Browsing by Author "Spicer, Leonard D"
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Item Open Access Binding of MetJ repressor to specific and nonspecific DNA and effect of S-adenosylmethionine on these interactions.(Biochemistry, 2010-04-20) Augustus, Anne M; Sage, Harvey; Spicer, Leonard DWe have used analytical ultracentrifugation to characterize the binding of the methionine repressor protein, MetJ, to synthetic oligonucleotides containing zero to five specific recognition sites, called metboxes. For all lengths of DNA studied, MetJ binds more tightly to repeats of the consensus sequence than to naturally occurring metboxes, which exhibit a variable number of deviations from the consensus. Strong cooperative binding occurs only in the presence of two or more tandem metboxes, which facilitate protein-protein contacts between adjacent MetJ dimers, but weak affinity is detected even with DNA containing zero or one metbox. The affinity of MetJ for all of the DNA sequences studied is enhanced by the addition of SAM, the known cofactor for MetJ in the cell. This effect extends to oligos containing zero or one metbox, both of which bind two MetJ dimers. In the presence of a large excess concentration of metbox DNA, the effect of cooperativity is to favor populations of DNA oligos bound by two or more MetJ dimers rather than a stochastic redistribution of the repressor onto all available metboxes. These results illustrate the dynamic range of binding affinity and repressor assembly that MetJ can exhibit with DNA and the effect of the corepressor SAM on binding to both specific and nonspecific DNA.Item Open Access Development of new approaches to NMR data collection for protein structure determination(2007-05-10T16:02:04Z) Coggins, Brian E.Multidimensional nuclear magnetic resonance (NMR) spectroscopy has become one of the most important techniques available for studying the structure and function of biological macromolecules at atomic resolution. The conventional approach to multidimensional NMR involves the sampling of the time domain on a Cartesian grid followed by a multidimensional Fourier transform (FT). While this approach yields high quality spectra, as the number of dimensions is increased the time needed for sampling on a Cartesian grid increases exponentially, making it impractical to record 4-D spectra at high resolution and impossible to record 5-D spectra at all. This thesis describes new approaches to data collection and processing that make it possible to obtain spectra at higher resolution and/or with a higher dimensionality than was previously feasible with the conventional method. The central focus of this work has been the sampling of the time domain along radial spokes, which was recently introduced into the NMR community. If each radial spoke is processed by an FT with respect to radius, a set of projections of the higher-dimensional spectrum are obtained. Full spectra at high resolution can be generated from these projections via tomographic reconstruction. We generalized the lower-value reconstruction algorithm from the literature, and later integrated it with the backprojection algorithm in a hybrid reconstruction method. These methods permit the reconstruction of accurate 4-D and 5- D spectra at very high resolution, from only a small number of projections, as we demonstrated in the reconstruction of 4-D and 5-D sequential assignment spectra on small and large proteins. For nuclear Overhauser spectroscopy (NOESY), used to measure interproton distances in proteins, one requires quantitative reconstructions. We have successfully obtained these using filtered backprojection, which we found was equivalent to processing the radially sampled data by a polar FT. All of these methods represent significant gains in data collection efficiency over conventional approaches. The polar FT interpretation suggested that the problem could be analyzed using FT theory, to design even more efficient methods. We have developed a new approach to sampling, using concentric rings of sampling points, which represents a further improvement in efficiency and sensitivity over radial sampling.Item Open Access FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus.(Biochemical and biophysical research communications, 2020-05) Juvvadi, Praveen R; Bobay, Benjamin G; Gobeil, Sophie MC; Cole, D Christopher; Venters, Ronald A; Heitman, Joseph; Spicer, Leonard D; Steinbach, William JThe 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.Item Open Access Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents.