Guanine-rich DNA sequences tend to form four-stranded G-quadruplex structures. Characteristic glycosidic conformational patterns along the G-strands, such as the 5′-syn-anti-syn-anti pattern observed with the Oxytricha nova telomeric G-quadruplexes, have been well documented. However, an explanation for these featured glycosidic patterns has not emerged. This work presents MD simulation and free energetic analyses for simplified two-quartet [d(GG)]4 models and suggests that the four base pair step patterns show quite different relative stabilities: syn-anti > anti-anti > anti-syn > syn-syn. This suggests the following rule: when folding, anti-parallel G-quadruplexes tend to maximize the number of syn-anti steps and avoid the unfavorable anti-syn and syn-syn steps. This rule is consistent with most of the anti-parallel G-quadruplex structures in the Protein Databank (PDB). Structural polymorphisms of G-quadruplexes relate to these glycosidic conformational patterns and the lengths of the G-tracts. The folding topologies of G2- and G4-tracts are not very polymorphic because each strand tends to populate the stable syn-anti repeat. G3-tracts, on the other hand, cannot present this repeating pattern on each G-tract. This leads to smaller energy differences between different geometries and helps explain the extreme structural polymorphism of the human telomeric G-quadruplexes.

We provide theoretical predictions of the intrinsic stability of different arrangements of guanine quadruplex (G-DNA) stems. Most computational studies of nucleic acids have applied Molecular Mechanics (MM) approaches using simple pairwise-additive force fields. The principle limitation of such calculations is the highly approximate nature of the force fields. In this study we for the first time apply accurate QM computations (DFT-D3 with large atomic orbital basis sets) to essentially complete DNA building blocks, namely, seven different folds of the cation-stabilized 2-quartet G-DNA stem, each having more than 250 atoms. The solvent effects are approximated by COSMO continuum solvent. We reveal sizeable differences between MM and QM descriptions of relative energies of different G-DNA stems, which apparently reflect approximations of the DNA force field. Using the QM energy data, we propose correction to earlier free energy estimates of relative stabilities of different parallel, hybrid and antiparallel G-stem folds based on classical simulations. The new energy ranking visibly improves the agreement between theory and experiment. We predict the 5′-anti-anti-3′ GpG dinucleotide step to be the most stable one, closely followed by the 5′-syn-anti-3′ step. The results are in good agreement with known experimental structures of 2, 3 and 4-quartet G-DNA stems. Besides providing specific results for G-DNA, our study highlights basic limitations of force field modeling of nucleic acids. Although QM computations have their own limitations, mainly the lack of conformational sampling and the approximate description of the solvent, they can substantially improve quality of calculations currently relying exclusively on force fields.

The article reviews the application of biomolecular simulation methods to understand the structure, dynamics and interactions of nucleic acids with a focus on explicit solvent molecular dynamics simulations of guanine quadruplex (G-DNA and G-RNA) molecules. While primarily dealing with these exciting and highly relevant four-stranded systems, where recent and past simulations have provided several interesting results and novel insight into G-DNA structure, the review provides some general perspectives on the applicability of the simulation techniques to nucleic acids.

Many cancer cells display enhanced glycolysis and suppressed mitochondrial metabolism. This phenomenon, known as the Warburg effect, is critical for tumor development. However, how cancer cells coordinate glucose metabolism through glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle is largely unknown. We demonstrate here that phosphoglycerate kinase 1 (PGK1), the first ATP-producing enzyme in glycolysis, is reversibly and dynamically modified with O-linked N-acetylglucosamine (O-GlcNAc) at threonine 255 (T255). O-GlcNAcylation activates PGK1 activity to enhance lactate production, and simultaneously induces PGK1 translocation into mitochondria. Inside mitochondria, PGK1 acts as a kinase to inhibit pyruvate dehydrogenase (PDH) complex to reduce oxidative phosphorylation. Blocking T255 O-GlcNAcylation of PGK1 decreases colon cancer cell proliferation, suppresses glycolysis, enhances the TCA cycle, and inhibits tumor growth in xenograft models. Furthermore, PGK1 O-GlcNAcylation levels are elevated in human colon cancers. This study highlights O-GlcNAcylation as an important signal for coordinating glycolysis and the TCA cycle to promote tumorigenesis.

Cholesterol, an abundant membrane component in both lipid rafts and caveolae of cell membrane, plays a crucial role in regulating the function and organization of various G-protein coupled receptors (GPCRs). However, the underlying mechanism for cholesterol-GPCR interaction is still unclear. To this end, we performed a series of microsecond molecular dynamics (MD) simulations on β(2)-adrenergic receptor (β(2)AR) in the presence and absence of cholesterol molecules in the POPC bilayer. The unbiased MD simulation on the system with cholesterols reveals that cholesterol molecules can spontaneously diffuse to seven sites on the β(2)AR surfaces, three in the extracellular leaflet (e1-e3) and four in the intracellular leaflet (i1, i2, i4, and i5). The MD simulation identifies three cholesterol-binding sites (i2, e2, and e3) that are also observed in the crystal structures of several GPCRs. Cholesterol binding to site e1 lock Trp313(7.40) into a certain conformation that may facilitate ligand-receptor binding, and cholesterol binding to site i2 provides a structural support for the reported cholesterol-mediate dimeric form of β(2)AR (PDB code 2RH1). In addition, both competitive and cooperative effects between cholesterols and phospholipids in binding to β(2)AR were observed in our MD simulations. Together, these results provide new insights into cholesterol-GPCR interactions.

