John Tanner - US grants

This award provides partial support for purchase of a cryo-cooling device to be installed in a shared facility used for x-ray protein crystallography. Crystallographic studies now play a central role in biology as the sequencing of large stretches of DNA reveals more and more genes whose role in the cell are poorly understood. Cryo-cooling minimizes radiation damage to protein crystals during data collection, and is an essential feature of modern x-ray crystallography workstations. At least 90% of the current crystallography projects on campus require cryo-cooling, including structural studies of a thermostable glyceraldehyde-3-phosphate dehydrogenase, other NAD binding enzymes, human bactericidal/permeability-increasing protein, EF-hand calcium binding proteins, anti-DNA antibodies, and enzymes in the alginate biosynthetic pathway. Although one such device is already available to the PIs, usage of the X-ray facility is expected to increase significantly as a result of the recent establishment of a Structural Biology Core facility for which they are responsible.

The parvalbumins - containing two "EF-hand Ca2+-binding motifs - offer an attractive system for examining protein-ligand interactions. Despite extensive homology, parvalbumin (PV) isoforms exhibit disparate metal ion-binding properties. There is compelling experimental evidence that PV divalent ion affinity is influenced by structural features outside the EF-hand motifs. The PV molecule consists of a 70-residue ion-binding domain (the CD-EF domain) and a 40-residue N-terminal AB domain. Others have previously shown that the AB/CD-EF interaction in pike PV is Ca2+-dependent. This project will extend this observation, exploring the hypothesis that the AB domain is a primary modulator of divalent ion-binding behavior. Recombinant AB and CD-EF domains from several a and b parvalbumin isoforms - and select site-specific variants - will be purified and characterized. Following examination of the isolated domains, the energetics of the AB/CD-EF interaction will be delineated in the presence and absence of divalent ions. These issues will be addressed by diverse methods: x-ray crystallography, optical spectroscopy, NMR spectroscopy, analytical ultracentrifugation, surface plasmon resonance, 45Ca2+-binding assays, and titration and scanning calorimetries.

Broadly defined, protein-ligand interactions underlie all aspects of protein function - from structure to transport to regulation to catalysis. Importantly, the precise orientation of the coordinating groups in a ligand-binding site can be influenced by structural reorganization events distant from the ligand-binding site. These conformationally mediated "action at a distance" phenomena are among the most intriguing aspects of protein/enzyme action. The parvalbumin molecule - with its juxtaposition of a single EF-hand domain and an autonomous structural element - offers an elegant model system for examining the influence of remote determinants on ligand-binding events and, conversely, the propagation of a ligand-binding signal to neighboring structural elements. Thus, the relevance of these studies extends well beyond structure-affinity correlations in EF-hand proteins.

DESCRIPTION (provided by applicant): The goal of this project is to characterize structure-function relationships for the multifunctional flavoprotein, PutA from Escherichia coli. This remarkable protein is both a transcriptional repressor of the proline utilization (put) regulon and a membrane-associated proline catabolic enzyme. The three-dimensional structural basis for the versatility of PutA is unknown. The working hypothesis whereby PutA changes its intracellular location and function is that conformational changes governed by the flavin redox state control its macromolecular associations (i.e. DNA and membrane-binding). The proposed research addresses three fundamental outstanding questions related to PutA structure and function: (1) What is the three-dimensional structure of PutA? (2) How does PutA interact with DNA? and (3) What are the conformational changes that allow PutA to function as both a DNA-binding protein and a membrane bound enzyme? The first aim of this proposal is to determine the three-dimensional structure of PutA using X-ray crystallography. Crystallization of PutA is challenging due to its large size (1320 amino acid residues) therefore a "divide and conquer" strategy will be employed in which shorter polypeptides that retain one or more of the functions of PutA will be engineered and crystallized separately. These smaller structures will then be stitched together computationally to derive a model of the full-length protein. Good progress has already been made using this approach - the 2.0 A crystal structure of a protein corresponding to the first 669 residues of PutA has been solved. The second aim is to determine the structural basis for PutA-DNA interactions by solving the crystal structures of PutA and truncated PutA proteins complexed with well-defined DNA binding sites. The third aim is to explore the conformational changes induced by proline reduction of the flavin by determining the crystal structures of PutA and truncated PutA proteins in the proline-reduced state. These studies will contribute pivotal understanding into the regulatory mechanism of PutA and timely knowledge of its structure.

