General Interest I

Conference: 2020: 70th ACA Annual Meeting
08/03/2020: 12:00 PM  - 3:00 PM 
Oral Session 


General Interest sessions are the forum for topics of broad interest to the crystallographic community or for presentations that do not fit the specific theme of other sessions. All presentations are selected from submitted abstracts.


Incorporating HT-SAXS into Drug Discovery Pipelines

12:00 PM - 12:20 PM 
High-throughput (HT) methods for discovering single-target protein and nucleic acid ligands are well established and readily utilized for drug discovery. However, important biological outcomes are mediated by multi-component assemblies and dynamic macromolecular architectures. HT approaches to monitor ligand impact on functional assemblies and to assess chemical selection of architectural states remain underdeveloped. Here, we have incorporated HT-SAXS into a classic fragment screening pipeline and use this approach to identify chemical allosteric effectors targeting structural states of the mitochondrial and cell death protein, Apoptosis-Inducing Factor (AIF). AIF allosterically switches from monomer to dimer upon engaging NADH in a charge-transfer complex. This dimer-monomer exchange is proposed to regulate AIF's functional transition from supporting mitochondrial import to facilitating PARP-1-dependent cell death (parthanatos). Application of time-resolved HT-SAXS to lead fragment binders from differential scanning fluorimetry (DSF) screening identified and ranked three chemotypes allosterically stabilizing monomeric or dimeric AIF. Secondary screening with focused fragment libraries enriched with these chemotypes has produced optimized scaffolds targeting AIF's NADH binding site. Our results demonstrate how incorporation of HT-SAXS into fragment screening protocols customizes ligand development to macromolecular architecture, assembly, and allostery. 

View Abstract 338


Chris Brosey, MD Anderson Houston, TX 

Additional Author(s)

Runze Shen, Molecular and Cellular Oncology, M.D. Anderson Cancer Center Houston, TX 
Kathryn Burnett, Molecular Biophysics and Integrated Bioimaging Division (MBIB), Lawrence Berkeley National Laborator Berkeley, CA 
Greg Hura, Molecular Biophysics and Integrated Bioimaging Division (MBIB), Lawrence Berkeley National Laborator Berkeley, CA 
Davide Moiani, Molecular and Cellular Oncology, M.D. Anderson Cancer Center Houston, TX 
Darin Jones, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences Little Rock, AR 
John Tainer, MD Anderson

SAXS studies of X-ray induced disulfide bond damage: Engineering high-resolution insight into a low resolution technique

12:20 PM - 12:40 PM 
A significant problem in biological X-ray crystallography is the radiation chemistry caused by the incident X-ray beam. This produces both global and site-specific damage. Global damage manifests itself in the decay of the diffraction pattern and data processing parameters. Site specific damage targets glutamates, aspartates, methionines, and disulfide bonds. This damage can misdirect the biological interpretation of the structural models produced. Cryo-cooling crystals has been successful in mitigating damage but not eliminating it altogether; however, cryo-cooling can be difficult in some cases and has also been shown to limit functionally relevant protein conformations. Due to this, there has been an interest in the return to near-physiological temperature studies. The doses used for X-ray crystallography are typically in the kGy to MGy range. X-rays are used therapeutically at significantly lower doses. While, disulfide bonds are among the most significantly affected species in proteins in the crystalline state at both cryogenic and higher temperatures, there is limited information on their response to low X-ray doses. In this work we engineered a protein that dimerizes through a susceptible disulfide bond to relate the radiation damage processes seen in crystals to those closer to physiologic conditions. This approach enables a low-resolution technique, small angle X-ray scattering (SAXS), to detect and monitor a residue specific process. We monitored monomerization with SAXS detecting structural impact with doses a fraction of that required for crystallographic studies. A dose dependent fragmentation of the engineered protein was seen that can be explained by a dimer to monomer transition, from disulfide bond cleavage. This supports the crystallographically derived mechanism and demonstrates that results obtained crystallographically, can be usefully extrapolated to physiologic conditions. Fragmentation was pH dependent, providing information on mechanism and pointing to future routes for investigation and potential mitigation. The engineered protein approach generating a large-scale change through a site-specific interaction represents a promising tool for advancing radiation damage studies under solution conditions. 

