Abstract
Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA-protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.
Highlights
DNA-binding proteins are essential components of all biological systems, where they perform crucial roles
This study suggests that native intrinsic single-wavelength anomalous dispersion (SAD) methods can be used successfully and routinely on long-wavelength beamlines such as I23 to solve protein–nucleic acid structures de novo, eliminating the need for molecular replacement
The most likely oligomeric state, as suggested by previous studies (Ochiai et al, 2013), is that of an interferon regulatory factor 4 (IRF4) homodimer bound to one interferonstimulated response element (ISRE) element, suggesting a molecular weight for the complex of about 44.8 kDa
Summary
DNA-binding proteins are essential components of all biological systems, where they perform crucial roles. Deregulation or mutation of DNA-binding proteins, such as transcription factors, is closely associated with the pathogenesis of several human diseases, including cancer, making them attractive therapeutic targets (Lee & Young, 2013; Hudson & Ortlund, 2014). Structure solution of protein–DNA complexes provides the basis of our understanding of normal and pathogenic DNA metabolism and underpins attempts to develop novel drugs targeting disease-associated DNA-binding proteins (Bushweller, 2019). The last ten years have witnessed a step-change increase in the number of experimentally determined protein– nucleic acid complexes. More than two thirds of all structures of complexes deposited in the Protein Data Bank (PDB) as of April 2021 (6145 out of 9204) were solved in the last ten years. The number of protein–nucleic acid complex structures solved remains only a small part of the deposited structures as their experimental determination often remains challenging. Even when molecular replacement (MR) can be employed, DNA-binding proteins can be flexible and/or disordered
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