Browsing by Subject "Models, Molecular"
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Item Biochemical Reconstitution and Functional Characterization of the WAVE Regulatory Complex(2009-09-04) Ismail, Ayman Mohamed; Rosen, Michael K.Members of the Wiskott-Aldrich syndrome protein (WASP) family (WASP, N-WASP, WAVE 1-3) have a central role in the transmission of the extracellular signals to the actin cytoskeleton. These proteins use their C-terminal VCA domain to stimulate the actin-nucleating activity of Arp2/3 complex in response to upstream signals from the Rho family GTPases Cdc42 and Rac1. While WASP regulation by GTPases and kinases is well characterized both biochemically and structurally, little is known about WAVE regulation. WAVE exists as part of a five protein complex termed the WAVE Regulatory Complex (WRC). It consists of WAVE, Sra1, Nap1, Abi2 and HSPC300. Biochemical studies of WRC have been hampered by the difficulty of expressing WRC components in bacterial, insect or yeast expression systems. Baculoviruses yielding high expression of each component of WRC were obtained using a modified pFastBac vector, where translation is driven by the lobster tropomyosin promoter. Co-infection into Sf9 cells allowed efficient expression and purification of WRC and two sub-complexes, Sra-Nap and Abi2-WAVE1-HSPC300. We show that WRC is inactive toward Arp2/3 complex in pyrene based actin assembly assays. However, Abi2-WAVE1-HSPC300 heterotrimer is active and Sra1-Nap1 heterodimer inhibits it suggesting that WRC is autoinhibited. A modified WRC complex, where WAVE1 has a PreScission protease site between its VCA domain (the active domain) and its N-terminus and is lacking the proline rich domain, is also inactive toward Arp2/3 complex. However, upon digestion with PreScission protease, this modified complex becomes active. This suggests that the affinity between VCA and the Sra-Nap heterodimer is inherently weak and the heterodimer requires the linkage provided by the Abi2-WAVE1-HSPC300 heterotrimer to VCA to efficiently inhibit it from activating the Arp2/3 complex. The same results are obtained using all Drosophila components. Finally, Rac1-GTP is able to activate WRC towards Arp2/3. However, no dissociation of the complex is detected upon activation by Rac1. In addition to WRC regulation, we have established the mechanism for hyperactivation of VCA through dimerization. We found that a dimeric VCA construct binds Arp2/3 complex with a two VCAs to one Arp2/3 ratio. The affinity of dimeric VCA for Arp2/3 is at least 100 fold higher than monomeric VCA. That explains the potentiation of VCA toward Arp2/3 observed upon VCA dimerization, and provides a mechanistic framework for a new model of WASP regulation superimposed upon allostery. We have also demonstrated that N-WASP and WRC may be able to form a hetero-VCA dimer through the interaction of Abi2 SH3 domain and N-WASP PRD. Such interaction increases the complexity and the signal integration potential of WASp family proteins.Item A Fragile Native State Structure: An Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Variant Exhibits Slow Interconversion Kinetics Between two Different Folds(2009-09-04) Evans, Matthew Ryan; Gardner, Kevin H.The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways by forming heterodimeric transcriptional regulator complexes with several other bHLH-PAS proteins via the beta-sheet surfaces of its two PAS domains. The beta-sheets of PAS domains are involved in many intermolecular interactions with other proteins and natural cofactors in order to detect environmental changes in sensor PAS proteins. As part of a study of the HIF-2 alpha and ARNT PAS-B heterodimer, site-directed mutagenesis was performed on the ARNT PAS-B domain. Interestingly, one point mutation on the ARNT beta-sheet surface (Y456T) resulted in a new conformation of the domain that existed in equimolar concentrations with the wild-type conformation. Subsequent studies demonstrated that the two conformations are in equilibrium and that relative populations of the two conformations can be perturbed by additional mutations. Using solution NMR spectroscopy, we solved the high resolution solution structure of a mutant ARNT PAS-B domain in the new conformation, demonstrating that it contains a three-residue slip in register and accompanying inversion of the central beta-strand. In addition, this new conformer has a greater than hundred-fold reduction in affinity for HIF-2 alpha PAS-B, disrupting the hypoxia response pathway. Solution NMR measurements of the interconversion kinetics have let us establish that these two conformations interconvert slowly (40 min at RT) with a linear Arrhenius temperature-dependence of the interconversion rate. Stopped-flow unfolding experiments using GdmHCl on Y456T, revealed a similarly slow unfolding rate (25 min at RT) and an energy barrier to unfold of approximately 13 kcal/mol, which is nearly identical to that for the interconversion process. These data indicate that the protein must undergo a global unfolding process in order to interconvert between