(Nature communications, 2019-09) Juvvadi, Praveen R; Fox, David; Bobay, Benjamin G; Hoy, Michael J; Gobeil, Sophie MC; Venters, Ronald A; Chang, Zanetta; Lin, Jackie J; Averette, Anna Floyd; Cole, D Christopher; Barrington, Blake C; Wheaton, Joshua D; Ciofani, Maria; Trzoss, Michael; Li, Xiaoming; Lee, Soo Chan; Chen, Ying-Lien; Mutz, Mitchell; Spicer, Leonard D; Schumacher, Maria A; Heitman, Joseph; Steinbach, William JCalcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.Item Open Access Salmonella Typhi Vi capsule prime-boost vaccination induces convergent and functional antibody responses.(Science immunology, 2021-10) Dahora, Lindsay C; Verheul, Marije K; Williams, Katherine L; Jin, Celina; Stockdale, Lisa; Cavet, Guy; Giladi, Eldar; Hill, Jennifer; Kim, Dongkyoon; Leung, Yvonne; Bobay, Benjamin G; Spicer, Leonard D; Sawant, Sheetal; Rijpkema, Sjoerd; Dennison, S Moses; Alam, S Munir; Pollard, Andrew J; Tomaras, Georgia DVaccine development to prevent Salmonella Typhi infections has accelerated over the past decade, resulting in licensure of new vaccines, which use the Vi polysaccharide (Vi PS) of the bacterium conjugated to an unrelated carrier protein as the active component. Antibodies elicited by these vaccines are important for mediating protection against typhoid fever. However, the characteristics of protective and functional Vi antibodies are unknown. In this study, we investigated the human antibody repertoire, avidity maturation, epitope specificity, and function after immunization with a single dose of Vi-tetanus toxoid conjugate vaccine (Vi-TT) and after a booster with plain Vi PS (Vi-PS). The Vi-TT prime induced an IgG1-dominant response, whereas the Vi-TT prime followed by the Vi-PS boost induced IgG1 and IgG2 antibody production. B cells from recipients who received both prime and boost showed evidence of convergence, with shared V gene usage and CDR3 characteristics. The detected Vi antibodies showed heterogeneous avidity ranging from 10 μM to 500 pM, with no evidence of affinity maturation after the boost. Vi-specific antibodies mediated Fc effector functions, which correlated with antibody dissociation kinetics but not with association kinetics. We identified antibodies induced by prime and boost vaccines that recognized subdominant epitopes, indicated by binding to the de–O-acetylated Vi backbone. These antibodies also mediated Fc-dependent functions, such as complement deposition and monocyte phagocytosis. Defining strategies on how to broaden epitope targeting for S. Typhi Vi and enriching for antibody Fc functions that protect against typhoid fever will advance the design of high-efficacy Vi vaccines for protection across diverse populations.Item Open Access Interactions of the MetJ Repressor from E. Coli with DNA and SAM(2009) Augustus, Anne MarieTranscription regulators are proteins that bind to specific DNA sequences in order to control the expression of specific genes. Often the sequences that are bound are not identical, but contain deviations from a common "consensus" sequence. The proteins that recognize these non-consensus sites must be able to recognize a variety of related sequences. MetJ is the transcription regulator that controls the expression of genes involved in methionine biosynthesis and transport in E. coli and other related organisms. A consensus sequence is known, but almost all the naturally occurring binding sites for MetJ differ from this. The goal of this dissertation is to understand how MetJ recognizes its various target sites within the context of the genomic DNA in which they are embedded. This work uses a variety of biochemical and biophysical techniques to further our understanding of an important regulatory protein.
Chapter 2 describes the results of both in cell and in vitro NMR showing that MetJ associates with non-specific genomic DNA in the cell, and that specific DNA (containing the consensus sequence) can successfully compete with a large excess of non-specific DNA for MetJ binding.
Chapter 3 describes work performed with small-angle neutron scattering showing different modes of MetJ binding to DNA of variable length and sequence.
Chapter 4 extends the neutron-scattering results by using analytical ultracentrifugation to look at MetJ binding to a wide variety of DNA sequences, both in the presence and absence of its co-factor, S- adenosylmethionine (SAM). Evidence is presented for SAM-mediated binding to both specific and non-specific DNA, as well as the importance of cooperativity in binding multiple MetJ molecules to a single DNA.
