DNA controls the dimerization of the human FoxP1 forkhead domain.

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences and gating access to genes. Even when the binding of TFs and their cofactors to DNA is reversible, indicating a reversible control of gene expression, there is little knowledge about the molecular effect DNA has on TFs. Using single-molecule multiparameter fluorescence spectroscopy, molecular dynamics simulations, and biochemical assays, we find that the monomeric form of the forkhead (FKH) domain of the human FoxP1 behaves as a disordered protein and increases its folded population when it dimerizes. Notably, DNA binding promotes a disordered FKH dimer bound to DNA, negatively controlling the stability of the dimeric FoxP1:DNA complex. The DNA-mediated reversible regulation on FKH dimers suggests that FoxP1-dependent gene suppression is unstable, and it must require the presence of other dimerization domains or cofactors to revert the negative impact exerted by the DNA.

Domain tethering impacts dimerization and DNA-mediated allostery in the human transcription factor FoxP1.

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)-known to form coiled-coil dimers-and a Forkhead (FKH) domain-known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.

Out-of-Equilibrium Biophysical Chemistry: The Case for Multidimensional, Integrated Single-Molecule Approaches.

Out-of-equilibrium processes are ubiquitous across living organisms and all structural hierarchies of life. At the molecular scale, out-of-equilibrium processes (for example, enzyme catalysis, gene regulation, and motor protein functions) cause biological macromolecules to sample an ensemble of conformations over a wide range of time scales. Quantifying and conceptualizing the structure-dynamics to function relationship is challenging because continuously evolving multidimensional energy landscapes are necessary to describe nonequilibrium biological processes in biological macromolecules. In this perspective, we explore the challenges associated with state-of-the-art experimental techniques to understanding biological macromolecular function. We argue that it is time to revisit how we probe and model functional out-of-equilibrium biomolecular dynamics. We suggest that developing integrated single-molecule multiparametric force-fluorescence instruments and using advanced molecular dynamics simulations to study out-of-equilibrium biomolecules will provide a path towards understanding the principles of and mechanisms behind the structure-dynamics to function paradigm in biological macromolecules.

Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS.

CGG tandem repeat expansion in the 5'-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A.

The protein p27, a prominent regulatory protein in eukaryotes and an intrinsically disordered protein (IDP), regulates cell division by causing cell cycle arrest when bound in ternary complex with cyclin-dependent kinase (Cdk2) and cyclins (e.g., Cdk2/Cyclin A). We present an integrative study of p27 and its binding to Cdk2/Cyclin A complex by performing single-molecule multiparameter fluorescence spectroscopy, stopped-flow experiments, and molecular dynamics simulations. Our results suggest that unbound p27 adopts a compact conformation and undergoes conformational dynamics across several orders of magnitude in time (nano-to milliseconds), reflecting a multi-step mechanism for binding Cdk2/Cyclin A. Mutagenesis studies reveal that the region D1 in p27 plays a significant role in mediating the association kinetics, undergoing conformational rearrangement upon initial binding. Additionally, FRET experiments indicate an expansion of p27 throughout binding. The detected local and long-range structural dynamics suggest that p27 exhibits a limited binding surface in the unbound form, and stochastic conformational changes in D1 facilitate initial binding to Cdk2/Cyclin A complex. Furthermore, the post-kinase inhibitory domain (post-KID) region of p27 exchanges between distinct conformational ensembles: an extended regime exhibiting worm-like chain behavior, and a compact ensemble, which may protect p27 against nonspecific interactions. In summary, the binding interaction involves three steps: (i) D1 initiates binding, (ii) p27 wraps around Cdk2/Cyclin A and D2 binds, and (iii) the fully-formed fuzzy ternary complex is formed concomitantly with an extension of the post-KID region. An understanding of how the IDP nature of p27 underpins its functional interactions with Cdk2/Cyclin A provides insight into the complex binding mechanisms of IDPs and their regulatory mechanisms.

A B-Z junction induced by an A A mismatch in GAC repeats in the gene for cartilage oligomeric matrix protein promotes binding with the hZαADAR1 protein.

