Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

Abstract

Under severe or prolonged stress, bacteria produce a nonspecific DNA‐binding protein (Dps), which effectively protects DNA against damaging agents both in vitro and in vivo by forming intracellular biocrystals. The phenomenon of protective crystallization of DNA in living cells has been intensively investigated during the last two decades; however, the results of studies are somewhat contradictory, and up to now, there has been no direct determination of a Dps–DNA crystal structure. Here, we report the in vitro analysis of the vital process of Dps–DNA co‐crystallization using two complementary structural methods: synchrotron small‐angle X‐ray scattering in solution and cryo‐electron tomography. Importantly, for the first time, the DNA in the co‐crystals was visualized, and the lattice parameters of the crystalline Dps–DNA complex were determined.

Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

“…It was also demonstrated that Dps is unable to bind DNA directly and Dps-DNA complex formation relies on the ion bridges formed by Mg 2+ [32,33]. However, our recent results demonstrate a formation of highly ordered Dps-DNA complexes in solution in the absence of the metal ions [9].…”

“…It is well-known that histones being basic protein components of chromatin act as spools around which DNA winds. In our previous work, we proposed a Dps-DNA binding model based on the SAXS data where DNA is bent around Dps confirming histone-like nature of the protein [9]. Dps proteins have been found in the majority of bacterial groups, including archaea [10].…”

“…Recently, we have collected and analyzed the data of two complementary structural methods, cryo-electron tomography (CET) and synchrotron small-angle scattering (SAXS), and have demonstrated a formation of the central symmetric triclinic crystal structure of the Dps-DNA complex obtained in vitro [9]. It's worth noting that our measurements were performed at pH 8.0 in the buffer containing 0.5 mM EDTA (ethylenediaminetetraacetic acid).…”

Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

“…This technique has achieved subnanometer reconstructions of pleomorphic viruses with imperfect helical symmetry and could prove similarly useful for solving structures from tomograms of disordered nanocrystals [71]. In addition to extending the high-resolution limit of structure determination, the ability to spatially characterize disorder could provide insights into both the organization of proteins that form ordered arrays in vivo and defects in these biological crystals [72][73][74].…”

Protein phase separation and determinants of in cell crystallization

“…The observation of crystals in living cells, reported in the literature as in cellulo or in vivo crystals is known for decades and was repeatedly described as a natural phenomenon . Examples are crystals of storage proteins in seeds, insulin crystals within secretory granules, solid state catalysts, biocrystallization of the DNA‐binding protein (Dps) and DNA as a response to cellular damage and stress, or β‐hematin crystals produced as a detoxification strategy by malaria parasites . Although in cellulo crystallization still is a new exciting area in cell biology, many natively crystallizing proteins in living cells first function as storage such as vitellin yolk protein crystals from bullfrog oocytes, from leopard frogs, from A. aegypti oocytes, lipovitellin from bony fish oocytes, edestin from hemp plant, tobacco seed protein and Cry protein in B. thuringiensis , trichocyst matrix protein in Paramecium and food milk protein in Diplotera punctata .…”