View Abstract 120


Timothy Stachowski, Hauptman Woodward Institute Buffalo, NY 

Additional Author(s)

Elizabeth Snell, Hauptman Woodward Institute Buffalo, NY 
Edward Snell, Hauptman-Woodward Medical Research Institute/BioXFEL Buffalo, NY 

Structural Basis for the tight binding inhibition of E coli. CTP Synthase Inhibition by Gemcitabine and its analogues

12:40 PM - 1:00 PM 
CTP synthase (CTPS) is a vital metabolic enzyme which controls the pyrimidine nucleotide pool by converting UTP into CTP with the use of ATP and ammonia. The E coli. variant is tightly regulated by the available nucleotides, where it is allosterically modulated by GTP and CTP. CTP binding can induce inhibitory filament formation, whereas the human isozyme forms active filaments upon UTP binding. Due to the in vivo importance of CTPS, variable regulatory mechanism across species, and previous data suggesting it is vital to the maintenance of various disease states, selective inhibition of CTPS may be a fruitful avenue to develop treatments for some of these illnesses. Most recently, a series of fluorinated CTP analogues were characterized for their inhibition against CTPS. Of these molecules, a doublely fluorinated at the 2' position of the ribose moiety had an at least 30x increased inhibition over its monofluorinated counterparts. Here, we determined the crystal structures of the various fluorinated inhibitors of CTPS to determine the molecular interactions and conformational changes contributing to the potent inhibition of the enzyme by these nucleotide analogues. 

View Abstract 264


Matthew McLeod, Biology, Univ of Waterloo Waterloo

Additional Author

Todd Holyoak, Biology Dept, Univ of Waterloo

Coffee Break

1:00 PM - 1:20 PM 

Structure of the Fiber Core of Orb2A, A Functional Amyloid, Revealed by Micro-electron Diffraction

1:20 PM - 1:40 PM 
Amyloid protein aggregation is typically associated with neurodegenerative diseases such as Alzheimer's disease. However, not all amyloid proteins are pathogenic in their aggregated state. The neuronal cytoplasmic polyadenylation element binding (CPEB) protein has been shown to form "functional" amyloid aggregates in several model organisms. CPEB is a regulator of synaptic mRNA translation, and CPEB aggregation has been shown to be an important step in the formation of long-term memory. Our work is focused on the Drosophila CPEB homolog, termed Orb2A. The first 9 N-terminal amino acid residues of Orb2A are necessary for its aggregation, and this segment has been suggested to form the amyloid fiber core. Intriguingly, a single point mutation of the phenylalanine residue in the 5th position to tyrosine (F5Y) decreases Orb2A aggregation and impairs long-term memory formation in Drosophila. To understand the structural basis for Orb2A aggregation, we characterized this critical 9-residue segment of Orb2A, which we call M9I-WT. Using micro-electron diffraction, we determined the crystal structure of M9I-WT at a resolution of 1.0 Å. The segment forms an array of parallel in-register β-sheets, which are held together tightly by inter-strand aromatic and hydrophobic side chain interactions. We also compared the structural properties of the wild-type protein with interface-blocking mutants, and observed mutant segment fibrils are shorter and exhibit poorer ordering. Our model provides an explanation for the decreased aggregation observed for the F5Y mutant, and offers a hypothesis for how the addition of a single atom (the tyrosyl oxygen) can affect memory. Ultimately we aim to understand the differences between functional and pathological amyloids, and thus further our understanding of amyloid disease mechanisms, and improve therapeutic strategies. 

View Abstract 346


David Eisenberg, Univ Of California Los Angeles Los Angeles, CA 

Additional Author(s)

Jeannette Bowler, UCLA-DOE Institute Los Angeles, CA 
Michael Sawaya
David Boyer, UCLA-DOE Institute Los Angeles, CA 
Duilio Cascio, UCLA-DOE Institute Los Angeles, CA 
David Eisenberg, Univ Of California Los Angeles Los Angeles, CA 

Conformational switching of FMN and THF riboswitches

1:40 PM - 2:00 PM 
Flavin mononucleotide (FMN) and tetrahydrofolate (THF) are essential cofactors for a number of biosynthetic pathways in bacteria. As such, the FMN and THF RNA riboswitches, which depend on those cofactors for genetic regulation, present attractive targets for developing novel antimicrobial therapeutics. Riboswitches get their names from the conformational switching that takes place upon ligand binding, which enables control of transcription or translation of the gene to which the riboswitch is associated. The first step in understanding the structural basis for the switching mechanism of a riboswitch requires structure determination of both ligand-free and ligand-bound conformational states. We recently determined novel structures of the FMN and THF riboswitches in the absence of their respective ligands, illustrating the conformational differences from their ligand-bound conformers. In the case of FMN riboswitch, the flexible L4 loop adjacent to the FMN-binding site alters the structure and stability of the regulatory helix P1, depending on whether or not FMN is bound. Similarly, the apo structure of THF riboswitch illustrates a different configuration of the ligand binding pocket that subsequently alters the conformations of the helices P1 and P3. Together, these structures increase our understanding of the ligand-dependent control mechanisms of RNA riboswitches. 

