Browsing by Subject "Structural biology"

Bacteria recycle pre-existing peptidoglycan in order to minimize the de novo synthesis of peptidoglycan precursors. The recycling pathway is under study for its chemotherapeutic target potential. Anhydromuramic acid kinase (AnmK) is part of this recycling pathway and catalyzes the dual hydrolysis/phosphorylation of anhMurNAc to MurNAc-6-P. This enzyme has been discovered and introduced, but only minimally characterized. Therefore, the overarching goal of this work was to clone, express and purify AnmK to homogeneity; perform further kinetic characterization; solve the open, closed and transition state mimic-bound conformations of AnmK by x-ray crystallography; and develop a putative mechanism based on the accumulated research findings and 18O-labeling studies.

The anmK gene was successfully cloned as a hexahistidine fusion protein and overexpression was optimized. After exhaustive trials, a final purification scheme was designed to yield homogeneous AnmK in three chromatographic steps and in less than 36 hours. Additionally, the synthesis of both anhMurNAc and a pseudosubstrate (anhGlcNAc) were carried out in 35% and 77% overall yield, respectively. The synthesis of these compounds allowed for both kinetic characterization and structural studies.

To this end, the structure of de novo AnmK was solved using SAD and high-resolution (1.9 Å) data. Also, an ATP analog (ANP) and anhMurNAc substrate-bound, closed conformation structure (1.95 Å) was solved. These structures elucidated an 11° domain closure of the enzyme upon substrate binding and also revealed the active site geometry to be used to determine potential molecular determinants of specificity.

Insights into the kinetic mechanism of AnmK were then gathered using multiple techniques. First, the structure of AnmK (2.5 Å) was solved the with a known transition state analog, the MgADP-vanadate complex. Following this structure, which sheds light on the potential importance of a residue other than the catalytic base (Asp187), isotopic labeling was performed with H₂18O. Using NMR and MS, the regiochemical selectivity of AnmK hydrolysis to impart the solvent derived oxygen at C1 was established. Additionally, this was carried out with stereochemical preference to create the α-anomer of the carbohydrate product. This regiochemistry and stereospecificity drove the design of our putative concomitant hydrolysis/phosphorylation mechanism but we are not able to rule out the formation of a transient phosphoenzyme intermediate.

This research can be applied to the immediate goal of understanding the function of a single, novel enzyme with unique chemistry and the clarification of the AnmK mechanism will facilitate future investigation into enzymes with dual hydrolase/kinase functionality. Furthermore, this research contributes to understanding of the complex bacterial peptidoglycan layer in order to harness new ideas for developing antibiotics.

The overall goal of this project is to enhance the physical accuracy of individual models in macromolecular NMR (Nuclear Magnetic Resonance) structures and the realism of variation within NMR ensembles of models, while improving agreement with the experimental data. A secondary overall goal is to combine synergistically the best aspects of NMR and crystallographic methodologies to better illuminate the underlying joint molecular reality. This is accomplished by using the powerful method of all-atom contact analysis (describing detailed sterics between atoms, including hydrogens); new graphical representations and interactive tools in 3D and virtual reality; and structural bioinformatics approaches to the expanded and enhanced data now available.

The resulting better descriptions of macromolecular structure and its dynamic variation enhances the effectiveness of the many biomedical applications that depend on detailed molecular structure, such as mutational analysis, homology modeling, molecular simulations, protein design, and drug design.

Biomolecules such as proteins are highly dynamic, and undergo a wide variety of motions at different timescales. Movements as small as a bond vibration or as large as a domain rearrangement can be critical for the function of a protein, making consideration and investigation of protein dynamics necessary for understanding biological systems and developing therapeutics. In this work, we describe the development and implementation of novel techniques to study dynamics in proteins and protein-bound ligands, and discuss our investigation of the crucial role of dynamics in two disease-relevant systems.

First, we have expanded the utility of Chemical Exchange Saturation Transfer (CEST) NMR techniques to aid in the characterization of dynamics for nitrogen- and carbon-attached protons, as well as fluorine nuclei. Protons and fluorine nuclei can be exceptionally sensitive to their chemical environment, allowing detection and measurement of protein motions which may not be readily identified by conventional heteronuclear experiments. Additionally, we discovered the motion of a protein-bound ligand and utilized such information to improve the potency of an antibiotic molecule.

