Electronic structure calculations of all 10 unique base pair (bp) steps have been calculated to study the interaction energies of the bp steps, density of states (DOS), projected density of states (pDOS) using the density functional theory (DFT). Plane wave basis with ultrasoft pseudo-potential method has been used within the local density approximation (LDA) for the exchange correlation functional. Electron densities of the bp steps corresponding to HOMO and LUMO level have been calculated to understand the difference in stacking energies of the bp steps. The variation of HOMO–LUMO gap (g) of all possible bp steps on twist angle has been studied. We have observed that out of the 10 bp steps, 4 purine–purine bp steps (d(AA), d(GG), d(AG) and d(GA)), show significant variation of [Formula: see text] on twist angle. The observed variation on twist angle of d(AA) bp step has been explained by the calculated DOS and electron densities.

DNA within the living cells experiences a diverse range of temperature, ranging from freezing condition to hot spring water. How the structure, the mechanical properties of DNA, and the solvation dynamics around DNA changes with the temperature is important to understand the functionality of DNA under those acute temperature conditions. In that notion, we have carried out molecular dynamics simulations of a DNA oligomer, containing TATA-box sequence for three different temperatures (250K, 300K and 350K). We observed that the structure of the DNA, in terms of backbone torsion angles, sugar pucker, base pair parameters, and base pair step parameters, did not show any unusual properties within the studied range of temperatures, but significant structural alteration was noticed between BI and BII forms at higher temperature. As expected, the flexibility of the DNA, in terms of the torsional rigidity and the bending rigidity is highly temperature dependent, confirming that flexibility increases with increase in temperature. Additionally, the groove widths of the studied DNA showed temperature sensitivity, specifically, the major groove width decreases and the minor groove width increases, respectively, with the increase in temperature. We observed that at higher temperature, water around both the major and the minor groove of the DNA is less structured. However, the water dynamics around the minor groove of the DNA is more restricted as compared to the water around the major groove throughout the studied range of temperatures, without any anomalous behavior.

Phosphorylation of S17 in the N-terminal "lid" of MDM2 (residues 1-24) is proposed to regulate the binding of p53. The lid is composed of an intrinsically disordered peptide motif that is not resolved in the crystal structure of the MDM2 N-terminal domain. Molecular dynamics simulations of MDM2 provide novel insight into how the lid undergoes complex dynamics depending on its phosphorylation state that have not been revealed by NMR analyses. The difference in charges between the phosphate and the phosphomimetic 'Asp' and the change in shape from tetrahedral to planar are manifested in differences in strengths and durations of interactions that appear to modulate access of the binding site to ligands and peptides differentially. These findings unveil the complexities that underlie protein-protein interactions and reconcile some differences between the biochemical and NMR data suggesting that lid mutation or deletion can change the specific activity of MDM2 and provide concepts for future approaches to evaluate the effects of S17 modification on p53 binding.

The mechanism of coupling of ion pumping in the membrane-bound A(O) sector with ATP synthesis in the A(3)B(3) headpiece of the A(1) sector in the A(1)A(O) ATP synthase is a puzzle. Previously, crosstalk between the stalk and nucleotide-binding subunits F(Mm) and B(Mm) of the Methanosarcina mazei Gö1 A-ATP synthase has been observed by nucleotide-dependent cross-link formation of both subunits inside the enzyme. The recently determined NMR solution structure of F(Mm) depicts the protein as a two-domain structure, with a well-folded N-terminus having 78 residues and a flexible C-terminal part (residues 79-101), proposed to become structured after binding to its partner, B(Mm). Here, we detail the crucial interactions between subunits B(Mm) and F(Mm) by determining the NMR structure of the very C-terminus of F(Mm), consisting of 20 residues and hereafter termed F(Mm(81-101)), and performing molecular dynamics simulations on the resulting structure. These data demonstrate that the flexibility of the C-terminus enables F(Mm) to switch between an elongated and retracted state. Docking and MD in conjunction with previously conducted and published NMR results, biochemical cross-linking, and fluorescence spectroscopy data were used to reconstruct a model of a B(Mm)-F(Mm) assembly. The model of the B(Mm)-F(Mm) complex shows the detailed interactions of helices 1 and 2 of the C-terminal domain of B(Mm) with the C-terminal residues of F(Mm). Movements of both helices of B(Mm) accommodate the incoming C-terminus of F(Mm) and connect the events of ion pumping and nucleotide binding in the A(1)A(O) ATP synthase.

