Professional Education


  • Doctor of Philosophy, Hong Kong University Of Science & Technology (2015)
  • Bachelor of Science, Universidad Nacional Autonoma Mexico (2011)

Stanford Advisors


All Publications


  • 8-oxo-guanine DNA damage induces transcription errors by escaping two distinct fidelity control checkpoints of RNA polymerase II. The Journal of biological chemistry Konovalov, K. A., Pardo-Avila, F., Tse, C. K., Oh, J., Wang, D., Huang, X. 2019

    Abstract

    RNA polymerase II (Pol II) has an intrinsic fidelity control mechanism to maintain faithful genetic information transfer during transcription. 8-Oxo-guanine (8OG), a commonly occurring damaged guanine base, promotes misincorporation of adenine into the RNA strand. Recent structural work has shown that adenine can pair with the syn conformation of 8OG directly upstream of the Pol II active site. However, it remains unknown how 8OG is accommodated in the active site as a template base for the incoming ATP. Here, we used molecular dynamics (MD) simulations to investigate two consecutive steps that may contribute to the adenine misincorporation by Pol II. First, the mismatch is located in the active site, contributing to initial incorporation of adenine. Second, the mismatch is in the adjacent upstream position, contributing to extension from the mismatched base pair. These results are supported by an in vitro transcription assay, confirming that 8OG can induce adenine misincorporation. Our simulations further suggest that 8OG forms a stable base pair with the mismatched adenine in both the active site and the adjacent upstream position. This stability predominantly originates from hydrogen bonding between the mismatched adenine and 8OG in a noncanonical syn conformation. Interestingly, we also found that an unstable base pair present directly upstream of the active site, such as adenine paired with 8OG in the canonical anti conformation, largely disrupts the stability of the active site. Our findings have uncovered two main factors contributing to how 8OG induces transcriptional errors and escapes Pol II transcriptional fidelity control checkpoints.

    View details for DOI 10.1074/jbc.RA118.007333

    View details for PubMedID 30718278

  • De novo design of potent and selective mimics of IL-2 and IL-15. Nature Silva, D., Yu, S., Ulge, U. Y., Spangler, J. B., Jude, K. M., Labao-Almeida, C., Ali, L. R., Quijano-Rubio, A., Ruterbusch, M., Leung, I., Biary, T., Crowley, S. J., Marcos, E., Walkey, C. D., Weitzner, B. D., Pardo-Avila, F., Castellanos, J., Carter, L., Stewart, L., Riddell, S. R., Pepper, M., Bernardes, G. J., Dougan, M., Garcia, K. C., Baker, D. 2019; 565 (7738): 186–91

    Abstract

    We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor betagammac heterodimer (IL-2Rbetagammac) but have no binding site for IL-2Ralpha (also called CD25) or IL-15Ralpha (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rbetagammac with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Ralpha and IL-15Ralpha. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rbetagammac, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

    View details for DOI 10.1038/s41586-018-0830-7

    View details for PubMedID 30626941

  • Structural, thermodynamic and catalytic characterization of an ancestral triosephosphate isomerase reveal early evolutionary coupling between monomer association and function. The FEBS journal Schulte-Sasse, M., Pardo-Avila, F., Pulido-Mayoral, N. O., Vazquez-Lobo, A., Costas, M., Garcia-Hernandez, E., Rodriguez-Romero, A., Fernandez-Velasco, D. A. 2018

