Bacterial chromosome organization. I. Crucial role of release of topological constraints and molecular crowders.

We showed in our previous studies that just 3% cross-links (CLs), at special points along the contour of the bacterial DNA, help the DNA-polymer to get organized at micron length scales [T. Agarwal et al., J. Phys.: Condens. Matter 30, 034003 (2018) and T. Agarwal et al., EPL (Europhys. Lett.) 121, 18004 (2018)]. In this work, we investigate how does the release of topological constraints help in the "organization" of the DNA-polymer. Furthermore, we show that the chain compaction induced by the crowded environment in the bacterial cytoplasm contributes to the organization of the DNA-polymer. We model the DNA chain as a flexible bead-spring ring polymer, where each bead represents 1000 base pairs. The specific positions of the CLs have been taken from the experimental contact maps of the bacteria Caulobacter crescentus and Escherichia coli. We introduce different extents of ease of release of topological constraints in our model by systematically changing the diameter of the monomer bead. It varies from the value where chain crossing can occur freely to the value where chain crossing is disallowed. We also study the role of compaction of the chain due to molecular crowders by introducing an "effective" weak Lennard-Jones attraction between the monomers. Using Monte Carlo simulations, we show that the release of topological constraints and the crowding environment play a crucial role to obtain a unique organization of the polymer.

Bacterial chromosome organization. II. Few special cross-links, cell confinement, and molecular crowders play the pivotal roles.

Using a coarse-grained bead-spring model of bacterial chromosomes of Caulobacter crescentus and Escherichia coli, we show that just 33 and 38 effective cross-links in 4017 and 4642 monomer chains at special positions along the chain contour can lead to the large-scale organization of the DNA polymer, where confinement effects of the cell walls play a key role in the organization. The positions of the 33/38 cross-links along the chain contour are chosen from the Hi-C contact map of bacteria C. crescentus and E. coli. We represent 1000 base pairs as a coarse-grained monomer in our bead-spring flexible ring polymer model of the DNA polymer. Thus, 4017/4642 beads on a flexible ring polymer represent the C. crescentus/E. coli DNA polymer with 4017/4642 kilo-base pairs. Choosing suitable parameters from Paper I, we also incorporate the role of compaction of the polymer coil due to the presence of molecular crowders and the ability of the chain to release topological constraints. We validate our prediction of the organization of the bacterial chromosomes with available experimental data and also give a prediction of the approximate positions of different segments within the cell. In the absence of confinement, the minimal number of effective cross-links required to organize the DNA chains of 4017/4642 monomers was 60/82 [Agarwal et al., Europhys. Lett. 121, 18004 (2018) and Agarwal et al., J. Phys.: Condens. Matter 30, 034003 (2018)].

Structure function relations in PDZ-domain-containing proteins: Implications for protein networks in cellular signalling.

Protein scaffolds as essential backbones for organization of supramolecular signalling complexes are a recurrent theme in several model systems. Scaffold proteins preferentially employ linear peptide binding motifs for recruiting their interaction partners. PDZ domains are one of the more commonly encountered peptide binding domains in several proteins including those involved in scaffolding functions. This domain is known for its promiscuity both in terms of ligand selection, mode of interaction with its ligands as well as its association with other protein interaction domains. PDZ domains are subject to several means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to the effect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical and biophysical basis for this selectivity as well as promiscuity has been investigated and reviewed extensively. The present review focuses on the plasticity inherent in PDZ domains and its implications for modular organization as well as evolution of cellular signalling pathways in higher eukaryotes.

Role of special cross-links in structure formation of bacterial DNA polymer.

Using data from contact maps of the DNA-polymer of Escherichia coli (E. Coli) (at kilobase pair resolution) as an input to our model, we introduce cross-links between monomers in a bead-spring model of a ring polymer at very specific points along the chain. Via suitable Monte Carlo simulations, we show that the presence of these cross-links leads to a particular organization of the chain at large (micron) length scales of the DNA. We also investigate the structure of a ring polymer with an equal number of cross-links at random positions along the chain. We find that though the polymer does get organized at the large length scales, the nature of the organization is quite different from the organization observed with cross-links at specific biologically determined positions. We used the contact map of E. Coli bacteria which has around 4.6 million base pairs in a single circular chromosome. In our coarse-grained flexible ring polymer model, we used 4642 monomer beads and observed that around 80 cross-links are enough to induce the large-scale organization of the molecule accounting for statistical fluctuations caused by thermal energy. The length of a DNA chain even of a simple bacterial cell such as E. Coli is much longer than typical proteins, hence we avoided methods used to tackle protein folding problems. We define new suitable quantities to identify the large scale structure of a polymer chain with a few cross-links.