Item Open Access Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs.(mBio, 2021-12) Gobeil, Sophie M-C; Bobay, Benjamin G; Juvvadi, Praveen R; Cole, D Christopher; Heitman, Joseph; Steinbach, William J; Venters, Ronald A; Spicer, Leonard DCalcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.Item Open Access Proteins Inside Cells and On the Surface of Membranes: Developing Three Dimensional In-Cell NMR and Structural Characterization of a Trimeric Membrane Proximal External Region Construct from HIV-1 gp41(2011) Reardon, Patrick NealOur ability to understand complex biological processes is enhanced by studying the components of those processes at atomic resolution. Major advancements in technology and methodology have been made that continue to improve our ability to determine atomic resolution structures of proteins. These advancements have also enabled new ways to study difficult protein systems. In this dissertation, I will discuss our efforts to apply NMR spectroscopy and other biophysical techniques to better understand two problems. The first problem, and the focus of the first part of this work, is to understand the behavior of proteins in the complex milieu of a living cell. We have developed 3D In-Cell protein NMR and demonstrated its use to assign the backbone resonances of a protein in living E. coli cells. I will also discuss our application of In-Cell NMR to show that the methionine repressor, MetJ, is generally associated non-specifically with DNA inside living cells. In the second part of this dissertation I will discuss our efforts to better understand the biophysical behavior of the membrane proximal external region (MPER) of the HIV-1 envelope protein, gp41. This region is membrane associated and an important target for HIV-1 vaccine development because it contains the epitopes for several of the broadly neutralizing antibodies against HIV-1. In addition to studies of the monomeric peptide, we have developed a new trimeric MPER construct, designated gp41-M-MAT, that associates with detergent micelles and lipid bilayers. This construct is a stable trimer, which binds the broadly neutralizing antibodies 2F5 and 4E10, as shown by equilibrium analytical ultracentrifugation and surface plasmon resonance. Finally, we have solved the structure of this construct bound to detergent micelles using NMR spectroscopy. The structure shows that the MPER adopts an α-helix conformation and suggests that each helix in the symmetric trimer associates directly with the surface of detergents and lipid membranes. This association suggests that the MPER might contribute to the mechanism of viral fusion by inducing strain in the viral membrane.
Item Open Access 15N, 13C and 1H resonance assignments of FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus.(Biomolecular NMR assignments, 2019-04) Gobeil, Sophie MC; Bobay, Benjamin G; Spicer, Leonard D; Venters, Ronald AInvasive fungal infections are a leading cause of death in immunocompromised patients and remain difficult to treat since fungal pathogens, like mammals, are eukaryotes and share many orthologous proteins. As a result, current antifungal drugs have limited clinical value, are sometimes toxic, can adversely affect human reaction pathways and are increasingly ineffective due to emerging resistance. One potential antifungal drug, FK506, establishes a ternary complex between the phosphatase, calcineurin, and the 12-kDa peptidyl-prolyl isomerase FK506-binding protein, FKBP12. It has been well established that calcineurin, highly conserved from yeast to mammals, is necessary for invasive fungal disease and is inhibited when in complex with FK506/FKBP12. Unfortunately, FK506 is also immunosuppressive in humans, precluding its usage as an antifungal drug, especially in immunocompromised patients. Whereas the homology between human and fungal calcineurin proteins is > 80%, the human and fungal FKBP12s share 48-58% sequence identity, making them more amenable candidates for drug targeting efforts. Here we report the backbone and sidechain NMR assignments of recombinant FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus in the apo form and compare these to the backbone assignments of the FK506 bound form. In addition, we report the backbone assignments of the apo and FK506 bound forms of the Homo sapiens FKBP12 protein for evaluation against the fungal forms. These data are the first steps towards defining, at a residue specific level, the impacts of FK506 binding to fungal and mammalian FKBP12 proteins. Our data highlight differences between the human and fungal FKBP12s that could lead to the design of more selective anti-fungal drugs.