Parvalbumins are small Ca2+-binding proteins best known for their cytosolic calcium-buffering activity. The b branch of the parvalbumin (PV) family exhibits a spectrum of divalent ion-binding affinity, with avian thymic hormone (ATH) and rat b-PV at the high- and low-affinity extremes, respectively, and avian parvalbumin 3 (PV3) in the middle. The goal of this project is to elucidate the structural basis for these differing metal ion-binding signatures. Existing data suggest that ion affinity is influenced by determinants outside the metal ion-binding motifs per se; that the interaction between the N-terminal AB region of the molecule and the CD-EF metal ion-binding domain can modulate ion affinity; and that structural differences in the unliganded proteins are responsible in part for the observed variations in affinity. This project has four specific objectives: 1) to identify the amino acid residues in PV3 that attenuate divalent ion affinity relative to ATH; 2) to identify those residues in rat b that attenuate metal ion affinity relative to PV3; 3) to obtain high-resolution structural data on the Ca2+-free forms of rat a (a high-affinity isoform), rat b (a low-affinity isoform), chicken PV3, and, time permitting, chimeric PVs formed from the AB and CD-EF domains of rat a and rat b; and 4) to compare and contrast the backbone dynamics in this same set of proteins. This research is expected to provide substantial insight into protein-ligand interactions, illustrating how the unliganded form of the protein can shape the energetics of ligand binding. The results should have relevance to the nascent field of protein design and engineering.

This project will provide valuable training to graduate and undergraduate students. Individuals involved with this effort will receive exposure to rigorous thermodynamic analysis of protein-ligand interactions and cutting-edge methods for high-resolution protein structure analysis. The data will be integrated into a problem-based graduate physical biochemistry course offered annually by the PI. This ever-evolving course seeks to provide an accessible introduction to physical methods of biomolecular characterization to students with average math and physics skills; to provide these students with an appreciation of the power of these physical tools; to encourage them to consider ways to incorporate these tools into their own research and, ideally, to consider a career in biophysics.