Next, we undertook the investigation and optimization of an inhibitor targeting translesion synthesis, a process that cancer cells can employ to resist the killing action of chemotherapeutics. Early work on this project demonstrated that inhibition of Rev1, an important scaffold in the translesion synthesis process, by the compound JH-RE-06 sensitizes cancer cells to cisplatin chemotherapy and prevents drug resistance. Surprisingly, we found that this inhibition occurs through inhibitor-induced dimerization of Rev1, which masks the protein-protein interface required for assembly of the translesion machinery. We further investigated a transient conformational change in the C-terminal tail of Rev1 and validated dimerization in solution using NMR. Our structure- activity relationship investigation of JH-RE-06 yielded a number of insights into how to develop more potent inhibitors. Most significantly, we found that small changes in the chemical structure of the inhibitor resulted in improved inhibitory activity and also led to a novel dimer arrangement. Our combination of Rev1 crystal structures and dynamics studies has led to a deeper understanding of the inhibitory mechanism of JH-RE-06 and will guide the optimization of this potential chemotherapy adjuvant.

Finally, we have investigated a mechanism of resistance to beta-lactam antibiotics in Neisseria gonorrhoeae, which relies on modulation of conformational dynamics. Neisseria gonorrhoeae is a major growing health concern due to the rapid spread of multi-drug resistance. We have discovered conformational exchange in PBP2, the target of beta-lactam antibiotics in Neisseria gonorrhoeae, between a low-affinity state and a high- affinity state. A histidine residue was found to be the key mediator of interconversion between these states via a network of molecular interactions, and we found that drug resistance-conferring mutations shift the equilibrium toward the low-affinity state by modulating these interactions. This work describes a novel mechanism of drug resistance in bacteria in which conformational dynamics are restricted.

This document illustrates a small sample of the important roles molecular motions have in biology, and the power of dynamics studies in understanding protein function, developing drugs, and elucidating resistance mechanisms.

RNA is the molecular workhorse of nature, capable of doing many cellular tasks, from genetic data storage and regulation, to enzymatic synthesis--even to the point of self-catalyzing its own replication. While RNA can act as a catalyst on its own, as in the hammerhead ribozyme, the added efficiency of proteins is often a necessity; the ribosome--the large ribozyme responsible for peptide chain formation, is aided by proteins which ensure correct assembly and structural stability. These complexes of RNA and proteins feature in many essential cellular processes, including the RISC silencing complex and in the spliceosome. Despite its enormous utility, structural determination of RNA is notoriously difficult--particularly in the backbone, since a nucleotide standardly has 12 torsion angles (including χ) and 12 non-hydrogen atoms, compared to 4 torsions (including χ1) and 4 non-H atoms in a typical amino acid. The abundance of backbone atoms, their conformational flexibility, and experimental resolution limitations often result in systematic errors that can have a significant impact on the interpretation. False trails due to structural errors can lead to significant loss of time and effort, especially with such high-profile complexes as the ribosome and the RISC complex.

My research has focused on harnessing the recently discovered ribosome structures and the Richardsons' RNA dataset to find trends in RNA backbone conformations and motifs that were then used to develop structural validation techniques and provide improved diagnosis and correction techniques for RNA backbone. Methods for fixing RNA structure have been developed for both NMR and X-ray crystallography. For NMR structures, a method for assigning RNA backbone structure based on NOE data was developed, leading to improved identification and building of RNA backbone conformation in NMR ensembles. For crystallography, our method of diagnosing the correct ribose pucker from clear observables allows reliable assessment of pucker in validation or refinement. Observed differences in bond-lengths, bond-angles, and dihedrals have been categorized by sugar pucker in the PHENIX refinement package. I have shown that this improves the refinement behavior of both pucker and geometry.

There have also been improvements in identifying structural motifs. Many previously identified structural motifs have now been defined in terms of backbone suitestrings, a series of 2-character code divisions of RNA backbone that show the best clustering of dihedral angle correlations. Combined with a BLAST-like alignment program called SuiteAlign, these suitestrings were quickly and easily identified in a number of structures, eventually leading to the discovery of multiple instances of TψC-loop structures in the ribosome.

To facilitate error diagnosis and corrections in RNA-protein complexes, as well as to expand the knowledge base of the scientific community as a whole, a database of RNA-protein interaction motifs has been developed. This database is rooted in the quality-filtering, visualization, and analysis techniques of the Richardson lab, particularly those developed by Laura Murray specifically for RNA structures.

The consensus backbone conformers, pucker diagnosis, and all-atom contacts have been combined to develop first manual and then automated tools for RNA structure correction. I have applied all these techniques to improve the accuracy of a number of important RNA and RNA/protein complex structures.

Nucleosides are small polar biomolecules that play important roles in every aspect of cellular life. Due to their impermeability to lipid bilayers, dedicated integral membrane proteins are tasked with selective transport of nucleosides across biological membranes. In humans, two genetically distinct protein families mediate nucleoside membrane transport: the concentrative and equilibrative nucleoside transporter families. Owing to the roles played by nucleoside transporters in physiology, pathophysiology, and pharmacology of nucleoside-analog therapeutics, a mechanistic understanding of human nucleoside transporters would not only advance our understanding of transporter biology in general but would also uncover exploitable pharmacological features for future drug development efforts. In this work, we first determined X-ray crystal structures of the human equilibrative nucleoside transporter in complex with two different adenosine reuptake inhibitors. Using our structural data as a starting point, we then designed and synthesized a series of novel transporter inhibitors with improved pharmacological properties. We also interrogated the role of both concentrative and equilibrative nucleoside transporters in nucleoside-analog antiviral drug pharmacological properties using biochemical experiments, viral assays, and cryo-electron microscopy.