    Abstract

    Function, structure and stability are strongly coupled in obligated oligomers, such as Triosephosphate Isomerase (TIM). However, little is known about how this coupling evolved. To address this question, five ancestral TIMs (ancTIMs) in the opisthokont lineage were inferred. The encoded proteins were purified and characterized, and spectroscopic and hydrodynamic analysis indicated that all are folded dimers. The catalytic efficiency of ancTIMs is very high and all dissociate into inactive and partially unfolded monomers. The placement of catalytic residues in the three-dimensional structure, as well as the enthalpy-driven binding signature of the oldest ancestor (TIM63) resemble extant TIMs. Although TIM63 dimers dissociate more readily than do extant TIMs, calorimetric data show that the free ancestral subunits are folded to a greater extent than their extant counterparts are, suggesting that full catalytic proficiency was established in the dimer before the stability of the isolated monomer eroded. Notably, the low association energy in ancTIMs is compensated for by a high activation barrier, and by a significant shift in the dimer-monomer equilibrium induced by ligand binding. Our results indicate that before the animal and fungi lineages diverged, TIM was an obligated oligomer with substrate binding properties and catalytic efficiency that resemble that of extant TIMs. Therefore, TIM function and association have been strongly coupled at least for the last third of biological evolution on earth. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1111/febs.14741

    View details for PubMedID 30589511

  • Structure of the 30S ribosomal decoding complex at ambient temperature. RNA (New York, N.Y.) Dao, E. H., Poitevin, F., Sierra, R. G., Gati, C., Rao, Y., Ciftci, H. I., Aksit, F., McGurk, A., Obrinski, T., Mgbam, P., Hayes, B., DE Lichtenberg, C., Pardo-Avila, F., Corsepius, N., Zhang, L., Seaberg, M. H., Hunter, M. S., Liang, M., Koglin, J. E., Wakatsuki, S., Demirci, H. 2018

    Abstract

    The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New lightsources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 A resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.

    View details for DOI 10.1261/rna.067660.118

    View details for PubMedID 30139800

  • Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature. Nucleic acids research O'Sullivan, M. E., Poitevin, F., Sierra, R. G., Gati, C., Dao, E. H., Rao, Y., Aksit, F., Ciftci, H., Corsepius, N., Greenhouse, R., Hayes, B., Hunter, M. S., Liang, M., McGurk, A., Mbgam, P., Obrinsky, T., Pardo-Avila, F., Seaberg, M. H., Cheng, A. G., Ricci, A. J., DeMirci, H. 2018

    Abstract

    The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

    View details for DOI 10.1093/nar/gky693

    View details for PubMedID 30113694

  • Constructing Kinetic Network Models to Elucidate Mechanisms of Functional Conformational Changes of Enzymes and Their Recognition with Ligands COMPUTATIONAL APPROACHES FOR STUDYING ENZYME MECHANISM, PT B Zhang, L., Jiang, H., Sheong, F. K., Pardo-Avila, F., Cheung, P. H., Huang, X. 2016; 578: 343-371

    Abstract

    Enzymes are biological macromolecules that catalyze complex reactions in life. In order to perform their functions effectively and efficiently, enzymes undergo conformational changes between different functional states. Therefore, elucidating the dynamics between these states is essential to understand the molecular mechanisms of enzymes. Although experimental methods such as X-ray crystallography and cryoelectron microscopy can produce high-resolution structures, the detailed conformational dynamics of many enzymes still remain obscure. While molecular dynamics (MD) simulations are able to complement the experiments by providing structure-based dynamics at atomic resolution, it is usually difficult for them to reach the biologically relevant timescales (hundreds of microseconds or longer). Kinetic network models (KNMs), in particular Markov state models (MSMs), hold great promise to overcome this challenge because they can bridge the timescale gap between MD simulations and experimental observations. In this chapter, we review the procedure of constructing KNMs to elucidate the molecular mechanisms of enzymes. First, we will give a general introduction of MSMs, including the methods to construct and validate MSMs. Second, we will present the applications of KNMs to study two important enzymes: the human Argonaute protein and the RNA polymerase II. We conclude by discussing the future perspectives regarding the potential of KNMs to investigate the dynamics of enzymes' functional conformational changes.

    View details for DOI 10.1016/bs.mie.2016.05.026

    View details for Web of Science ID 000383908000016

    View details for PubMedID 27497174