Structural and DNA binding properties of mycobacterial integration host factor mIHF

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (similar to 300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA.

Functional Dissection of Intersubunit Interactions in the EspR Virulence Regulator of Mycobacterium tuberculosis

Benjamin Blasco, Stewart Cole, Matteo Dal Peraro, Giovanni Dietler, Aleksandre Japaridze, Florence Pojer, Marco Stenta, Basile Isidore Martin Wicky

The nucleoid-associated protein EspR, a chromosome organizer, has pleiotropic effects on expression of genes associated with cell wall function and pathogenesis in Mycobacterium tuberculosis. In particular, EspR binds to several sites upstream of the espACD locus to promote its expression, thereby ensuring full function of the ESX-1 secretion system, a major virulence determinant. The N terminus of EspR contains the helix-turn-helix DNA-binding domain, whereas the C-terminal dimerization domain harbors residues involved in intersubunit interactions. While direct binding to DNA appears to be mediated by an EspR dimer-of-dimers, where two helix-turn-helix motifs remain free for long-range interactions, the mechanism of EspR higher-order organization and its impact on chromosome structure and gene expression are not understood. To investigate these processes, we identified seven amino acid residues using molecular dynamics and replaced them with Ala in order to probe interactions at either the dimer or the dimer-of-dimers interfaces. Arg70, Lys72, and Arg101 were important for protein stability and optimal DNA-binding activity. Moreover, the Arg70 mutant showed decreased dimerization in a mycobacterial two-hybrid system. To correlate these defects with higher-order organization and transcriptional activity, we used atomic force microscopy to observe different EspR mutant proteins in complex with the espACD promoter region. In addition, complementation of an M. tuberculosis espR knockout mutant was performed to measure their impact on EspA expression. Our results pinpoint key residues required for EspR function at the dimer (Arg70) and the dimer-of-dimers (Lys72) interface and demonstrate that EspR dimerization and higher-order oligomerization modulate espACD transcriptional activity and hence pathogenesis.

Amer Soc Microbiology2014

From polymers to gene regulation

Aleksandre Japaridze

DNA molecule is the fundamental component of every living organism, since it encodes the hereditary information. Although the genetic code of almost 200 species has been sequenced, the direct connection between gene sequence, DNA structure and biological function remains poorly understood. The aim of this thesis was to investigate the connection between DNA function and structure. Throughout the thesis we will discuss experiments testing the link between different physical properties of DNA with its biological function. For this purpose we used atomic force microscopy (AFM), a high resolution imaging technique. The first chapter is devoted to explain the basic concepts of AFM technique, as well as the composition and structure of DNA molecules. We also describe some of the polymer physics models of DNA, defining basic quantities like the persistence and the critical exponent. In chapter 2 we discuss experiments, in which we studied changes in the physical properties of DNA molecules, when bound to the substrate surface with various methods. We show that, when DNA is strongly bound to the surface, it retains its physiological B-form. On contrary, when weakly bound, a conformational B-to-A-form transition takes place. We also discuss a novel experimental method, combining nanometer sized PDMS slits with AFM, for studying DNA in geometrical confinement. When DNA is weakly confined, DNA persistence length increases, molecules elongate and adopt more anisotropic shapes. Under stronger confinement, DNA hairpins form. In chapter 3 we study binding of staining dyes, proteins and RNA polymerase (RNAP) to DNA molecules. First we show that DNA physical properties are significantly affected when stained with dyes. Depending on the dye binding mode, either the contour length, or the persistence length or both simultaneously change. We move further and investigate the role of protein amino acids and DNA sequence in the formation of nucleoprotein complexes. On the example of EspR protein, a key virulence regulator in Mycobacterium tuberculosis (MTB), we show that single amino acid mutations, lead to lower DNA binding affinity. These mutations also impair the formation of higher order protein-DNA complexes, silencing the MTB virulence. Afterwards, on the examples of RNAP and H-NS protein, we show the importance of DNA sequence for the binding and higher order oligomerization. By introducing DNA mutations disrupting the helical phasing between RNAP binding sites in the fis promoter sequence, we significantly lower the RNAP binding affinity. The helical phasing of the binding sites somehow coordinates the cooperative binding of RNAP to the promoter. Finally on the model of H-NS protein, we show how different spatial arrangements of strong binding sites for an architectural protein in the DNA, determine the final 3D structure of the assembled nucleoprotein complex. The final chapter, is fully devoted to the study of a completely new type of DNA organisation, which we call Hyperplectonemes. Hyperplectonemes are very ordered DNA structures, formed by large supercoiled molecules, in the presence of attractive DNA-DNA potential. First, we describe their structure and its dependence on various environmental factors, than their binding to nucleoid associated proteins. We argue that this emerging DNA organisation is the basic structure of the bacterial chromatin, which is further modulated by numerous DNA binding and condensing proteins in vivo.

EPFL2015

Exploring on Protein-DNA interactions

Alexandra Styliani Kalantzi

A variety of DNA-binding proteins organizes the chromosomal DNA and regulates gene transcription, and DNA replication and recombination. In particular, for gene regulation there is a category of DNA-binding proteins, the transcription factors, which can detect and bind to a specific set of DNA motifs. For the protein-DNA complexes there are some solved structures, but for some cases, it is difficult to define experimentally these structures at the atomistic level. However, there are various in silico methods, that alone or in combination with experimental techniques, are capable of predicting proteins structures with high accuracy. DNA-binding proteins contain DNA-binding domains and unfortunately, there are few protein-DNA complexes that have been characterized at atomistic resolution. Here we have developed artificial neural networks to predict the binding sites and the interface between proteins and the DNA, based on a structure-based approach. Specifically, we used different interface descriptors and we included a variety of protein-DNA complexes, in order to predict the correct localizations of the DNA on a protein. A large group of proteins that can recognize specific DNA sequences is the KRAB-ZFP family. DNA recognition by zinc finger proteins (ZFPs) plays an important role in gene regulation. However, the molecular determinants defining the recognition of specific DNA nucleotides by ZFP finger repeats has not yet been decoded. Here, we present a method that can predict which ZF repeats specifically bind to a DNA target sequence and what is the most probable DNA target sequence for these repeats. Our method is based on the structural analysis of the binding network of resolved protein DNA complexes, and is validated on a benchmark set of solved ZFP-DNA complexes. We also characterized the binding specificity of two KRAB-ZFPs (ZFP14 and ZNF145) integrating our predictions with SMiLE-seq (Selective Microfluidics-based Ligand Enrichment followed by sequencing) data. Subsequently, we use our recognition code on ChIP-exo data, in order to give an insight into the poly-ZF domains binding patterns. We determined the most probable binding motifs and we separated the ZFPs in two groups: the ones that have a potential canonical and non-canonical binding. Altogether, this work gives an insight into the proteins that interact with the DNA, through a better understanding of the various protein-DNA interfaces and the predictions of these complexes at the atomistic level.

EPFL2019