conformations. Lastly, these relative populations of Y456T can be affected by compound preferentially binding into the core of one of these conformations. This discovery highlights the malleability of PAS beta-sheets and suggests ARNT may act as a regulatory switch to control different biological pathways. Furthermore, this system presents a great opportunity to further understand the structural and kinetic impact of beta-strand slips observed in nature.Item A New Approach to Optimize a Protein Energy Function on a Folding Pathway Using Gō-Like Potential and All-Atom, Ab Initio Monte Carlo Simulations(2016-01-19) Safronova, Aleksandra; Goldsmith, Elizabeth J.; Grishin, Nick V.; Otwinowski, Zbyszek; Rice, Luke M.Prediction of a protein structure is important for understanding the function of a protein. The process of protein structure prediction employs the approximation of a protein free energy that guides protein folding to the protein's native state. A function with a good approximation of the protein free energy should allow estimation of the structural distance of the evaluated candidate structure to the protein native state. Currently the energy optimization process relies on the correlation between the energy and the similarity to the native structure. The energy function is presented as a weighted sum of components which are designed by human experts with the use of statistical analysis of solved protein strictures. Values of the weights are derived through the procedure that maximizes the correlation between the energy and the similarity to the native structure measured by a root mean square deviation between coordinates of the protein backbone. Two major components are required for a successful ab initio modelling: (1) an effective energy function that discriminates the native protein structure out of all possible decoy structures; (2) an efficient sampling algorithm that quickly searches for the low-energy states. In this dissertation a new method for energy optimization is proposed. The method relies on a fast sampling algorithm and targets successful protein folding. The weights for energy components are optimized on a found with the Gō potential energy fast folding pathway. The Lennard-Jones potential, the Lazaridis-Karplus solvation potential, hydrogen bonding potential are used in the optimization algorithm. The optimized weights successfully predict all α and α/β proteins. The proposed strategy is conceptually different from the existing methods that optimize the energy on solved protein structures. The developed algorithm is a novel concept that allows the optimization of a more complex functional combination of the energy components that would improve the prediction quality.Item Protein Composition and Subcellular Localization of the De Novo Lipogenic Metabolon(2016-04-18) McKean, William Bennion, Jr.; DeBose-Boyd, Russell A.; Horton, Jay D.; Russell, David W.; Uyeda, KosakuFatty acids are the major components of triglycerides, phospholipids, and sphingolipids. Production of palmitate, the most abundant saturated fatty acid, involves the stepwise actions of three enzymes: ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase. Canonically each enzyme catalyzes discrete reactions, and it is thought that they localize diffusely in cellular cytoplasm separate from one another. If true, transfer of metabolic intermediates must occur through passive diffusion from one lipogenic enzyme to another. Such a model proposes an extremely inefficient and potentially hazardous method of palmitate production. We demonstrated that two related proteins - designated MIG12 and Spot 14 - modulate fatty acid synthesis and triglyceride production by regulating the polymerization and activity of acetyl-CoA carboxylase. To better characterize the relationship between these three proteins, biochemical properties of purified recombinant MIG12, Spot 14, and MIG12:Spot 14 heterodimer were assayed in combination with acetyl-CoA carboxylase. We found that Spot 14 abrogates the ability of MIG12 to polymerize and activate acetyl-CoA carboxylase. Co-immunoprecipitation studies using Spot 14 in rat liver revealed Spot 14 exists in a complex with fatty acid synthase and acetyl-CoA carboxylase. MIG12 and Spot 14 co-immunoprecipitation also revealed that ATP citrate lyase was in association with the complex, suggesting that these proteins can function as scaffolds for the three enzymes required for palmitate synthesis. Studies of the subcellular localization of these lipogenic proteins corroborated a functional interaction between these proteins. Confocal images of MIG12 and acetyl-CoA carboxylase in primary hepatocytes show filamentous structures that are immunofluorescent along junctions between the endoplasmic reticulum and mitochondria. Under high carbohydrate dietary conditions in which lipogenesis is stimulated, these structures expand to include fatty acid synthase, ATP citrate lyase, and Spot 14. They also co-localize around lipid droplets - storage organelles for excess triglycerides. Finally, the structural integrity of this lipogenic complex is shown to require