Molecular Mechanism Underlying ATP-Induced Conformational Changes in the Nucleoprotein Filament of Mycobacterium smegmatis RecA.

RecA plays a central role in bacterial DNA repair, homologous recombination, and restoration of stalled replication forks by virtue of its active extended nucleoprotein filament. Binding of ATP and its subsequent recognition by the carboxamide group of a highly conserved glutamine (Gln196 in MsRecA) have been implicated in the formation of active RecA nucleoprotein filaments. Although the mechanism of ATP-dependent structural transitions in RecA has been proposed on the basis of low-resolution electron microscopic reconstructions, the precise sequence of events that constitute these transitions is poorly understood. On the basis of biochemical and crystallographic analyses of MsRecA variants carrying mutations in highly conserved Gln196 and Arg198 residues, we propose that the disposition of the interprotomer interface is the structural basis of allosteric activation of RecA. Furthermore, this study accounts for the contributions of several conserved amino acids to ATP hydrolysis and to the transition from collapsed to extended filament forms in Mycobacterium smegmatis RecA (MsRecA). In addition to their role in the inactive compressed state, the study reveals a role for Gln196 and Arg198 along with Phe219 in ATP hydrolysis in the active extended nucleoprotein filament. Finally, our data suggest that the primary, but not secondary, nucleotide binding site in MsRecA isomerizes into the ATP binding site present in the extended nucleoprotein filament.

Crystallization and preliminary X-ray studies of the C-terminal domain of Mycobacterium tuberculosis LexA.

The C-terminal domain of Mycobacterium tuberculosis LexA has been crystallized in two different forms. The form 1 and form 2 crystals belonged to space groups P3(1)21 and P3(1), respectively. Form 1 contains one domain in the asymmetric unit, while form 2 contains six crystallographically independent domains. The structures have been solved by molecular replacement.

Functionally important movements in RecA molecules and filaments: studies involving mutation and environmental changes.

The crystal structures of mutants of Mycobacterium smegmatis RecA (MsRecA) involving changes of Gln196 from glutamine to alanine, asparagine and glutamic acid, wild-type MsRecA and several of their nucleotide complexes have been determined using mostly low-temperature and partly room-temperature X-ray data. At both temperatures, nucleotide binding results in a movement of Gln196 towards the bound nucleotide in the wild-type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 19 crystal structures reported here, together with 11 previously reported MsRecA structures, provide further elaboration of the relation between the pitch of the ;inactive' RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low-temperature structures define one extreme of the range of positions the C-terminal domain can occupy. The movement of the C-terminal domain is correlated with those of the LexA-binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the ;active' filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron-microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. The work reported here contributes to the description of the early stages of this trajectory and provides insight into structures relevant to the later stages.

Snapshots of RecA protein involving movement of the C-domain and different conformations of the DNA-binding loops: crystallographic and comparative analysis of 11 structures of Mycobacterium smegmatis RecA.

Mycobacterium smegmatis RecA and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals reported here and five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P6(1). They include one obtained by reducing relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 6(1) screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported for Escherichia coli RecA, the variation in the pitch among the three forms correlates well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue, which is believed to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue similar to that caused by nucleotide binding. The ordering of the DNA-binding loops, which present ensembles of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide binding, presumably on account of the movement of the switch residue that forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications for intermolecular communications within the RecA filament. The structures resulting from different orientations of the C domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA at different phases of activity, and provide insights into the mechanism of action of RecA.

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery.

RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102-105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide.

Structure of Mycobacterium smegmatis single-stranded DNA-binding protein and a comparative study involving homologus SSBs: biological implications of structural plasticity and variability in quaternary association.

The structure of Mycobacterium smegmatis single-stranded DNA-binding protein (SSB) has been determined using three data sets collected from related crystals. The structure is similar to that of its homologue from Mycobacterium tuberculosis, indicating that the clamp arrangement that stabilizes the dimer and the ellipsoidal shape of the tetramer are characteristic features of mycobacterial SSBs. The central OB fold is conserved in mycobacterial SSBs as well as those from Escherichia coli, Deinococcus radiodurans and human mitochondria. However, the quaternary structure exhibits considerable variability. The observed plasticity of the subunit is related to this variability. The crystal structures and modelling provide a rationale for the variability. The strand involved in the clamp mechanism, which leads to higher stability of the tetramer, appears to occur in all high-G+C Gram-positive bacteria. The higher stability is perhaps required by these organisms. The mode of DNA binding of mycobacterial SSBs is different from that of E. coli SSB partly on account of the difference in the shape of the tetramers. Another difference between the two modes is that the former contains additional ionic interactions and is more susceptible to salt concentration.