[unreadable] DESCRIPTION (provided by applicant): Metabolism is built of complex networks of enzymes that form links by sharing substrate and product molecules. Some of these shared molecules, which are often reactive, are not freely diffusing, but rather, their motion is directed, or channeled from one enzyme to another. In general, the mechanisms by which reactive molecules are passed between enzyme active sites are poorly understood. The goal of this project is to understand how the intermediates of proline catabolism are channeled from proline dehydrogenase (PRODH) to ?1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH). PRODH is a flavoenzyme that catalyzes the oxidation of proline to P5C. P5C is a reactive molecule that forms a nonenzymatic, pH-dependent equilibrium with the reactive carbonyl species glutamic semialdehyde (GSA). P5CDH is an NAD+dependent enzyme that catalyzes the oxidation of GSA to glutamate. The intermediate P5C/GSA is common to both the proline catabolic and synthetic pathways, and to arginine biosynthesis. P5C/GSA also influences many biological processes, including apoptosis, reactive oxygen species generation and RNA translation initiation. This project will use the bacterial bifunctional enzyme, Proline utilization A (PutA), as a model to understand channeling in detail. In PutAs, PRODH and P5CDH are fused into a single, large protein. The recent crystal structure of a PutA has revealed a unique system of internal cavities and tunnels that is hypothesized to function as both a reaction chamber for the hydrolysis of P5C to GSA and a protected pathway that facilitates transport of GSA to the P5CDH active site. Steady-state and rapid reaction kinetic data reported here also support a channeling mechanism for PutA. These initial observations furthermore suggest the hypothesis that monofunctional PRODH and P5CDH enzymes, such as those found in humans, interact and engage in intermolecular channeling. Channeling in bacterial homologs of the human enzymes will also be studied. This proposal has three aims: 1. Establish the structural and kinetic framework underlying substrate channeling in PutAs. 2. Investigate the mechanism of substrate channeling in PutA. 3. Explore substrate channeling and protein-protein interactions in monofunctional PRODH and P5CDH. Completion of these aims will provide a comprehensive, yet detailed, understanding of substrate channeling in proline catabolism. PUBLIC HEALTH RELEVANCE: This project proposes detailed biochemical and structural studies of the enzymes that recycle the amino acid proline by oxidizing it to glutamate. Genetic defects in these enzymes lead to hyperprolinemia disorders, which can be associated with mental retardation, higher frequency of febrile seizures and increased susceptibility to the disabling brain disorder schizophrenia. Also, one of the enzymes, proline dehydrogenase, helps reduce carcinogenesis in humans by serving as a reactive oxygen species generator in the cell death cascade mediated by tumor suppressor p53. The proposed research will examine how reactive intermediates are passed between proline recycling enzymes in a process known as substrate channeling. It is proposed that channeling is a fundamental aspect of the proline oxidation process. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Metabolic enzymes in cells rarely function in isolation. Often their activities are coordinated by physical association with each other and cellular structures. A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu but rather are channeled between enzymes. Despite the widespread recognition that protein-protein interactions are ubiquitous, the mechanisms of substrate channeling remain relatively understudied and thus poorly understood. We help close this knowledge gap by exploring substrate channeling within and between the enzymes of proline catabolism. Proline catabolism comprises two enzymes and an intervening hydrolysis step. The flavoenzyme proline dehydrogenase (PRODH) catalyzes the oxidization of L-proline to ?1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C yields L-glutamate-?-semialdehyde, which is oxidized to L-glutamate by the NAD+-dependent enzyme P5C dehydrogenase (P5CDH). These enzymes have been implicated in many aspects of human health and disease, including tumor suppression, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life- span extension, and the virulence of fungal and bacterial pathogens. In some organisms, PRODH and P5CDH are combined into a single polypeptide chain known as proline utilization A (PutA). The packaging of sequential enzymes from a metabolic pathway into a single protein not only implies substrate channeling but also the possibility of protein-protein interactions between the monofunctional enzymes. Thus, proline catabolism affords an excellent opportunity to compare substrate channeling within and between enzymes. The next phase of this project builds upon three major accomplishments made during the previous funding cycle: determination of the first crystal structures of PutA proteins, discovery of a novel hysteretic substrate channeling kinetic mechanism, and uncovering the first evidence for inter-enzyme substrate channeling between monofunctional PRODH and P5CDH enzymes. The specific aims are to (1) elucidate the diverse structural solutions to substrate channeling that have evolved in the PutA family, (2) determine the structural basis and conservation of the hysteretic channeling mechanism, and (3) study substrate channeling in biological context by examining PRODH - P5CDH interactions in whole cells and determining the phenotypic consequences of disrupting proline metabolic channeling.

? DESCRIPTION (provided by applicant): Knowledge of three-dimensional protein structure is indispensable in biomedical research. Protein structure and function are intimately linked, and thus structure facilitates drug discovery, aids investigations of protein-protein interactions, informs mutagenesis analysis, guides protein engineering and the design of new proteins, and provides a foundation for understanding the molecular basis of disease. However, the number of protein sequences available in the genomic era far exceeds the capacity of the main experimental structure determination techniques of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, resulting in a substantial sequence- structure gap. We address this ever-widening gap by developing and disseminating novel protein structure modeling tools. This renewal project is a new collaboration between experts in computational modeling (Cheng) and experimental structural biology (Tanner). We plan to develop innovative, integrated machine learning (e.g., deep learning), data mining and statistical modeling methods to address major challenges in both template-based structure modeling and template-free (ab initio) structure modeling. We will apply these tools to enzymes in the aldehyde dehydrogenase (ALDH) superfamily, a group of enzymes that are involved in numerous important biological processes and implicated in many diseases due to mutations. The ALDH models will be experimentally validated using X-ray crystallography and biochemical assays. Furthermore, we will combine the modeling power of our structural Input-Output hidden Markov model with experimental small- angle X-ray scattering (SAXS) to predict the tertiary structures of large multi-domain proteins. The integration of computational and experimental sciences in this project positions us uniquely in structure modeling space.