microtubules. Blockade of microtubule formation inhibits proper formation of acetyl-CoA carboxylase structure and decreases total fatty acid synthesis in cells. Combined, these findings support the existence of a functional metabolon complex which facilitates the efficient channeling of fatty acid synthesis intermediates through an enzyme cascade that results in the production of palmitate at functionally relevant locations within the cell.Item Protein Structure and Ion Binding in Potassium Selective Channels(2013-01-17) Sauer, David Bryant; Jiang, YouxingPotassium channels play a central role in a number of biological processes, most classically the action potential of excitable cells in multicellular organisms. These channels are defined by their selective conduction of potassium to the exclusion of other monovalent ions as governed by a common sequence and structural motif, the selectivity filter. This structure, made of backbone carbonyls and threonine side chains, directly coordinates the ions as they diffuse through the channel and appears central to this sub-angstrom discrimination between cations. Utilizing the non-selective Sodium and Potassium conducting channel (NaK) as a structural scaffold the mechanisms of both ion selectivity and formation of this selective structure are examined.Item Structural and Mechanistic Studies of Two Regulatory Factors in Actin Cytoskeletal Signaling: Vav and VopL(2011-12-15) Yu, Bingke; Rosen, Michael K.Proper control of actin cytoskeletal dynamics is essential for cell survival. The goals of my thesis work have been to characterize the structural and biophysical properties of two regulatory proteins in actin cytoskeletal rearrangement pathway: Vav and Vibrio outer protein L (VopL). Vav proteins are guanine nucleotide exchange factors for Rho family GTPases. They play key roles in actin regulatory pathways and control diverse cellular processes like T cell maturation and activation, cell migration and phagocytosis. They belong to a group of multi-domain signaling proteins which display complex behaviors because of the collective regulation from multiple domains. Previous work has shown that Vav is autoinhibited in the resting state through the cooperative suppression of N-terminal Calponin domain and Acidic region, with the physical mechanism yet to be determined. Here through structural, energetic and biochemical studies, I demonstrate that the Calponin homology domain of Vav binds to the Pleckstrin homology domain, restrains the inhibitory helix in the Acidic region, and shifts the Dbl homology domain - inhibitory helix equilibrium to a more closed state. This construction enables strong suppression and an efficient activation process. The energetic basis of Vav autoinhibition may turn out to be widespread in multi-domain systems. VopL, a pathogenic effector from Vibrio parahaemalyticus, is an actin nucleation factor that induces stress fibers during bacterial infection. It contains three N-terminal Wiskott-Aldrich Homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). It potently promotes actin filament nucleation in vitro. However, the physical basis of VopL mediated nucleation has not been understood. Here I performed structural and biochemical studies to investigate the mechanism of actin filament nucleation by VopL. I found that both the WH2 element and VCD are required for VopL activity. The crystal structure of VCD revealed a U-shaped dimer that is stabilized by a terminal coiled-coil. Dimerization of the WH2 motifs as well as contacts between VCD and actin contribute to the nucleation activity of VopL. My studies suggest the formation of a structurally organized actin cluster involving lateral contacts during nucleation. Stabilization of these lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.Item Towards Prediction of Phenotype from Genotype(2017-04-14) Cong, Qian; Otwinowski, Zbyszek; Grishin, Nick V.; Hobbs, Helen H.; Deisenhofer, JohannPredicting phenotype from genotype represents the epitome of biological questions. As a multiscale problem, it starts from predicting exons and culminates with modeling of whole organisms. Focusing on the molecular level, I studied the relationship between sequences and protein spatial structures and analyzed proteins with similar sequences but different structures. To aid the assessment of structure prediction, I developed a method to rank the predictions of proteins with new folds, a very challenging problem that was previously addressed by expert inspection. Then, I developed a set of computer programs and scripts to predict various structural and functional properties of proteins from their sequences and implemented them as a public web-server. I applied these methods to important agricultural (citrus disease) and medical (Ebolavirus) problems. Moving on to organismal level predictions, I sequenced, annotated and analyzed complete genomes of butterflies and suggested hypotheses about genetic determinants of their behavior and other phenotypic traits. Taken together, these applications highlight the achievements possible today and challenges that lie ahead.