View Abstract 255


Jason Stagno, National Cancer Institute Frederick, MD 

Additional Author(s)

Haley Wilt, MCL Frederick, MD 
Kemin Tan, Argonne National Laboratory Argonne, IL 
Ping Yu, National Cancer Institute Frederick, MD 
Yun-Xing Wang, Structural Biophysics Laboratory, National Cancer Institute

Structural insights into the recognition of mono- and di-acetylated histones by the ATAD2B bromodomain

2:00 PM - 2:20 PM 
Bromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In this study, we examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain selects for mono- and di-acetylated histones, and structural analysis identified key residues in the acetyllysine binding pocket that dictate ligand binding specificity. The X-ray crystal structure of the ATAD2B bromodomain in complex with an ATAD2 bromodomain inhibitor was solved at 2.4 Å resolution. This structure demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized a variant of the ATAD2B bromodomain that through alternative splicing loses critical amino acids required for histone ligand and inhibitor coordination. Altogether our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism important for regulating the interaction of the ATAD2B protein with chromatin. 

View Abstract 256


Karen Glass, Pharmaceutical Sciences, Albany College of Pharmacy & Health Sciences Colchester, VT 

Toledo Crystallization Box: Microgravity Crystallization of Perdeuterated S. typhimurium Tryptophan Synthase for Neutron Diffraction

2:20 PM - 2:40 PM 
As ubiquitous proteins with significant metabolic functions, PLP-dependent enzymes are attractive targets for specific inhibitor design. Tryptophan synthase (TS) is a model for -elimination catalyzed by a PLP-dependent enzyme. In TS, an indole is coupled to a PLP- activated serine, forming tryptophan. Because it lacks a human or animal homolog, TS is an attractive target for inhibition of pathogenic organisms, such as S. aureus. Neutron diffraction of TS will provide positions of hydrogens or deuteriums in the structure, not only allowing for a complete visualization of the active site electronic state but also providing more accurate input models for high order computation. We designed the Toledo Crystallization Box (TCB) to perform capillary dialysis crystallization of perdeuterated TS in microgravity. Our design allows for modulation of equilibration times and sampling of a large variation in crystallization conditions while being highly cost effective. Here, we report the development and employment of the TCB for CASIS PCG 12 and 15 aboard the International Space Station (ISS) for 1 month and 6 months duration, respectively. Preliminary neutron diffraction of perdeuterated TS crystals from PCG 15 exhibited improved neutron diffraction resolution compared to ground control experiments. 

View Abstract 334


Victoria Drago, University of Toledo Columbiana, OH 

Additional Author(s)

Timothy Mueser, Dept of Chemistry and Biochemistry, Univ. Of Toledo
Constance Schall, University of Toledo Toledo, OH 

Structural basis of ribosomal RNA transcription regulation

2:40 PM - 3:00 PM 
Ribosomal RNA (rRNA) is the most highly expressed gene in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. To reveal the mechanism of highly regulated rRNA transcription, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme at different steps of rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element of rRNA promoter using the dimerized α subunit carboxyl-terminal domain and scrunches the template DNA with the σfinger and β'lid to select a transcription start site favorable for rRNA expression. Promoter DNA binding to RNAP induces conformational change of the σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding to RNAP also opens the DNA loading gate, but it is not coupled to σ1.1 ejection and impedes the open complex formation of the rRNA promoter due to its G+C rich discriminator sequence. Mutations in σ1.1 or the β'lid stabilize the RNAP and rRNA promoter complex and decrease its sensitivity to DksA/ppGpp. These results provide a molecular basis for exceptionally active rRNA transcription and for its vulnerability to DksA/ppGpp. ( 

View Abstract 247


Katsuhiko Murakami, Penn State Univ University Park, PA