Doctor of Philosophy, Hong Kong University Of Science & Technology (2015)
Bachelor of Science, Universidad Nacional Autonoma Mexico (2011)
Michael Levitt, Postdoctoral Faculty Sponsor
8-oxo-guanine DNA damage induces transcription errors by escaping two distinct fidelity control checkpoints of RNA polymerase II.
The Journal of biological chemistry
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 2019; 565 (7738): 186-+
De novo design of potent and selective mimics of IL-2 and IL-15.
2019; 565 (7738): 186–91
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 PubMedID 30626941
Structural, thermodynamic and catalytic characterization of an ancestral triosephosphate isomerase reveal early evolutionary coupling between monomer association and function.
The FEBS journal
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
The shape of the ribosome exit tunnel affects cotranslational protein folding.
The E.coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Dloop ribosomes, while two ~100-residue protein normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Dloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.
View details for PubMedID 30475203
Structure of the 30S ribosomal decoding complex at ambient temperature.
RNA (New York, N.Y.)
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 PubMedID 30139800
Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature.
Nucleic acids research
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 PubMedID 30113694
Comprehensive computational design of ordered peptide macrocycles
2017; 358 (6369): 1461–66
Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with l-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of l- and d-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.
View details for PubMedID 29242347
View details for PubMedCentralID PMC5860875
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
2016; 578: 343-371
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
Millisecond dynamics of RNA polymerase II translocation at atomic resolution
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (21): 7665-7670
Transcription is a central step in gene expression, in which the DNA template is processively read by RNA polymerase II (Pol II), synthesizing a complementary messenger RNA transcript. At each cycle, Pol II moves exactly one register along the DNA, a process known as translocation. Although X-ray crystal structures have greatly enhanced our understanding of the transcription process, the underlying molecular mechanisms of translocation remain unclear. Here we use sophisticated simulation techniques to observe Pol II translocation on a millisecond timescale and at atomistic resolution. We observe multiple cycles of forward and backward translocation and identify two previously unidentified intermediate states. We show that the bridge helix (BH) plays a key role accelerating the translocation of both the RNA:DNA hybrid and transition nucleotide by directly interacting with them. The conserved BH residues, Thr831 and Tyr836, mediate these interactions. To date, this study delivers the most detailed picture of the mechanism of Pol II translocation at atomic level.
View details for DOI 10.1073/pnas.1315751111
View details for Web of Science ID 000336411300044
View details for PubMedID 24753580
View details for PubMedCentralID PMC4040580