The tissue specificity of mitochondrial tRNA mutations remains largely elusive. In this study, we demonstrated the deleterious effects of tRNA Thr 15927G>A mutation that contributed to pathogenesis of coronary artery disease. The m.15927G>A mutation abolished the highly conserved base-pairing (28C-42G) of anticodon stem of tRNA Thr . Using molecular dynamics simulations, we showed that the m.15927G>A mutation caused unstable tRNA Thr structure, supported by decreased melting temperature and slower electrophoretic mobility of mutated tRNA. Using cybrids constructed by transferring mitochondria from a Chinese family carrying the m.15927G>A mutation and a control into mitochondrial DNA (mtDNA)-less human umbilical vein endothelial cells, we demonstrated that the m.15927G>A mutation caused significantly decreased efficiency in aminoacylation and steady-state levels of tRNA Thr . The aberrant tRNA Thr metabolism yielded variable decreases in mtDNA-encoded polypeptides, respiratory deficiency, diminished membrane potential and increased the production of reactive oxygen species. The m.15927G>A mutation promoted the apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 3, 7, 9 and PARP. Moreover, the lower wound healing cells and perturbed tube formation were observed in mutant cybrids, indicating altered angiogenesis. Our findings provide new insights into the pathophysiology of coronary artery disease, which is manifested by tRNA Thr mutation-induced alterations.

The lengths of G-tracts and their connecting loop sequences determine G-quadruplex folding and stability. Complete understanding of the sequence–structure relationships remains elusive. Here, single-loop G-quadruplexes were investigated using explicit solvent molecular dynamics (MD) simulations to characterize the effect of loop length, loop sequence, and G-tract length on the folding topologies and stability of G-quadruplexes. Eight loop types, including different variants of lateral, diagonal, and propeller loops, and six different loop sequences [d0 (i.e., no intervening residues in the loop), dT, dT2, dT3, dTTA, and dT4] were considered through MD simulation and free energy analysis. In most cases the free energetic estimates agree well with the experimental observations. The work also provides new insight into G-quadruplex folding and stability. This includes reporting the observed instability of the left propeller loop, which extends the rules for G-quadruplex folding. We also suggest a plausible explanation why human telomere sequences predominantly form hybrid-I and hybrid-II type structures in K+ solution. Overall, our calculation results indicate that short loops generally are less stable than longer loops, and we hypothesize that the extreme stability of sequences with very short loops could possibly derive from the formation of parallel multimers. The results suggest that free energy differences, estimated from MD and free energy analysis with current force fields and simulation protocols, are able to complement experiment and to help dissect and explain loop sequence, loop length, and G-tract length and orientation influences on G-quadruplex structure.

Nuclear modifier gene(s) was proposed to modulate the phenotypic expression of mitochondrial DNA mutation(s). Our previous investigations revealed that a nuclear modifier allele (A10S) in TRMU (methylaminomethyl-2-thiouridylate-methyltransferase) related to tRNA modification interacts with 12S rRNA 1555A→G mutation to cause deafness. The A10S mutation resided at a highly conserved residue of the N-terminal sequence. It was hypothesized that the A10S mutation altered the structure and function of TRMU, thereby causing mitochondrial dysfunction. Using molecular dynamics simulations, we showed that the A10S mutation introduced the Ser dynamic electrostatic interaction with the Lys residue of helix 4 within the catalytic domain of TRMU. The Western blotting analysis displayed the reduced levels of TRMU in mutant cells carrying the A10S mutation. The thermal shift assay revealed the value of mutant TRMU protein, lower than that of the wild-type counterpart. The A10S mutation caused marked decreases in 2-thiouridine modification of U34 of tRNA, tRNA and tRNA However, the A10S mutation mildly increased the aminoacylated efficiency of tRNAs. The altered 2-thiouridine modification worsened the impairment of mitochondrial translation associated with the m.1555A→G mutation. The defective translation resulted in the reduced activities of mitochondrial respiration chains. The respiratory deficiency caused the reduction of mitochondrial ATP production and elevated the production of reactive oxidative species. As a result, mutated TRMU worsened mitochondrial dysfunctions associated with m.1555A→G mutation, exceeding the threshold for expressing a deafness phenotype. Our findings provided new insights into the pathophysiology of maternally inherited deafness that was manifested by interaction between mtDNA mutation and nuclear modifier gene.