? DESCRIPTION (provided by applicant): Metabolic enzymes in cells rarely function in isolation. Often their activities are coordinated by physical association with each other and cellular structures. A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu but rather are channeled between enzymes. Despite the widespread recognition that protein-protein interactions are ubiquitous, the mechanisms of substrate channeling remain relatively understudied and thus poorly understood. We help close this knowledge gap by exploring substrate channeling within and between the enzymes of proline catabolism. Proline catabolism comprises two enzymes and an intervening hydrolysis step. The flavoenzyme proline dehydrogenase (PRODH) catalyzes the oxidization of L-proline to ?1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C yields L-glutamate-?-semialdehyde, which is oxidized to L-glutamate by the NAD+-dependent enzyme P5C dehydrogenase (P5CDH). These enzymes have been implicated in many aspects of human health and disease, including tumor suppression, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life- span extension, and the virulence of fungal and bacterial pathogens. In some organisms, PRODH and P5CDH are combined into a single polypeptide chain known as proline utilization A (PutA). The packaging of sequential enzymes from a metabolic pathway into a single protein not only implies substrate channeling but also the possibility of protein-protein interactions between the monofunctional enzymes. Thus, proline catabolism affords an excellent opportunity to compare substrate channeling within and between enzymes. The next phase of this project builds upon three major accomplishments made during the previous funding cycle: determination of the first crystal structures of PutA proteins, discovery of a novel hysteretic substrate channeling kinetic mechanism, and uncovering the first evidence for inter-enzyme substrate channeling between monofunctional PRODH and P5CDH enzymes. The specific aims are to (1) elucidate the diverse structural solutions to substrate channeling that have evolved in the PutA family, (2) determine the structural basis and conservation of the hysteretic channeling mechanism, and (3) study substrate channeling in biological context by examining PRODH - P5CDH interactions in whole cells and determining the phenotypic consequences of disrupting proline metabolic channeling.

? DESCRIPTION (provided by applicant): Metabolic enzymes in cells rarely function in isolation. Often their activities are coordinated by physical association with each other and cellular structures. A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu but rather are channeled between enzymes. Despite the widespread recognition that protein-protein interactions are ubiquitous, the mechanisms of substrate channeling remain relatively understudied and thus poorly understood. We help close this knowledge gap by exploring substrate channeling within and between the enzymes of proline catabolism. Proline catabolism comprises two enzymes and an intervening hydrolysis step. The flavoenzyme proline dehydrogenase (PRODH) catalyzes the oxidization of L-proline to ?1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C yields L-glutamate-?-semialdehyde, which is oxidized to L-glutamate by the NAD+-dependent enzyme P5C dehydrogenase (P5CDH). These enzymes have been implicated in many aspects of human health and disease, including tumor suppression, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life- span extension, and the virulence of fungal and bacterial pathogens. In some organisms, PRODH and P5CDH are combined into a single polypeptide chain known as proline utilization A (PutA). The packaging of sequential enzymes from a metabolic pathway into a single protein not only implies substrate channeling but also the possibility of protein-protein interactions between the monofunctional enzymes. Thus, proline catabolism affords an excellent opportunity to compare substrate channeling within and between enzymes. The next phase of this project builds upon three major accomplishments made during the previous funding cycle: determination of the first crystal structures of PutA proteins, discovery of a novel hysteretic substrate channeling kinetic mechanism, and uncovering the first evidence for inter-enzyme substrate channeling between monofunctional PRODH and P5CDH enzymes. The specific aims are to (1) elucidate the diverse structural solutions to substrate channeling that have evolved in the PutA family, (2) determine the structural basis and conservation of the hysteretic channeling mechanism, and (3) study substrate channeling in biological context by examining PRODH - P5CDH interactions in whole cells and determining the phenotypic consequences of disrupting proline metabolic channeling.

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Pablo Sobrado from Virginia Tech and John. J. Tanner from the University of Missouri to investigate the structure and chemistry of enzymes known as flavin-dependent-N-monooxygenases (NMOs). NMOs play integral parts in how microorganisms make numerous natural compounds for a variety of purposes. These compounds can also have useful bioactivities that benefit society. While many flavin-dependent enzymes are well known and have been thoroughly characterized, the NMOs are a relatively recently discovered branch on the family tree, and do not resemble the other members in common ways. This leads to the hypothesis that new and interesting structures and chemistries are likely to be at play in the reactions catalyzed by the NMOs. The investigators choose several examples of NMOs for in depth examination in order to compare and contrast their behaviors. Further, a highly multidisciplinary approach is proposed to provide the broadest possible understanding of the enzymes. The proposed experimental approach will provide a platform for the training of undergraduate and graduate students in modern enzymology and biophysical chemistry. The laboratories are also committed to strategies to enhance diversity in the scientific research workforce.

