Escherichia coli NikR is a metal-responsive transcription factor that controls nickel uptake by regulating expression of a nickel-specific transporter, NikABCDE. NikR contains a high-affinity nickel-binding site, and nickel must occupy this site in order to activate DNA binding. Stoichiometric amounts of nickel bind to this site and stabilize the α3 helix and the preceding loop (residues 63 – 79). The α3 helix forms non-specific electrostatic interactions with the DNA. The current hypothesis is that nickel activates NikR-DNA binding by organizing α3 helix and activating non-specific DNA contacts, allowing NikR to scan along the DNA until it reaches the nik promoter. This presentation will examine this hypothesis by generating a nickel independent NikR through mutagenesis.

Nedd4-1 and Nedd4-2 are closely related E3 ubiquitin protein ligases that contain a C2 domain, 3-4 WW domains, and a catalytic ubiquitin ligase HECT domain. The WW domains of Nedd4 proteins recognize substrates for ubiquitination by binding the sequence L/PPxY (the PY-motif) found in target proteins. Nedd4-2 functions as a suppressor of the epithelial Na+ channel (ENaC), which interacts with Nedd4-2 WW domains via PY-motifs located at its C-terminus. The importance of Nedd4-2 mediated ENaC regulation is highlighted by the fact that mutations affecting the ENaC PY-motifs cause Liddle syndrome, a hereditary hypertension. Since all Nedd4 family members recognize the same core sequence in their target proteins, the question was raised of how substrate specificity for Nedd4 family members is achieved. Using intrinsic tryptophan florescence to measure the binding affinity of Nedd4-1/-2 WW domains for their substrate PY-motifs, we demonstrate the importance of both PY-motif and WW domain residues, outside the core binding residues, in determining the specificity of WW domain-ligand interactions. Little was known about regulation of catalytic activity for this family of E3 ligases, and hence was the second focus of my work. Notably, Nedd4-2 contains a PY-motif within its HECT domain, raising the possibility that its catalytic activity is regulated by an interaction between its WW domains and HECT domain. Here I present evidence supporting a model in which a low-affinity interaction between the Nedd4-2 WW domains and its HECT domain regulate Nedd4-2 stability by preventing self-ubiquitination and subsequent degradation. Furthermore, evidence is presented suggesting that interaction between Nedd4-2 and the RING-E3 ligase Rnf11, a Nedd4-2 substrate, may also serve to regulate Nedd4-2 stability, as this interaction leads to decreased Nedd4-2 self-ubiquitination. Collectively, the studies presented here further our understanding of the substrate specificity and regulation of Nedd4-1 and Nedd4-2.

Nedd4-1 and Nedd4-2 are closely related E3 ubiquitin protein ligases that contain a C2 domain, 3-4 WW domains, and a catalytic ubiquitin ligase HECT domain. The WW domains of Nedd4 proteins recognize substrates for ubiquitination by binding the sequence L/PPxY (the PY-motif) found in target proteins. Nedd4-2 functions as a suppressor of the epithelial Na+ channel (ENaC), which interacts with Nedd4-2 WW domains via PY-motifs located at its C-terminus. The importance of Nedd4-2 mediated ENaC regulation is highlighted by the fact that mutations affecting the ENaC PY-motifs cause Liddle syndrome, a hereditary hypertension. Since all Nedd4 family members recognize the same core sequence in their target proteins, the question was raised of how substrate specificity for Nedd4 family members is achieved. Using intrinsic tryptophan florescence to measure the binding affinity of Nedd4-1/-2 WW domains for their substrate PY-motifs, we demonstrate the importance of both PY-motif and WW domain residues, outside the core binding residues, in determining the specificity of WW domain-ligand interactions. Little was known about regulation of catalytic activity for this family of E3 ligases, and hence was the second focus of my work. Notably, Nedd4-2 contains a PY-motif within its HECT domain, raising the possibility that its catalytic activity is regulated by an interaction between its WW domains and HECT domain. Here I present evidence supporting a model in which a low-affinity interaction between the Nedd4-2 WW domains and its HECT domain regulate Nedd4-2 stability by preventing self-ubiquitination and subsequent degradation. Furthermore, evidence is presented suggesting that interaction between Nedd4-2 and the RING-E3 ligase Rnf11, a Nedd4-2 substrate, may also serve to regulate Nedd4-2 stability, as this interaction leads to decreased Nedd4-2 self-ubiquitination. Collectively, the studies presented here further our understanding of the substrate specificity and regulation of Nedd4-1 and Nedd4-2.