GAC repeat expansion from five to seven in the exonic region of the gene for cartilage oligomeric matrix protein (COMP) leads to pseudoachondroplasia, a skeletal abnormality. However, the molecular mechanism by which GAC expansions in the COMP gene lead to skeletal dysplasias is poorly understood. Here we used molecular dynamics simulations, which indicate that an A … A mismatch in a d(GAC)6·d(GAC)6 duplex induces negative supercoiling, leading to a local B-to-Z DNA transition. This transition facilitates the binding of d(GAC)7·d(GAC)7 with the Zα-binding domain of human adenosine deaminase acting on RNA 1 (ADAR1, hZαADAR1), as confirmed by CD, NMR, and microscale thermophoresis studies. The CD results indicated that hZαADAR1 recognizes the zigzag backbone of d(GAC)7·d(GAC)7 at the B-Z junction and subsequently converts it into Z-DNA via the so-called passive mechanism. Molecular dynamics simulations carried out for the modeled hZαADAR1-d(GAC)6d(GAC)6 complex confirmed the retention of previously reported important interactions between the two molecules. These findings suggest that hZαADAR1 binding with the GAC hairpin stem in COMP can lead to a non-genetic, RNA editing-mediated substitution in COMP that may then play a crucial role in the development of pseudoachondroplasia.

3D-NuS: A Web Server for Automated Modeling and Visualization of Non-Canonical 3-Dimensional Nucleic Acid Structures.

The inherent conformational flexibility of nucleic acids facilitates the formation of a range of conformations such as duplex, triplex, quadruplex, etc. that play crucial roles in biological processes. Elucidation of the influence of non-canonical base pair mismatches on DNA/RNA structures at different sequence contexts to understand the mismatch repair, misregulation of alternative splicing mechanisms and the sequence-dependent effect of RNA-DNA hybrid in relevance to antisense strategy demand their three-dimensional structural information. Furthermore, structural insights about nucleic acid triplexes, which are generally not tractable to structure determination by X-ray crystallography or NMR techniques, are essential to establish their biological function(s). A web server, namely 3D-NuS (http://iith.ac.in/3dnus/), has been developed to generate energy-minimized models of 80 different types of triplexes, 64 types of G-quadruplexes, left-handed Z-DNA/RNA duplexes, and RNA-DNA hybrid duplex along with inter- and intramolecular DNA or RNA duplexes comprising a variety of mismatches and their chimeric forms for any user-defined sequence and length. It also generates an ensemble of conformations corresponding to the modeled structure. These structures may serve as good starting models for docking proteins and small molecules with nucleic acids, NMR structure determination, cryo-electron microscope modeling, DNA/RNA nanotechnology applications and molecular dynamics simulation studies.

Key diffusion mechanisms involved in regulating bidirectional water permeation across E. coli outer membrane lectin.

Capsular polysaccharides (CPSs) are major bacterial virulent determinants that facilitate host immune evasion. E. coli group1 K30CPS is noncovalently attached to bacterial surface by Wzi, a lectin. Intriguingly, structure based phylogenetic analysis indicates that Wzi falls into porin superfamily. Molecular dynamics (MD) simulations further shed light on dual role of Wzi as it also functions as a bidirectional passive water specific porin. Such a functional role of Wzi was not realized earlier, due to the occluded pore. While five water specific entry points distributed across extracellular &periplasmic faces regulate the water diffusion involving different mechanisms, a luminal hydrophobic plug governs water permeation across the channel. Coincidently, MD observed open state structure of "YQF" triad is seen in sugar-binding site of sodium-galactose cotransporters, implicating its involvement in K30CPS surface anchorage. Importance of Loop 5 (L5) in membrane insertion is yet another highlight. Change in water diffusion pattern of periplasmic substitution mutants suggests Wzi's role in osmoregulation by aiding in K30CPS hydration, corroborating earlier functional studies. Water molecules located inside ß-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence.

Twisting right to left: A A mismatch in a CAG trinucleotide repeat overexpansion provokes left-handed Z-DNA conformation.

Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders that arise from overexpansion of trinucleotide repeats located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington's disorder and spinocerebellar ataxias. Nonetheless, DNA hairpin stem structure that generally embraces B-form with canonical base pairs is poorly understood in the context of periodic noncanonical A…A mismatch as found in CAG repeat overexpansion. Molecular dynamics simulations on DNA hairpin stems containing A…A mismatches in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. Transition from B-to-Z is due to the mechanistic effect that originates from its pronounced nonisostericity with flanking canonical base pairs facilitated by base extrusion, backbone and/or base flipping. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A…A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.