This project expands our knowledge of the diversity of flavoenzymes through the structural and biochemical characterization of new N-monooxygenases. Because these enzymes have low similarity to proteins already in the Protein Data Bank, the structures are expected to exhibit novel features and reveal unanticipated relationships to other enzymes. This further advances our understanding of the evolutionary relatedness of these enzymes. Also addressed are key knowledge gaps in our understanding of flavoenzyme mechanisms by characterizing ?double-hydroxylating? NMOs, exploring a newly discovered flavin ?flapping? motion in Group B NMOs, and trapping heretofore-elusive flavoenzyme-ligand complexes in crystallo for structure determination.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary A Chirascan V100 circular dichroism (CD) spectrometer is requested for the University of Missouri. CD is a widely applicable biochemical method for probing the secondary- and tertiary structures of proteins and nucleic acids in solution. It is also useful for evaluating macromolecular stability, monitoring the effects of various perturbants (pH, temperature, etc.) and can be used to study ligand-binding events. Data obtained from the proposed instrument will benefit the entire life sciences community on our campus, including the 6 major and 5 minor NIH-funded users on this proposal. In particular, research programs in structural biology and virology will be heavy users of this instrument. The Chirascan V100 will provide a state-of-the-art instrument with new features and accessories that are otherwise unavailable on campus. Among its advantages are superior sensitivity and accuracy, automatic calibration, an inexpensive air-cooled illumination source, and low nitrogen consumption. The Chirascan V100 is easy to use, compact, and requires minimal consumables. We request the following accessories to meet the needs of our user community: sample titrator with pH meter; fluorometer and fluorescence anisotropy detector; and a multi-cell sample turret. Justification for each of these is detailed in the proposal. The requested instrument will replace a non-functional, >25 year old Aviv spectrometer owned by the Biochemistry Department, which was the only shared CD on campus. Although painstakingly maintained for many years, this instrument was retired in 2018, due to an accumulation of problems that are beyond repair. The Chirascan V100 will be housed and administered in the Molecular Interactions research core facility in the centrally located Life Sciences Center. Instrument operation, supervision, and user training will be conducted by the Assistant Director of the core, a highly qualified research scientist. Detailed information on a financial plan, institutional commitment, and an external advisory committee for the instrument is provided. The Chirascan V100 will maximize recent campus investments in structural biology, and greatly benefit the overall growth and development of the life sciences community on our campus.

Modified Project Summary/Abstract Proline biosynthesis and catabolism share a common intermediate, ?1-pyrroline-5-carboxylate (P5C), and the enzymatic interconversion of proline and P5C is known as the ?proline cycle?. The first enzyme of catabolism, proline dehydrogenase (PRODH), catalyzes the FAD-dependent oxidation of proline to P5C, while the last enzyme of biosynthesis, P5C reductase (PYCR1), catalyzes the NAD(P)H-dependent reduction of P5C to proline. Together, PRODH and PYCR1 form the proline cycle, a novel pathway that effects the net transfer of electrons from NAD(P)H in the cytoplasm to the synthesis of ATP in mitochondria. Recent studies have shown that metastatic breast cancer cells alter their metabolism to harness the proline cycle for energy production, suggesting the hypothesis that PRODH and PYCR1 are potential cancer therapy targets. This idea is supported by in vivo data showing that the inhibition of PRODH by a proline analog impairs the formation of lung metastases in orthotopic mouse models of breast cancer. These results motivate this short-term project to develop chemical probes against PRODH and PYCR1 using focused and high-throughput screening approaches. The set of probes to be developed will enable future studies to mechanistically dissect the role of proline metabolism in cancer progression and assess the tractability of the proline cycle as a cancer therapy target. We expect that this knowledge will result in the long-term in new therapeutic strategies against cancer.