Macromolecular complexes are important in almost all cellular processes. The bacteriophage head-tail connector complex offers a model with which to study the mechanisms that control their formation and the interactions that govern their assembly. The head-tail connector joins DNA-filled heads with mature tails. This thesis describes the structures, functions, and evolution of two connector proteins, HK97 gp6 and Lambda gpFII. Middle-ring connector proteins act as head-stabilizing proteins after the packaging of DNA in the heads. I found that gp6 is the middle-ring connector protein of bacteriophage HK97. I determined that gp6 is part of a large family of middle-ring connector proteins that share common sequence and structure elements. I also show that the mechanism for the addition of gp6 to the connector is mediated by limiting gp6 expression by strictly controlling translation initiation. I also describe a three-dimensional model for the assembly of the head-tail joining protein of bacteriophage Lambda, gpFII. This model correctly predicts regions of the protein predicted to interact with the Lambda head and tail. It also provides evidence for the description of a new family of proteins that evolved from a tail tube protein. This family includes members from both contractile and non-contractile phage tails and the bacterial type VI secretion system.

ClpX is an ATPase that belongs to a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX functions alone or in conjunction with a cylindrical serine protease ClpP. ClpX consists of an N-terminal domain and a C-terminal AAA+ ATP-binding domain. The chaperone oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the N-termini extending out of the base. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) which forms a very stable dimer. ZBD is essential for promoting the degradation of some established ClpXP substrates such as lambdaO and MuA. Experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Furthermore, analysis of the binding preferences of the ZBD and AAA+ domains revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of SspB on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C-terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB towards ZBD and the geometry of the SspB-ZBD complex were also investigated. Analysis of ClpX conformational changes during its functional cycle indicated that the ZBDs in ClpX undergo a large nucleotide-dependent block movement into the AAA+ ring. Hence, we propose that ClpX switches between a capture and a feeding conformation. Based on all of these results, the ZBD in ClpX clearly plays a major role in substrate binding and cofactor recognition, as well as in substrate translocation into the ClpP chamber.

ClpX is an ATPase that belongs to a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX functions alone or in conjunction with a cylindrical serine protease ClpP. ClpX consists of an N-terminal domain and a C-terminal AAA+ ATP-binding domain. The chaperone oligomerizes into a hexamer with the AAA+ domains forming the base of the hexamer and the N-termini extending out of the base. Here we demonstrate that the N-terminal domain of ClpX is a C4-type zinc binding domain (ZBD) which forms a very stable dimer. ZBD is essential for promoting the degradation of some established ClpXP substrates such as lambdaO and MuA. Experiments indicate that ZBD contains a primary binding site for the lambdaO substrate and for the cofactor SspB. Furthermore, analysis of the binding preferences of the ZBD and AAA+ domains revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of SspB on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C-terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB towards ZBD and the geometry of the SspB-ZBD complex were also investigated. Analysis of ClpX conformational changes during its functional cycle indicated that the ZBDs in ClpX undergo a large nucleotide-dependent block movement into the AAA+ ring. Hence, we propose that ClpX switches between a capture and a feeding conformation. Based on all of these results, the ZBD in ClpX clearly plays a major role in substrate binding and cofactor recognition, as well as in substrate translocation into the ClpP chamber.

Macromolecular complexes are important in almost all cellular processes. The bacteriophage head-tail connector complex offers a model with which to study the mechanisms that control their formation and the interactions that govern their assembly. The head-tail connector joins DNA-filled heads with mature tails. This thesis describes the structures, functions, and evolution of two connector proteins, HK97 gp6 and Lambda gpFII. Middle-ring connector proteins act as head-stabilizing proteins after the packaging of DNA in the heads. I found that gp6 is the middle-ring connector protein of bacteriophage HK97. I determined that gp6 is part of a large family of middle-ring connector proteins that share common sequence and structure elements. I also show that the mechanism for the addition of gp6 to the connector is mediated by limiting gp6 expression by strictly controlling translation initiation. I also describe a three-dimensional model for the assembly of the head-tail joining protein of bacteriophage Lambda, gpFII. This model correctly predicts regions of the protein predicted to interact with the Lambda head and tail. It also provides evidence for the description of a new family of proteins that evolved from a tail tube protein. This family includes members from both contractile and non-contractile phage tails and the bacterial type VI secretion system.

Master of Science / Department of Biochemistry / Michal Zolkiewski / Early-onset dystonia (EOTD, also known as DYT1 or Oppenheim’s dystonia is the most severe and common form of hereditary dystonia, a neurological disorder characterized by abnormalities in the control of movement. It is linked to the deletion of a single GAG codon in the gene DYT1 that leads to the loss of a single glutamic acid residue in the C-terminal region of the protein torsinA (ΔE-torsinA). It is not known how the GAG deletion alters the torsinA structure and function. In this thesis, the expression and purification of recombinant torsinA variants from E. coli is reported. Wild type torsinA is not soluble after its expression in E. coli, possibly due to misfolding caused by cysteine. We produced Cys-less torsinA, and established a purification procedure to produce this mutant torsinA. Furthermore, because of the critical role likely to be played by the C-terminal domain of torsinA that contains the glutamate deletion, we produced fragments encoding the C-terminal domain of torsinA, and attempted to purify it. However, we failed to obtain appreciable amount of active proteins by both of the strategies. A novel SUMO fusion technology was also used in this study. We demonstrated that SUMO, when fused with torsinA variants, was able to enhance its expression and solubility in E. coli. A satisfactory yield of the fusion protein was successfully purified. Once we get appreciable quantities of folded torsinA variants, it is our future goal to study their function by using biochemical and high-resolution structural approaches.

Proper progression through the cell cycle is critical for cell growth and survival. Disruption of cell cycle progression can lead to cell cycle arrest and cell death. In addition, uncontrolled cell cycle progression can lead to cancer. Cells have evolved complex mechanisms to regulate each phase of the cell cycle to ensure proper cell cycle progression. In the presence of cellular stress, cells will respond promptly to arrest the cell cycle and allow repair. In order to study this complex process, it is important to identify the complete complement of proteins involved. I took two large-scale approaches to study the cell cycle. First, I down-regulated the majority of essential genes in Saccharomyces cerevisiae, and determined how depletion of individual gene product affected progression through the cell cycle. I determined that over 65% of essential genes I tested are most important at a specific cell cycle phase. In addition, I found that two genes, Smc4 and Cse1, have novel roles in S-phase of the cell cycle. In the second approach, I discovered two anti-proliferative compounds. Both compounds caused cell cycle delay in G1 phase of the cell cycle. Chemical genetic screens in yeast allowed me to determine the pathways most sensitive to each of these two compounds. By studying the response of cells to these compounds, I confirmed that compound 13 causes mitochondrial dysfunction in cells and compound 15 causes nuclear DNA damage. Furthermore, I found that both compounds are toxic in mammalian cells and that the responses that they elicit in mammalian cells are similar to those observed in yeast cells.

Vts1 is a member of the Smaug protein family, a group of sequence-specific RNA-binding proteins that regulate mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Using RNA reporters that recapitulate Vts1-mediated decay in vivo as well as endogenous mRNA transcripts, I show that Vts1 regulates the degradation of target mRNAs in Saccharomyces cerevisiae. In Chapter Two, I focus on the mechanism of Vts1-mediated mRNA decay. I demonstrate that Vts1 initiates mRNA degradation through deadenylation mediated by the Ccr4-Pop2-Not deadenylase complex. I also show that Vts1 interacts with the Ccr4-Pop2-Not deadenylase complex suggesting that Vts1 recruits the deadenylase machinery to target mRNAs, resulting in transcript decay. Following poly(A) tail removal, Vts1 target transcripts are decapped and subsequently degraded by the 5’-to-3’ exonuclease Xrn1. Taken together these data suggest a mechanism of mRNA degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs. Previous work in Drosophila melanogaster demonstrated that Smg interacts with the Ccr4-Pop2-Not complex to regulate mRNA stability, suggesting Smaug family members employ a conserved mechanism of mRNA decay. In Drosophila, Smg also regulates mRNA translation through a separate mechanism involving the eIF4E-binding protein Cup. In Chapter Three, I identify the eIF4E-associated protein Eap1 as a component of Vts1-mediated mRNA decay in yeast. Interestingly Cup and Eap1 share no significant homology outside of the seven amino acid eIF4E-binding motif. In eap1 cells mRNAs accumulate as deadenylated capped species, suggesting that Eap1 stimulates mRNA decapping. I demonstrate that the Eap1 eIF4E-binding motif is required for efficient degradation of Vts1 target mRNAs and that this motif enables Eap1 to mediate an interaction between Vts1 and eIF4E. Together these data suggest Vts1 influences multiple steps in the mRNA decay pathway through interactions with the Ccr4-Pop2-Not deadenylase and the decapping activator Eap1.