Nucleoid-Associated Proteins HU and IHF: Oligomerization in Solution and Hydrodynamic Properties

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Liubov A. Dadinova¹, Maxim V. Petoukhov¹, Alexander M. Gordienko¹, Valentin A. Manuvera^2,3, Vassili N. Lazarev^2,3, Tatiana V. Rakitina^4,5, Andrey A. Mozhaev^1,4, Georgy S. Peters⁵, and Eleonora V. Shtykova^1,a*

¹Shubnikov Institute of Crystallography, Federal Scientific Research Centre “Crystallography and Photonics”, Russian Academy of Sciences, 119333 Moscow, Russia

²Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, 119435 Moscow, Russia

³Moscow Institute of Physics and Technology (State University), 141701 Dolgoprudny, Moscow Region, Russia

⁴Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

⁵National Research Centre “Kurchatov Institute”, 123182 Moscow, Russia

^* To whom correspondence should be addressed.

Received December 28, 2022; Revised March 10, 2023; Accepted March 23, 2023

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Structure and function of bacterial nucleoid is controlled by the nucleoid-associated proteins (NAP). In any phase of growth, various NAPs, acting sequentially, condense nucleoid and facilitate formation of its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, Dps protein, is strongly expressed, and DNA–protein crystals are formed that transform nucleoid into a static, transcriptionally inactive structure, effectively protected from the external influences. Discovery of crystal structures in living cells and association of this phenomenon with the bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes formation of the protective DNA–Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying structure of proteins in solution, and dynamic light scattering as a complementary one. To interpret the SAXS data, various approaches and computer programs were used (in particular, the evaluation of structural invariants, rigid body modeling and equilibrium mixture analysis in terms of the volume fractions of its components were applied), which made it possible to determine macromolecular characteristics and obtain reliable 3D structural models of various oligomeric forms of HU and IHF proteins with ~2 nm resolution typical for SAXS. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. An analysis of the experimental and published data made it possible to hypothesize that just before the Dps expression, it is IHF that forms toroidal structures previously observed in vivo and prepares the platform for formation of DNA–Dps crystals. The results obtained are necessary for further investigation of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome resistance of various pathogens to external conditions.

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KEY WORDS: nucleoid-associated proteins, histone-like proteins HU and IHF, oligomerization, small-angle X-ray scattering, dynamic light scattering

DOI: 10.1134/S0006297923050073

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