12 research outputs found
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 β-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence
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 MD 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. MD simulations carried out for the modeled hZαADAR1−d(GAC)6.d(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 the COMP that may then play a crucial role in the development of pseudoachondroplasia
New structural insights into unusual nucleic acid conformations in relevance to mechanisms and pathogeneses of trinucleotide repeat expansion disorders
Trinucleotide repeats belong to the family of microsatellites (a tract of 1 to 6 repetitive nucleotides)
that are commonly observed in eukaryotes and exhibit repeat length polymorphism. The inherent
ability of trinucleotide repeats to undergo abnormal expansion (viz., increase in repeat length) leads
to many incurable genetic disorders, the best known are, Huntington's disease (CAG repeat), fragile
X syndrome (CGG repeat), myotonic dystrophy 1 (CTG repeat), Friedreich’s ataxia (GAA repeat)
and spinocerebellar ataxias (CAG repeat) that are mainly neurodegenerative. Severity of these
disorders is proportional to the number of expanded repeats. Overexpansion of these repeats results
in the formation of unusual nucleic acid secondary structures such as hairpins, triplexes, tetraplexes
etc. Although many investigations have been carried out to understand the mechanism(s) behind
these disorders, the therapeutics of trinucleotide repeat expansion disorders are not well developed.
Thus, to facilitate the therapeutics of aforementioned neurological and neuromuscular disorders, the
impact of such an overexpansion on DNA & RNA conformations and the concomitant functional
implications have to be investigated.
We have carried out molecular dynamics simulations (MD), circular dichroism (CD) and
electrophoretic mobility shift assay (EMSA) to determine (i) the secondary structural characteristics
of DNA hairpin stems that contain CAG (A...A mismatch), GAC (A...A mismatch), CCG (C...C
mismatch) & CGG repeat sequences and (ii) the complex model of CAG repeats containing RNA
duplex with alternative pre-mRNA splicing regulator muscle blind like protein 1 (MBNL1). MD
results revealed that A...A mismatch containing CAG/GAC dictates local B- to Z-DNA
transformation irrespective of the starting glycosyl conformation, in sharp contrast to the canonical
DNA duplex. This result is further confirmed by CD studies. MD and CD studies further showed
that more than four C...C mismatches cannot be accommodated in a RNA duplex formed by CCG
repeat, instead, these favor i-motif structure. In contrast, CCG can form duplex structure at the
DNA level irrespective of the number of C...C mismatches. Strikingly, CGG repeats prefer to form
quadruplex structure both at DNA and RNA levels. The current investigation also explored the
binding mode of MBNL1 with RNA duplex that contain CAG repeats. Our study revealed for the
first time that MBNL1 binds to the minor groove of the RNA duplex. Thus, the conformational
preference for CNG (N=A/C/G) and GAC repeats presented here along with the minor groove
binding preference for MBNL1 with CAG repeats containing RNA duplex may facilitate the
understanding of trinucleotide repeat expansion mechanism(s) and pathogeneses as well as expedite
the drug discovery process to treat these diseases
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
Z-DNA sandwich structure formed by A<sub>8</sub>…A<sub>23</sub> mismatch with <i>+syn…anti</i> starting <i>glycosyl</i> conformation (Fig. 1A) promotes intercalation between the mismatched bases.
<p>(Top) Snapshots of central 7mer illustrating the formation of intercalated A<sub>8</sub>&A<sub>23</sub> (colored magenta) during 133-144ns and (Bottom) the associated interaction between A<sub>8</sub> (magenta) & A<sub>23</sub> (green). Note the loss of hydrogen bond at 133.6ns following which, A<sub>8</sub>&A<sub>23</sub> move out of plane with each other (133.8ns). Subsequently, A<sub>8</sub> stacks onto A<sub>23</sub> completely ~142ns and stays in the same conformation till the end of the simulation.</p
Periodic B-Z junction induced by recurring A…A mismatches in CAG repeat expansion.