The noncoding RNA (ncRNA) revolution has revealed myriad RNA species that play critical roles at all stages of life, including embryogenesis and disease progression. For example, three long ncRNA (lncRNA), Hox Transcript Antisense Intergenic RNA (HOTAIR; ~2.5 kb), Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1; ~6.7 kb) and Second Chromosome Locus Associated with Prostate-1 (SChLAP1; ~1.5 kb), are basally expressed in normal prostate tissue but are dysregulated in prostate cancer. HOTAIR scaffolds the PRC2 and LSD1 protein complexes to selectively methylate and demethylate, respectively, histone proteins, thereby regulating downstream gene expression. MALAT1 acts in trans at nuclear speckles during mRNA post-transcriptional processing while SChLAP1 acts in cis to influence oncogenic gene expression. Initial work on HOTAIR developed technical skills that were then applied to the lncRNAs MALAT and SChLAP1 to learn more about their role in prostate cancer. There is compelling evidence that the 3′–end of MALAT1 is a triple helix structure that acts as a molecular knot, driving transcript accumulation in cancer cells and furthering their metastatic potential, but we currently lack any biophysical data detailing the relationship between SChLAP1 structure and function. In general, the relationship among lncRNA structure, dynamics, and function is not well understood; for example, even with high-resolution structures of the MALAT1 triple helix, questions remain regarding the role of intrinsic dynamics in transcript stability or protein binding. As lncRNA represent an underexplored therapeutic avenue, this work aims to investigate the role of lncRNA structure and dynamics in driving prostate cancer metastasis. This work uses biophysical and biochemical methods including chemical probing, NMR, SAXS, and native gels to study the two lncRNA MALAT1 and SChLAP1 and learn more about their respective structure-function relationships. From this work, we have found a discrete structure within the lncRNA SChLAP1 that is highly structured and implicated in driving metastasis via protein recognition. Additionally, our preliminary studies regarding MALAT1 support the presence of non-triplex states that require further characterization. Overall, this work supports a deeper understanding of lncRNA structure as it relates to their function in cancer and provides examples for the biophysical analysis of large and/or structurally complex RNA.

Proteins are among the most complex entities known to science. Composed of just 20 fundamental building blocks arranged in simple linear strings, they nonetheless fold into a dizzying array of architectures that carry out the machinations of life at the molecular level.

Despite this central role in biology, we cannot reliably predict the structure of a protein from its sequence, and therefore rely on time-consuming and expensive experimental techniques to determine their structures. Although these methods can reveal equilibrium structures with great accuracy, they unfortunately mask much of the inherent molecular flexibility that enables proteins to dynamically perform biochemical tasks. As a result, much of the field of structural biology is mired in a static perspective; indeed, most attempts to naively model increased structural flexibility still end in failure.

This document details my work to validate alternative protein conformations beyond the primary or equilibrium conformation. The underlying hypothesis is that more realistic modeling of flexibility will enhance our understanding of how natural proteins function, and thereby improve our ability to design new proteins that perform desired novel functions.

During the course of my work, I used structure validation techniques to validate conformational alternatives in a variety of settings. First, I extended previous work introducing the backrub, a local, sidechain-coupled backbone motion, by demonstrating that backrubs also accompany sequence changes and therefore are useful for modeling conformational changes associated with mutations in protein design. Second, I extensively studied a new local backbone motion, helix shear, by documenting its occurrence in both crystal and NMR structures and showing its suitability for expanding conformational search space in protein design. Third, I integrated many types of local alternate conformations in an ultra-high-resolution crystal structure and discovered the combinatorial complexity that arises when adjacent flexible segments combine into networks. Fourth, I used structural bioinformatics techniques to construct smoothed, multi-dimensional torsional distributions that can be used to validate trial conformations or to propose new ones. Fifth, I participated in judging a structure prediction competition by using validation of geometrical and all-atom contact criteria to help define correctness across thousands of submitted conformations. Sixth, using similar tools plus collation of multiple comparable structures from the public database, I determined that low-energy states identified by the popular structure modeling suite Rosetta sometimes are valid conformations likely to be populated in the cell, but more often are invalid conformations attributable to artifacts in the physical/statistical hybrid energy function.

Unified by the theme of validating conformational alternatives by reference to high-quality experimental structures, my cumulative work advances our fundamental understanding of protein structural variability, and will benefit future endeavors to design useful proteins for biomedicine or industrial chemistry.