferroxidation and iron entry. We conclude that the increased flexibility of aspartates in the presence of iron facilitates its movement from the entry site to the ferroxidaton site, and the two active sites are stabilized by the interactions of a conserved arginine residue R73.

A Mutation Directs the Structural Switch of DNA Binding Proteins under Starvation to a Ferritin-like Protein Cage.

Proteins of the ferritin family are ubiquitous in living organisms. With their spherical cage-like structures they are the iron storehouses in cells. Subfamilies of ferritins include 24-meric ferritins and bacterioferritins (maxiferritins), and 12-meric Dps (miniferritins). Dps safeguards DNA by direct binding, affording physical protection and safeguards from free radical-mediated damage by sequestering iron in its core. The maxiferritins can oxidize and store iron but cannot bind DNA. Here we show that a mutation at a critical interface in Dps alters its assembly from the canonical 12-mer to a ferritin-like 24-mer under crystallization. This structural switch was attributed to the conformational alteration of a highly conserved helical loop and rearrangement of the C-terminus. Our results demonstrate a novel concept of mutational switch between related protein subfamilies and corroborate the popular model for evolution by which subtle substitutions in an amino acid sequence lead to diversification among proteins.

Nonspecific interaction between DNA and protein allows for cooperativity: a case study with mycobacterium DNA binding protein.

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA-protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA-protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA-protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased. We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA-protein interactions under the same set of conditions.

The mycobacterial MsDps2 protein is a nucleoid-forming DNA binding protein regulated by sigma factors sigma and sigma.

The Dps (DNA-binding protein from starved cells) proteins from Mycobacterium smegmatis MsDps1 and MsDps2 are both DNA-binding proteins with some differences. While MsDps1 has two oligomeric states, with one of them responsible for DNA binding, MsDps2 has only one DNA-binding oligomeric state. Both the proteins however, show iron-binding activity. The MsDps1 protein has been shown previously to be induced under conditions of starvation and osmotic stress and is regulated by the extra cellular sigma factors sigma(H) and sigma(F). We show here, that the second Dps homologue in M. smegmatis, namely MsDps2, is purified in a DNA-bound form and exhibits nucleoid-like structures under the atomic force microscope. It appears that the N-terminal sequence of Dps2 plays a role in nucleoid formation. MsDps2, unlike MsDps1, does not show elevated expression in nutritionally starved or stationary phase conditions; rather its promoter is recognized by RNA polymerase containing sigma(A) or sigma(B), under in vitro conditions. We propose that due to the nucleoid-condensing ability, the expression of MsDps2 is tightly regulated inside the cells.