Conformational Capture Research Articles

Regulation of reversible conformational change, size switching, and immunomodulation of RNA nanocubes.

In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility in structure, diversity in function, and adaptability in a configuration similar to proteins. In the past, the work on the investigation of conformational change mainly focused on protein. The induced-fit and conformational capture in RNA have also been explored, such as in the study of riboswitches. Herein, we report the engineering of three-dimensional RNA nanocubes and demonstrated the operation and regulation for its configuration. We demonstrate the operation of reconfigurable RNA nanocubes whose shapes change precisely and reversibly in response to a specific trigger strand. The shape, size, and conformation can be regulated precisely and reversibly in response to the specific triggering signals. The shape and conformational conversion were observed by cryo-EM and gel electrophoresis, respectively. Harnessing the size, shape, conformation, and self-assembly capabilities of the RNA nanocube can provide a new potential use of this technology as nanocarriers for the treatment of various diseases.

Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recognition by Damage Recognition Protein Rad4

How DNA-binding proteins search for and identify their target sites buried amid a vast excess of non-target sites remains a central question in biology. This study is aimed at elucidating the mechanism by which DNA damage recognition protein XPC recognizes diverse DNA lesions, and initiates DNA repair. Structural studies of Rad4 (yeast ortholog of XPC) bound to model lesions showed that DNA is unwound and bent at the lesion site and the damaged nucleotides are flipped out. Previous studies using laser T-jump perturbation approaches indicated that Rad4 interrogates potential sites by unwinding DNA on ~100-500 μs and eventually flips out damaged nucleotides on ~5-10 ms. However, these ‘ensemble’ measurements could not capture intrinsic DNA deformability that is potentially critical in the ability of Rad4/XPC to distinguish damaged from undamaged sites. Previous studies were also limited to measurements on short, linear DNA oligomers, while DNA in our cells is typically bent and supercoiled, which is expected to have a profound impact on DNA damage recognition. In this study, I employed an innovative fluorescence lifetime approach with fluorescent probes exquisitely sensitive to DNA twisting/bending fluctuations and mapped the range of conformations accessible to mismatched DNA substrates of varying Rad4-binding specificities. My results reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity mismatched DNA, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound. Conversely, nonspecific mismatched DNA retained a largely homogeneous, B-DNA-like conformation. Surprisingly, even mismatched-DNA specifically bound to Rad4 remained highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. Using T-jump, I unveiled the rates of intrinsic DNA conformational dynamics in mismatched DNA. Remarkably, these rates remained unaltered in the presence of Rad4. Finally, I examined the effect of DNA bending strain on the intrinsic deformability at the mismatched sites by extending the fluorescence lifetime studies in the context of DNA minicircles. These studies revealed amplified distortions for both matched and mismatched sites in DNA minicircles, with further distortions detected upon Rad4 binding to mismatched sites. Taken together, my results strongly suggest that intrinsic DNA deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.

Immunity: Conformational Capture

Immunity: Conformational Capture

Highly Modular Structure and Ligand Binding by Conformational Capture in a Minimalistic Riboswitch

Be prepared: The structure of an synthetic neomycin riboswitch RNA (N1) is investigated by NMR spectroscopy. A largely disordered free structural ensemble also contains a compact conformation that resembles the ligand-bound state thus suggesting a binding mechanism by conformational capture (see scheme, red=ligand).

Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging

Many nucleic acid-binding proteins and the AAA+ family form hexameric rings, but the mechanism of hexamer assembly is unclear. It is generally believed that the specificity in protein/RNA interaction relies on molecular contact through a surface charge or 3D structure matching via conformational capture or induced fit. The pRNA of bacteriophage phi29 DNA-packaging motor also forms a ring, but whether the pRNA ring is a hexamer or a pentamer is under debate. Here, single molecule studies elucidated a mechanism suggesting the specificity and affinity in protein/RNA interaction relies on pRNA static ring formation. A combined pRNA ring-forming group was very specific for motor binding, but the isolated individual members of the ring-forming group bind to the motor nonspecifically. pRNA did not form a ring prior to motor binding. Only those RNAs that formed a static ring, via the interlocking loops, stayed on the motor. Single interlocking loop interruption resulted in pRNA detachment. Extension or reduction of the ring circumference failed in motor binding. This new mechanism was tested by redesigning two artificial RNAs that formed hexamer and packaged DNA. The results confirmed the stoichiometry of pRNA on the motor was the common multiple of two and three, thus, a hexamer.

Molecular dynamics simulation studies of induced fit and conformational capture in U1A-RNA binding: do molecular substates code for specificity?

Molecular dynamics (MD) simulations on stem loop 2 of U1 small nuclear RNA and a construct of the U1A protein were carried out to obtain predictions of the structures for the unbound forms in solution and to elucidate dynamical aspects of induced fit upon binding. A crystal structure of the complex between the U1A protein and stem loop 2 RNA and an NMR structure for the uncomplexed form of the U1A protein are available from Oubridge et al. (Nature, 1994, Vol. 372, pp. 432-438) and Avis et al. (Journal of Molecular Biology, 1996, Vol. 257, pp. 398-411), respectively. As a consequence, U1A-RNA binding is a particularly attractive case for investigations of induced fit in protein-nucleic acid complexation. When combined with the available structural data, the results from simulations indicate that structural adaptation of U1A protein and RNA define distinct mechanisms for induced fit. For the protein, the calculations indicate that induced fit upon binding involves a non-native thermodynamic substate in which the structure is preorganized for binding. In contrast, induced fit of the RNA involves a distortion of the native structure in solution to an unstable form. However, the RNA solution structures predicted from simulation show evidence that structures in which groups of bases are favorably oriented for binding the U1A protein are thermally accessible. These results, which quantify with computational modeling recent proposals on induced fit and conformational capture by Leuillot and Varani (Biochemistry, 2001, Vol. 40, pp. 7947-7956) and by Williamson (Nature Structural Biology, 2000, Vol. 7, pp. 834-837) suggest an important role for intrinsic molecular architecture and substates other than the native form in the specificity of protein-RNA interactions.

Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and conformational capture.

Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and conformational capture.