<p>(A) 18mer DNA duplex with 6 A…A mismatches used in the present MD simulation. (B) Time vs RMSD profile showing the significant conformational change from the starting model. (C-H) Histogram corresponding to: <i>glycosyl</i> conformation of (C) A’s & (D) G’s, twist angles at all the (E) CA (F) AG & (G) GC steps and epsilon at (H) CA step over 291-300ns. (I) Cartoon diagram showing the conformational changes from B- to Z-DNA during the simulation. Note that terminal 2 base pairs on either ends are not included due to end fraying effect.</p
Local Z-DNA formation by ‘base flipping’ mechanism in CAG repeat with periodic occurrence of A…A mismatches.
<p>(Top) Snapshots of A<sub>23</sub>…A<sub>14</sub> mismatch site illustrating base flipping of A<sub>23</sub> from <i>+syn</i> to -<i>syn glycosyl</i> conformation through <i>cis glycosyl</i> conformation that occurs at ~162ns. Adoption of -<i>syn glycosyl</i> conformation by both A<sub>14</sub> (black) & A<sub>23</sub> (red) beyond 160ns can be seen in Time vs <i>chi</i> profile (Top row, Right most corner). (Middle) Snapshots of A<sub>26</sub>…A<sub>11</sub> mismatch site indicating <i>+syn</i> to -<i>syn glycosyl</i> conformation of A<sub>26</sub> through <i>trans glycosyl</i> conformation ~205ns. Both A<sub>11</sub> (black) & A<sub>26</sub> (red) assume -<i>syn glycosyl</i> conformation beyond 205ns as seen in Time vs <i>chi</i> profile (Middle row, Right most corner). (Bottom) Cartoon diagram illustrating the formation of local Z-DNA concomitant with the above mentioned base extrusion & base flipping (A…A mismatches colored maroon). Positions of A<sub>23</sub>…A<sub>14</sub> and A<sub>26</sub>…A<sub>11</sub> mismatches are indicated by arrow (Bottom row, Right most corner). Compact B-form structure at 0.001ns and the extended Z-DNA like structure at 300ns can be clearly visualized (Bottom). Note that in all the figures the time associated with each snapshot is indicated.</p
Z-DNA sandwich structure formed by A<sub>8</sub>…A<sub>23</sub> mismatch with <i>+syn…anti</i> starting <i>glycosyl</i> conformation for sequence given in Fig. 1A.
<p>(A) Time vs RMSD profile showing three different ensembles during the 300ns simulation. (B) Cartoon diagram of representative average structures (calculated over 100ps) corresponding to the 4 different time intervals. Note the increase in the Z-DNA stretch (marked by square bracket) with respect to time. (C) 2D plot showing the conformational transformation occurring in <i>chi</i> at A<sub>8</sub>. (D) Time vs helical twist profile showing the preference for low twist at C<sub>7</sub>A<sub>8</sub>, A<sub>8</sub>G<sub>9</sub>, G<sub>9</sub>C<sub>10</sub> and C<sub>10</sub>A<sub>11</sub> steps due to the local Z-DNA formation.</p
Twisting Right to Left: A…A Mismatch in a CAG Trinucleotide Repeat Overexpansion Provokes Left-Handed Z-DNA Conformation
<div><p>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 <i>glycosyl</i> 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.</p></div
2D plots indicating backbone conformational preference for d(CAG)<sub>2</sub>CAG(CAG)<sub>2</sub>.d(CTG)<sub>2</sub>CAG(CTG)<sub>2</sub> duplex with A<sub>8</sub>…A<sub>23</sub> in <i>anti…anti</i> starting <i>glycosyl</i> conformation during the last 10ns.
<p>(ε&ζ) and (α&γ) 2D plots corresponding to the first strand is given in 2<sup>nd</sup> column along with the appropriate step marked in the 1<sup>st</sup> column. (ε&ζ) and (α&γ) 2D plots corresponding to the second strand is given in 4<sup>th</sup> column along with the appropriate step marked in the 3<sup>rd</sup> column. 2D plots of (ε&ζ) and (α&γ) are marked in black & red respectively. Note that ε&α are represented in X-axis and ζ&γ are represented in Y-axis.</p