Serge L. Smirnov

An N-terminal, 830 residues intrinsically disordered region of the cytoskeleton-regulatory protein supervillin contains Myosin II- and F-actin-binding sites

Supervillin, the largest member of the villin/gelsolin family, is a cytoskeleton regulating, peripheral membrane protein. Supervillin increases cell motility and promotes invasive activity in tumors. Major cytoskeletal interactors, including filamentous actin and myosin II, bind within the unique supervillin amino terminus, amino acids 1–830. The structural features of this key region of the supervillin polypeptide are unknown. Here, we utilize circular dichroism and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). Our combined data indicate that the N-terminus of human and bovine supervillin sequences (positions 1–830) represents an IDR, which is the largest IDR known to date in the villin/gelsolin family. Moreover, this result suggests a potentially novel mechanism of regulation of myosin II and F-actin via the intrinsically disordered N-terminal region of hub protein supervillin.

Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5‐methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8‐oxoguanine (oxoGua), which is repaired by 8‐oxoguanine‐DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite‐base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.

8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme

8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew—Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar—phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein—DNA interface.

Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics and enzymatic demethylation

Abstract DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3′ to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.

Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin.

Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences. It is unknown to what extent the plant villin headpiece structure and function resemble those of the well-studied vertebrate counterparts. Here we present the first solution NMR structure and backbone dynamics characterization of a headpiece from plants, villin isoform 4 from Arabidopsis thaliana. The villin 4 headpiece is a 63-residue domain (V4HP63) that adopts a typical headpiece fold with an aromatics core and a tryptophan-centered hydrophobic cap within its C-terminal subdomain. However, V4HP63 has a distinct N-terminal subdomain fold as well as a novel, high mobility loop due to the insertion of serine residue in the canonical sequence that follows the variable length loop in headpiece sequences. The domain binds actin filaments with micromolar affinity, like the vertebrate analogues. However, the V4HP63 surface charge pattern is novel and lacks certain features previously thought necessary for high-affinity F-actin binding. Utilizing the updated criteria for strong F-actin binding, we predict that the headpiece domains of all other villin isoforms in A. thaliana have high affinity for F-actin.

Gelsolin-like Activation of Villin: Calcium Sensitivity of the Long Helix in Domain 6

Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1-V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villins functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-Kd calcium binding in V6 (Kd1 of 22 μM and Kd2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism.