Browsing by Department "Biochemistry"

The bacterial protein FtsZ plays a vital role in cytokinesis in prokaryotes as it polymerizes to form an FtsZ ring (Z ring) at the division septum midcell. FtsZ exhibits a GTP hydrolysis activity and attempts have been made to model the kinetics of this process. There is a major discrepancy, however, over the concentration of GTP needed for activity. The dissociation constant (KD) between wild-type FtsZ protein and GTP was measured to be 30 nM using isothermal titration calorimetry. In contrast, several research groups have reported that GTP hydrolysis required GTP concentrations in the millimolar range. They used Michaelis-Menten kinetics to model the GTP concentration dependence and obtained an apparent binding constant (Km) in the range of 300-1,000 µM GTP. Km and KD are not identical measures of binding given that they differ by the kinetic constant governing catalysis, kcat, but we suggest that a five order of magnitude difference between the values is unprecedented and this was a problem that needed investigating.

My overall goal in this work has been to perform a comprehensive in vitro study of the FtsZ GTP hydrolysis activity using an enzyme-coupled regenerating system. With this system rates of GTP hydrolysis by FtsZ are obtained spectrophotometrically. I have confirmed for wild-type FtsZ that GTP hydrolysis rates show little to no dependence on GTP concentrations in the range of 50-3,000 µM, contradicting the high values of Km reported in some previous studies. Since we have failed to reproduce the high Km with three different preparations of FtsZ protein, we cannot propose a definitive mechanism for the previous results.

I also measured the GTP hydrolysis of several mutants of FtsZ: E238A, L169R, and FtsZ84 (G105S). I investigated each of these mutants to see if they had a high apparent Km. FtsZ84 had a low overall hydrolysis rate, but did show a large increase in hydrolysis rates when GTP was increased from 50-3,000 µM. We hypothesize that a lower affinity for GTP is not a Michaelis-Menten Km, but likely a reflection of a weak binding of GTP by FtsZ84, giving KD in the millimolar range.

A DNA-protein crosslink is a covalent bond between DNA and a protein. It is a type of DNA damage that is relatively understudied. This study reports on the identification of a set of transposon mutants sensitive to 5-azacytidine, a DNA- protein crosslink induction agent that induces a crosslink between DNA and a DNA methyltransferase protein. The screen showed that certain recombination, DNA repair, and tRNA modification mutants are hypersensitive to 5-azacytidine. These included the recombination recA, recC, and recG mutants. Since the recombination mutants consistently show high sensitivity to aza-C, it suggests a role for recombination in DNA-protein crosslink repair. Western blots for the levels of methyltransferase protein showed that mutants have similar levels of methyltransferase protein compared to wild type cells, arguing that the mutants' hypersensitivity to aza-C is not because of increased methyltransferase levels. Western blots for the levels of SsrA tagging in the presence of 5-azacytidine showed that the tRNA modification transposon mutants miaA, mnmE, and mnmG are all defective in SsrA tagging, which likely explains their hypersensitivity. The SsrA tag Western blots also unexpectedly showed that the recA transposon mutant had reduced levels of SsrA tagging when treated with 5-azacytidine.

Since RNA Polymerase II (RNAPII) transcribes much of the genome, it is well situated to encounter and initiate a response to various types of DNA damage. However, to date very little is known about any response of RNAPII to DNA damage outside of Transcription Coupled Nucleotide Excision Repair (TC-NER). A link between DNA damage response mechanisms and the C-terminal domain of RNAPII (CTD) is suggested by an overlap between proteins that bind the CTD and genes required for resistance to DNA damaging agents. In this thesis, I show that proper deployment of CTD associated proteins is required to respond to DNA damaging agents. Furthermore, I show that a CTD associated protein (Set2) is required for response to DNA damage, but its catalytic activity is not. Finally, I show that the recombinational ability of strains lacking the CTD kinase, Ctk1, is deficient. Based on these lines of evidence, I propose a novel CTD Associated DNA Damage Response (CAR) system of proteins that is required for proper response to DNA damaging agents.

Addition and removal of phosphate is an important post-translational modification involved in cellular signaling. The enzymes responsible for removing this phosphorylation mark, called phosphatases, play a vital role in the cellular decision making processes. In this work we discuss two discoveries, a novel enzyme for a known signaling function involving control of transcription and a novel target for an important cellular stress response enzyme.

In the first project we sought to determine a novel enzyme responsible for dephosphorylating the C-terminal domain of RNA polymerase II. This domain serves as a vital signaling platform for transcription of mammalian genes, with the ability to recruit cofactors that bind to specific patterns of phosphorylation throughout its repeating amino acid sequence. Using a functional assay for phosphatase activity at the Thr4 position we biochemically isolated the unknown enzyme and identified it as PP1 and validated its function in vitro and in vivo.

The second phosphatase studied in this dissertation is MESH1—a mammalian ortholog of the bacterial stringent response protein SpoT that dephosphorylates ppGpp. Because ppGpp is absent in mammalian cells MESH1 lacks a viable target. We established NADPH as a substrate of MESH1 biochemically and corroborated these results by determining the substrate bound structure. Our results reveal a novel regulatory role of MESH1 in a pathway that resembles the bacterial stringent response.

One of the most significant discoveries from mutational analysis of protein interfaces is that often a large percentage of interface residues negligibly perturb the binding energy upon mutation, while residues in a few critical "hotspots" drastically reduce affinity when mutated. The organization of protein interfaces into hotspots has a number of important implications. For example, small interfaces can have high affinity, and when multiple binding partners are generated to the same protein, they are predisposed to binding the same regions and often have the same hotspots. Even small molecules that bind to interfaces and disrupt protein-protein interactions (PPIs) tend to bind at hotspots. This suggests that some hotspot-forming sites on protein surfaces are intrinsically more apt to form protein interfaces. These observations paint a hotspot-centric picture of PPI energetics, and present a question of fundamental importance which remains largely unanswered: why are hotspots hot?

In order to gain insight into the nature of hotspots I experimentally examined the small, but high-affinity interface between the synthetically evolved ankyrin repeat protein Off7 with E. coli maltose binding protein by characterization of mutant variants and redesigned interfaces. In order to characterize many mutants, I developed two high-throughput assays to measure protein-protein binding that integrate with existing technology for the high-throughput fabrication of genes. The first is an ELISA-based method using in vitro expressed protein for semi-quantitative analysis of affinity. Starting from DNA encoding protein partners, binding data is obtained in just a few hours; no exogenous purification is required. For the second assay, I develop data fitting methods and thermodynamic framework for determination of binding free energies from binding-induced shifts in protein thermal stability monitored with Sypro Orange.

Analysis of Off7/MBP variants using these methods reveals that conservative mutagenesis or local computational repacking is tolerated for many residues in the interface without drastic loss of affinity, except for a single essential hotspot. This hotspot contains a Tyr-His-Asp hydrogen bonding network reminiscent of a common catalytic motif. Substitution of the tyrosine with phenylalanine shows that a single hydrogen bond across the interface is critical for binding. Analysis of the protein database by structural bioinformatics shows that, although rare, this motif is present in other naturally evolved interfaces. Such a triad was found in the homodimeric interface of PH0642 from Pyrococcus horikoshii, and is conserved between many homologues in the nitrilase superfamily, meeting one of the key criteria by which potential hotspots can be identified. This analysis supports a number of analogies between hotspot residues and catalytic residues in enzyme active sites, and raises the intriguing possibility that hotspots may be associated with other structural motifs that could be used for identification or design of PPIs.

Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.

In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.

We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.

To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.

The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.

β-Adrenergic receptors (βAR) are one of the key modulators of cardio-pulmonary functions and belong to a large family of membrane proteins, termed as G-protein coupled receptors (GPCRs). β-blockers (βAR antagonists) and βAR agonists are the mainstay treatments for heart failure and asthma respectively, which reflects the significance of βARs as therapeutic targets. The binding of catecholamines (e.g. adrenaline) to βARs activates intracellular transducer proteins such as hetero trimeric GTP binding proteins (G-proteins) or β-arrestins (βarr), which results in the regulation of cardiac output and bronchodilation.

The bifurcated signaling pathways initiated by G-protein and β-arrestin downstream of βAR, as well as other members in the GPCR family can be selectively activated, a phenomenon termed as ‘biased agonism’. Biased ligands, which can pharmacologically separate these pathways, are of major therapeutic interest due to their potential for improving the specificity of drug actions. For βAR, biased agonism towards β-arrestin is expected to render cardo-protective benefits, while selective activation of G proteins is hypothesized to subdue major side effects from current asthma therapy. Therefore, elucidation of how βARs can preferentially interact with their transducers is at the core of developing the next generation therapeutics, beyond conventional β-blockers and agonists.

Thus far, the exact mechanism behind GPCR biased agonism remains obscure. The leading hypothesis in the field is that GPCRs adopt distinct conformations that preferentially couple to G proteins or β-arrestins. In order to test this hypothesis, we developed and established a G protein biased mutant β2AR (Chapter 2), since efficacious biased ligands for this receptor are yet to be found. Subsequent assessment of GPCR kinase (GRK)-mediated phosphorylation states of this mutant receptor and phosphorylation rescue experiments revealed unexpected findings that contradict the initial hypothesis (Chapter 3). Next, we initiated a biophysical characterization of this mutant β2AR (Chapter 4) to comprehend the conformational and structural basis for its apparent biased phenotype. The cumulative insight gained from experiments described in chapters 2-4 highlight the underappreciated role of GRKs in determining GPCR biased agonism – the mutant β2AR is biased towards G protein due to conformational selection against GRKs, rather than β-arrestins. Furthermore, to obtain a comprehensive understanding of biased agonism, we devised a strategy to map the interface between β2AR-β-arrestin, which can also be used to form stable complexes for further biophysical characterizations (Chapter 5). In summary, this dissertation improves the current understanding of the molecular mechanism behind biased agonism at the prototypical GPCR, β2AR.

The outer-leaflet of the outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS), which is attached to the membrane via a hexa-acylated saccharolipid called lipid A. The fourth step of lipid A biosynthesis involves the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN to form lipid X; this step is carried out by LpxH in E. coli and the majority of Gamma- and Beta-Proteobacteria. LpxH has been previously characterized, however sample impurity and non-optimized assay conditions hindered meaningful conclusions. The enzyme was suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes, however the extent of biochemical data fails to demonstrate a significant level of metal activation in LpxH assays. We report cloning, purification, and detailed enzymatic characterization with a highly purified sample of H. influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of activity in the presence of Mn2+. Furthermore, EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Overall, through optimized purification and assay methods, our work unambiguously establishes LpxH as a membrane-associating CLP containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.

Several species of Gram-negative bacteria lack LpxH orthologs, yet retain other lipid A biosynthetic enzymes and still produce lipid A. An unrelated protein, LpxI, is responsible for UDP-DAGn hydrolysis is several such organisms. Interestingly, some bacteria, such as the human pathogen Chlamydia trachomatis, have neither LpxH nor LpxI orthologs, suggesting the presence of a third UDP-DAGn hydrolase. Through implantation of a novel complementation screen that used a C. trachmatis genomic library and a conditional-lethal lpxH mutant E. coli strain, we were able to identify an open reading frame encoding an new enzyme capable of lipid X production. Due to its ability to complement UDP-DAGn hydrolase function in vivo and catalyze the formation of lipid X in vitro, we have designated the enzyme LpxG. Further biochemical analysis with purified LpxG revealed it facilitates hydrolysis through attack on the alpha phosphate of its substrate and is activated by Mn2+ in vitro. LpxG is in the same CLP superfamily as LpxH, however it shows very little homology to LpxH or LpxI. Identification of LpxG improves our understanding of the lipid A biosynthetic pathway in C. trachomatis. More broadly, as limited genetic tools are available for the study of the prevalent pathogen, it provides an advantageous method for the functional screening of other C. trachomatis genes.

As bacterial resistance to multiple antibiotics continues to become a growing problem across the globe, the imperativeness for understanding mechanisms of antibiotic and multidrug resistance is increasingly apparent. Currently, the gram-negative bacteria Neisseria gonorrhoeae and Escherichia coli are considered urgent public health threats due to the rise in multidrug resistant strains. Mechanisms by which these bacteria become resistant to antibiotics include the overexpression of multidrug efflux systems. Prior to the introduction of antibiotics, the primary purpose of these multidrug efflux systems was to protect the bacteria from cytotoxins in the environment including innate host defense molecules or toxic molecules produced by the bacteria. Overtime, the bacteria have adapted these efflux systems to protect against clinically relevant antibiotics used to clear these bacterial infections. These multidrug efflux systems are energetically expensive to synthesize; thus, they are often tightly regulated at the transcription level by transcription repressors or activators. Many multidrug efflux systems are regulated by transcriptional regulators with proximal genes that specifically regulate a single multidrug efflux system. However, the expression of a few multidrug efflux systems is controlled by unique repressors that act as global regulators, which have a larger role in regulating complex virulence and stress response systems. Specifically, examples of multidrug efflux regulators in N. gonorrhoeae and E. coli that act as global regulators within their respective genomes include N. gonorrhoeae MtrR and E. coli MprA. Understanding the global regulatory activities of these two transcription regulators will broaden our understanding of the regulatory mechanisms that enable bacterial survival during host infection, the mechanisms that contribute to antibiotic resistance, as well as the fundamentals of bacterial transcription regulation. To provide novel insight into the global regulatory activities and function of N. gonorrhoeae MtrR, this dissertation expounds a series of original structural, biochemical, and in vivo studies identifying cytotoxin and DNA recognition mechanisms of MtrR. Previous work showed that MtrR represses directly the mtrCDE efflux transporter genes by binding an operator between the mtrR and mtrC genes; additionally, MtrR represses directly the rpoH oxidative stress response sigma factor. MtrR-mediated repression of the mtrCDE genes had been shown to be relieved upon exposure of gonococci to toxic hydrophobic agents and detergents (i. e. MtrR is “induced” by these toxic molecules). However, physiologically relevant innate host molecules recognized by MtrR had not been identified. In this work, we identify bile salts present at extra-urogenital gonococcal infection sites that MtrR directly binds, to result in derepression of the mtrCDE genes in vitro and in vivo. Furthermore, we use x-ray crystallography to solve structures of MtrR in its induced form and bound to the mtrCDE and rpoH operators. With these structures, we determined the structural mechanism of induction of MtrR. In addition, the MtrR-operator structures reveal a degenerate consensus sequence to which MtrR binds within the mtrCDE and rpoH operators. Mechanisms for cytotoxin and DNA recognition were confirmed by structure-guided site-directed mutagenesis studies and a combination of biochemical binding assays utilizing isothermal titration calorimetry (ITC) or fluorescence polarization (FP). Importantly, this structural and biochemical work also reveals the mechanisms by which common mutations in multidrug resistant strains of N. gonorrhoeae confer resistance. To elucidate the function of E. coli MprA and realize its potential as a drug target, this dissertation also includes research describing the ligand-binding mechanisms of MprA. MprA (formerly EmrR) represses directly the EmrAB efflux pump in E. coli. Previously published work identified MprA as the molecular target of a small molecule inhibitor (DU011) of the biosynthesis of an important virulence factor in E. coli, the polysaccharide capsule. This lead molecule has the potential for optimization for drug development and reveals a novel function of MprA as a regulator of polysaccharide capsule synthesis. We characterized the interaction between MprA and DU011 and compared this to the binding between MprA and other previously identified ligands including salicylate and 2,4-dinitrophenol (DNP) utilizing ITC assays. Through these studies, we revealed a novel binding mode for MprA and laid the groundwork for future structural studies and drug optimization. Collectively, this work provides important insight into the breadth of regulatory functions of N. gonorrhoeae MtrR and E. coli MprA, two key global regulators from highly prevalent multidrug resistant pathogens. Specifically, the original research presented here provides a biochemical evaluation of the bacterial stress response mechanisms controlled by MtrR and MprA and their contribution to antibiotic resistance. Indeed, the biochemical and structural characterization of these two regulators will inform future work to combat multidrug resistance.

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.

Human Exonuclease 1 (Exo1) plays important roles in numerous DNA metabolic/repair pathways including DNA mismatch repair, DNA double strand break repair, Okazaki fragment maturation etc. The nuclease activity of Exo1 is tightly regulated in vivo. The regulation of Exo1 in different pathways is achieved by interactions with different protein partners. The focus of this dissertation will be on characterization of Exo1 interactions with traditional protein partners and providing experimental evidences for new Exo1 interactions.

Molecular cloning, biochemical assays, collaborative nuclear magnetic resonance and X-ray crystallography have been employed to study Exo1 interactions with protein partners. This work contains: (i) the experimental evidence for new Exo1 interactions, and (ii) the detailed characterization of Exo1 interactions with PCNA, MLH1 and MutSα/β.

Taken together, the research progress presented in this dissertation further advances our understanding of traditional Exo1 interaction network and probably provides new insights to new functions and new regulations of Exo1.

The lipid A component of lipopolysaccharide (LPS) in the nitrogen-fixing plant endosymbionts Rhizobium leguminosarum and Rhizobium etli is strikingly different when compared to that of enteric bacteria such as Escherichia coli. The Rhizobium species produce several unique enzymes that process the lipid A biosynthetic intermediate Kdo₂-lipid IV_A. These enzymes include a 1-phosphatase (LpxE), a 4´-phosphatase (LpxF), a 3-O-deacylase (PagL), and a lipid A oxidase (LpxQ). The biological functions and enzymological properties of many of the modification enzymes have remained unconfirmed and/or unknown. The purpose of these studies was to confirm the activities of these enzymes and to explore the functional significance of the resulting lipid A modifications.

To confirm the proposed biological functions of the enzymes in vivo, homologs of the lipid A phosphatases, LpxE and LpxF, from Francisella novicida and the lipid A oxidase LpxQ, were expressed heterologously in combination in E. coli. The resulting novel lipid A hybrids were analyzed by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (ESI-MS).

The lipid A oxidase LpxQ, was characterized further biochemically. Two new purification procedures and a new in vitro assay were developed to analyze the properties of the enzyme. Purified LpxQ was shown to be dependent on oxygen and divalent cations for activity. Hydrogen peroxide was found to be a product of lipid A oxidation. A new fluorescence-based assay based on the detection of hydrogen peroxide was developed to monitor oxidation. LpxQ did not co-purifiy with any discernable cofactors, suggesting that it may employ a unique mechanism for the oxidation of lipid A.

The biological roles of LpxE and LpxF in plant nodulation were analyzed. Deletion mutants of the two phosphatases were generated in R. etli. The mutant strains accumulated the expected structures, confirming the specificity of the enzymes. Single and double phosphatase mutants were able to fix nitrogen in planta. Antimicrobial susceptibility testing indicated that dephosphorylation of lipid A increases resistance to cationic antimicrobials.

The biological role of the 3-O-deacylase, PagL, was also investigated. The pagL gene was identified using systematic homology searches. PagL was shown to be stimulated by calcium. A deletion mutant of the enzyme in R. etli was constructed and analyzed. The deletion mutant was found to be viable and unaltered in its ability to fix nitrogen. In conclusion, these studies have confirmed the roles of LpxE, LpxF, PagL, and LpxQ in Rhizobium lipid A biosynthesis and contributed new knowledge regarding the biochemical properties of LpxQ.

In this work, I develop computational and visual tools for analyzing and manipulating the backbone of macromolecules, and I demonstrate that these tools support building better structures than currently done. These visualization and analysis tools belong to an "Intelligence Amplification" (IA) tradition (rather than complete Artificial Intelligence (AI) automation), empowering users to improve structures.

Proteins and nucleic acids are among the most important molecules in biology, mediating the majority of biochemical processes that comprise a living organism. Therefore, these macromolecules are important targets, both for basic research to improve understanding of how life works, and for medical research as possible drug targets.

The function of these macromolecules is largely determined by their 3D structure. Although these macromolecules are chemically fairly simple, made up of linear sequences of a few possible subunits, they physically fold into complex, compact structures. Overall, structural biology aims to determine the general relationship between sequence and structure of these macromolecules, through determination of the positions of the atoms within individual macromolecules.

Because it is currently impossible to directly see the position of atoms in a molecule, all structural determination techniques, including X-ray crystallography, NMR, and homology modeling, result in an interpreted model of a structure. Nearly all of these models contain mistakes, in which atoms are fit in incorrect or impossible positions. These mistakes, especially at a functionally-important location in a structure, can mislead both basic and medical research, making it critical for structural biologists to build the highest quality models possible.

This document details how my dissertation work enables the building of better macromolecular structure models. This work follows an iterative development cycle, where visual analysis of models spurs development of better tools, which in turn improves the analysis. First, I describe how my analysis of protein loops from X-ray crystal structures reveals that the traditional definition of loop endpoints is too restrictive. Second, I create a protein backbone analysis and modeling tool, using a new peptide-centric division system. I show how this tool makes it easier to study protein loops, and also how it improves an algorithm for calculating core protein models from NMR residual dipolar coupling (RDC) data. Third, I describe how 3D visualization of RDCs in their structural context improves understanding of RDCs and validates NMR models in a novel way. Fourth, I describe how local quality analysis can diagnose problems in homology models. Fifth, I demonstrate that local quality analysis can be successfully used in conjunction with model rebuilding software to correct errors in low resolution structures. The various tools and software packages I created during the course of my work are freely available and have already made a positive impact on structures being generated by the community.

Archive versions of several of these software packages (JiffiLoop, RDCvis, and KiNG) should be included with this document; current versions can be downloaded from http://kinemage.biochem.duke.edu.

Repetitive Ca2+ transients in dendritic spines induce various forms of synaptic plasticity by transmitting information encoded in their frequency and amplitude. CaMKII plays a critical role in decoding these Ca2+ signals to initiate long-lasting synaptic plasticity. However, the properties of CaMKII that mediate Ca2+ decoding in spines remain elusive. Here, I measured CaMKII activity in spines using fast-framing two-photon fluorescence lifetime imaging. Following each repetitive Ca2+ elevations, CaMKII activity increased in a stepwise manner. This signal integration, at the time scale of seconds, critically depended on Thr286 phosphorylation. In the absence of Thr286 phosphorylation, only by increasing the frequency of repetitive Ca2+ elevations could high peak CaMKII activity or plasticity be induced. In addition, I measured the association between CaMKII and Ca2+/CaM during spine plasticity induction. Unlike CaMKII activity, association of Ca2+/CaM to CaMKII plateaued at the first Ca2+ elevation event. This result indicated that integration of Ca2+ signals was initiated by the binding of Ca2+/CaM and amplified by the subsequent increases in Thr286-phosphorylated form of CaMKII. Together, these findings demonstrate that CaMKII functions as a leaky integrator of repetitive Ca2+ signals during the induction of synaptic plasticity, and that Thr286 phosphorylation is critical for defining the frequencies of such integration.

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.

Epilepsy is a syndrome that affects about 65 million people around the world. About 150,000 new cases of epilepsy are reported in the United States every year. Temporal lobe epilepsy (TLE) is the most common form of human epilepsy. It is a chronic neurological disorder characterized by recurrent seizures that are devastating due to a lack of effective treatment. TLE is resistant to anticonvulsants and one-third of patients diagnosed with TLE are refractory to medication.

Excessive activation of tropomyosin receptor kinase B (TrkB) promotes TLE. The importance of phospholipase Cγ1 (PLCγ1) as a major downstream signaling effector of TrkB was first identified by the McNamara lab at Duke. Thus, selective inhibition of the PLCγ1-TrkB interaction constitutes a promising avenue for new drugs.

As a proof-of-concept, the McNamara lab engineered a novel 14-mer peptide pY816 that effectively inhibits epilepsy and prevents anxiety-like behavior induced by continuous seizure activity (status epilepticus), in a dose- and time-dependent manner. Despite their promising therapeutic effectiveness, the molecular details of the engagement of these inhibitors with PLCγ1 have remained elusive.

In this study, we propose x-ray crystallography and solution NMR studies to elucidate the binding mode of the peptide to PLCγ1 tandem SH2 domains. This will ultimately facilitate the development of novel therapeutics targeting the TrkB-PLCγ1 interaction.

Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA, but also coordinates transcription-related processes through the post-translational modification of its unique C-terminal repeat domain (CTD). The CTD is an RNAPII specific extension of the enzyme's largest subunit and consists of multiple repeating heptads with the consensus sequence Y₁S₂P₃T₄S₅P₆S₇. In Saccharomyces cerevisiae (Sc), RNAPII committed to productive elongation is phosphorylated at the S₂ positions of the CTD, primarily by CTDK-I (composed of the CDK-like Ctk1, the cyclin-like Ctk2, and Ctk3) the principal elongation-phase CTD kinase in Sc. Although responsible for the bulk of S₂ phosphorylation in vivo, Ctk1 coexists with the essential kinase Bur1 which also contributes to S₂ phosphorylation during elongation. In higher eukaryotes there appears to be only one CTD S₂ kinase: P-TEFb, which had been suggested to reconstitute the activity of both of the Sc S₂ CTD kinases. Based on comparative genomics, we hypothesized that the previously-unstudied Drosophila CDK12 (dCDK12) and little-studied human CDK12 and CDK13 (hCDK12 and hCDK13) proteins are CTD elongation-phase kinases, the metazoan orthologs of yeast Ctk1. Using fluorescence microscopy we show that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNAPII, but is distinct from that of P-TEFb. Chromatin immunoprecipitation experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes in a pattern reminiscent of a S₂ CTD kinase. Appropriately, we show that dCDK12, hCDK12, and hCDK13 purified from nuclear extracts manifest CTD kinase activity in vitro and associate with CyclinK, implicating it as the cyclin subunit of the kinase. Most importantly we demonstrate that RNAi knockdown of dCDK12 in Drosophila cell culture and hCDK12 in human cell lines alters the phosphorylation state of the CTD. In an effort to further characterize the transcriptional roles of human CDK12/CyclinK we overexpress, purify to near homogeneity, and characterize, full-length hCDK12/CyclinK. Additionally, we also identify hCDK12 associated proteins via mass spectrometry, revealing interactions with multiple RNA processing factors, and attempt to engineer an analog sensitive CDK12 human cell line. Overall, these results demonstrate that CDK12 is a major elongation-associated CTD kinase, the ortholog of yCtk1. Our findings clarify the relationships between two yeast CDKs, Ctk1 and Bur1, and their metazoan homologues and draw attention to major metazoan CTD kinase activities that have gone unrecognized and unstudied until now. Furthermore, the results suggest that hCDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.

LpxC, the deacetylase that catalyzes the second and committed step of lipid A biosynthesis in E. coli, is an essential enzyme for virtually all Gram-negative bacteria and one of the most promising novel antibiotic targets for the treatment of multidrug-resistant Gram-negative infections. Here, we report the characterization of two novel LpxC inhibitors that have apparent binding affinities for E. coli LpxC in the picomolar range. Furthermore, these compounds display broad spectrum activity against a plethora of Gram-negative pathogens.

In anticipation for the advancement of LpxC inhibitors in clinical trials, we undertook studies to probe potential bacterial resistance mechanisms to these compounds. In this study, we report a two-step isolation of spontaneously resistant E. coli mutants that have > 200-fold resistance to LpxC inhibitors. These mutants have two chromosomal point mutations that account for resistance additively and independently: one in fabZ, a dehydrase in fatty acid biosynthesis, and the other in thrS, the Thr-tRNA ligase.

For both enzymes, the isolated mutations result in reduced enzymatic activities in vitro. Most unexpectedly, we observed a decreased level of LpxC in bacterial cells harboring fabZ mutations, suggesting that the biosyntheses of fatty acids and lipid A are tightly regulated to maintain balance between phospholipid and lipid A. Additionally, we show that the mutation in thrS slows protein production and cellular growth, providing the first example that reduced protein biosynthesis confers a suppressive effect on inhibition of membrane biosynthesis. Altogether, our studies reveal an impressive compensatory ability of bacteria to overcome inhibition of lipid A biosynthesis by rebalancing cellular homeostasis, a unique mechanism of antibiotic resistance.

Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids while the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine-based saccharolipid that is further modified with core and O-antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria; in most species, a single copy of each bio-synthetic gene is present. The exception is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives lack an lpxH orthologue, yet these species must possess an activity analogous to that of LpxH. We used bioinformatics approaches to identify a candidate gene, designated lpxI, encoding this activity in the model organism Caulobacter crescentus. We then demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase activity in vitro. We have developed high-yield purification schema for Caulobacter crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI, which has no known homologues, consists of two novel domains connected by a linker. Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant to 3.0 Å; preliminary comparison with the product-complexed model reveals striking differences. The findings described herein set the stage for further mechanistic and structural characterization of this novel enzyme.

In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface. E. coli LpxB partitions with membranes, but this interaction is partially abolished in high-salt conditions, suggesting that a significant component of LpxB's membrane association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic activity is detected. Our data support the hypothesis that LpxB performs catalysis at the cytoplasmic surface of the inner membrane, and provide a rational starting-point for structural studies. This work contributes to knowledge of the small but growing set of structurally and mechanistically characterized enzymes which perform chemistry upon lipids.