Crystal structures of inactive PAK1(K299R) and the activation (A)-loop phospho-mimetic PAK1(T423E) have suggested that the kinase domain is in an active state regardless of activation loop status. Contrary to a large body of literature, we find that neither is PAK1(T423E) active in cells, nor does it exhibit significant activity in vitro. To explain these discrepancies all-atom molecular dynamics (MD) simulations of PAK1(phospho-T423) in complex with ATP and substrate were performed. These simulations point to a key interaction between PAK1 Lys308, at the end of the ccC helix, and the pThr423 phosphate group, not seen in X-ray structures. The orthologous PAK4 Arg359 fulfills the same role in immobilizing the LC helix. These in silico predictions were validated by experimental mutagenesis of PAK1 and PAK4. The simulations explain why the PAK1 A-loop phosphomimetic is inactive, but also point to a key functional interaction likely found in other protein kinases.

The spliceosome, consisting of different snRNPs and numerous non-U snRNP factors, catalyzes the splicing reaction. To activate the spliceosome and enable the first step of splicing to begin, the extensively base-paired U4, U6 snRNAs in the U4/U6 snRNP dissociate from each other. The U4, U6 snRNAs are associated to the common core proteins of the spliceosome namely the Sm and LSm proteins, as well as the specific proteins including 15.5K, hPrp31, and the CypH/hPrp4/hPrp3 protein tri-complex. The knowledge on these proteins is of paramount importance for the understanding of the dramatic structural rearrangement of the U4/U6 snRNA duplex prior to splicing. In this work, effort was made to understand the interactions between two of these specific proteins namely 15.5K and hPrp31 in the context of the U4 snRNP. Using HSQC titrations and cross-saturation experiments on the hPrp31-15.5K-U4 5"-SL and hPrp3178-333 -15.5K-U4 5"-SL complexes, we defined the interaction surface on 15.5K in complex with hPrp31. Combining the NMR and biochemical data, we successfully generated a 3D model of the ternary complex using comparative modelling and the HADDOCK2.0 docking program. From these results, we characterized the Nop domain as a bona fide RNP binding domain. The role of the assembly-initiating 15.5K is, therefore, not restricted to inducing or stabilizing a hPrp31 binding site in the RNA; rather 15.5K itself provides approximately half of the contact surface for the Nop domain of hPrp31. In the docking model, the interaction surfaces on 15.5K and hPrp31 showed charge complement. From our docking model we could also demonstrate that the elongation of stem II in U4 snRNA is highly unfavourable due to the physical barrier provided by the Nop domain. As in box C/D snoRNAs the length of the stem II naturally exceeds the required length, hPrp31 is not recruited into the box C/D snoRNPs. A surface mutant of 15.5K, which contains mutations outside the interaction surface between 15.5K and hPrp31, was previously shown to strongly reduce the binding of hPrp31. Using HSQC titration and RDC refinement, we could demonstrate that the structure of this mutant does not significantly vary from the wild type and therefore, the effect of this 15.5K mutant on hPrp31 binding could arise from subtle changes in the charge property of the interaction surface. Furthermore, the structural changes of 15.5K in complexes with different box C/D snoRNA constructs were studied using HSQC titrations to address the question whether the structure of 15.5K also contributes to the selectivity of these primary RNPs. The chemical shift perturbation data showed that the structure of 15.5K does not differ dramatically in this primary RNPs. Therefore, the selectivity arises primarily from the differences in the RNAs and the secondary binding proteins.

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.