Nitric oxide (NO) is a well-established signaling molecule and cytotoxic agent in mammals. NO is synthesized by nitric oxide synthase (NOS) by macrophages at high concentrations as a key part of the host immune response, and at low concentrations in endothelial and neuronal cells as a signaling agent. In endothelial cells, the primary NO receptor is soluble guanylate cyclase (sGC), which contains a heme-nitric oxide/oxygen binding domain (H-NOX). Selective binding of NO to the H-NOX domain is responsible for activation of sGC. Thus, the mammalian NO signaling system involves NO synthesis by NOS, and NO sensing by the H-NOX domain of sGC.

NOS and H-NOX proteins have also been identified in a number of bacterial species, including pathogens. Putative roles for bacterial NOS proteins include protection against oxidative stress and antibiotics, while bacterial H-NOX proteins have been shown to govern processes such as biofilm formation and bioluminescence via interactions with signaling proteins such as diguanylate cyclases (DGC) or histidine kinases (HK). Here, various aspects of NO signaling from three different organisms are characterized: the marine alphaproteobacterium Silicibacter sp. TrichCH4B; the soil-dwelling gammaproteobacterium Shewanella oneidensis, and the marine cyanobacterium Synechococcus sp. PCC 7335. This work and other recent studies seek to understand not only the diverse roles for NO in bacteria, but also the molecular mechanisms of bacterial NO signaling.

Silicibacter sp. TrichCH4B is the first bacterial organism discovered to contain both an NOS and H-NOX, thus capable of both NO synthesis and sensing, analogous to mammalian systems. The H-NOX protein from Silicibacter is found in an operon adjacent to an HK, forming part of a two-component phospho-relay signaling network. The response regulator of the network was identified to be a diguanylate cyclase (DGC), which is inactivated upon phosphorylation and establishes the link between NO and intracellular cyclic-di-GMP levels, and consequently biofilm formation. It was also determined that Silicibacter NOS activity is stimulated by a signaling protein from an algal symbiont, Trichodesmium erythraeum, which is a major marine nitrogen fixer. Thus, in the presence of Trichodesmium, the increase in NOS activity results in Silicibacter biofilm formation and poising the two species for nutrient exchange, revealing a novel role for NO in interspecies communication and symbiosis.

Given the diverse processes governed by NO/H-NOX signaling, it is crucial to understand the molecular mechanism by which H-NOX regulates HK autophosphorylation activity, the most common outcome of a NO-bound H-NOX. Here, the interaction and signal transduction between the H-NOX-HK signaling pair from Shewanella oneidensis are characterized. Binding kinetics measurements and analytical gel filtration revealed that NO-bound H-NOX has a tighter affinity for the HK, compared with H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with a panel of binding-deficient H-NOX mutants further reveal that while formation of the H-NOX-HK protein complex is required to stabilize the HK, H-NOX conformational changes upon NO binding are necessary for HK inhibition.

Characterization of H-NOX proteins has led to an increased understanding of bacterial NO sensing. However, NO production in bacteria is less well-understood, and here the NOS protein from Synechococcus sp. PCC 7335 is characterized. Mammalian NOS proteins are comprised of a P450-like heme/oxidase domain responsible for catalysis, and a reductase domain responsible for electron transfer. While most bacterial NOS proteins discovered to date contain only the heme/oxidase domain, Synechococcus NOS contains both the oxidase and reductase domains, and additionally contains a predicted globin domain resembling bacterial flavohemoglobins. Spectroscopic and biochemical characterization of the globin indicated a possible role in redox communication in this novel class of bacterial NOS enzymes.

The gaseous free radical nitric oxide (NO) is firmly established as a unique signaling agent in nature. Eukaryotes employ this freely diffusible molecule in low transient concentrations as a cardiovascular signaling agent and neurotransmitter. Macrophages produce higher, cytotoxic concentrations of NO to serve as an integral piece of the host-defense against pathogens. NO signaling in vertebrates is well characterized and involves the Heme-Nitric oxide/Oxygen binding (H-NOX) domain of soluble guanylate cyclase (sGC) as a selective NO sensor. H-NOX domains are also present in many bacteria including a number of pathogens. Bacterial H-NOX proteins are often found in the same operon as signaling proteins such as histidine kinases, suggesting a role for H-NOX proteins as sensors in prokaryotic NO signaling pathways.

H-NOX-dependent control of histidine kinase autophosphorylation has been demonstrated, but little was known about the biological role or output of H-NOX two-component signaling pathways in bacteria. Here, molecular details of a bacterial H-NOX signaling network in Shewanella oneidensis are presented. NO regulates biofilm formation by controlling the levels of the bacterial secondary messenger cyclic diguanosine monophosphate (cyclic-di-GMP) through an unusually complex multi-component signaling network. Homologous pathways exist in the pathogen Vibrio cholerae and in additional gammaproteobacteria. This work and other recent studies highlight a more general role for NO in restructuring bacterial communal behavior, influencing motility and biofilm formation, host-symbiont interactions, and quorum sensing.

The connectivity of the signaling network in S. oneidensis was mapped by phosphotransfer profiling, which demonstrated signal integration from two histidine kinases and branching to three response regulators: HnoB, HnoC, and HnoD. Phosphodiesterase assays showed that a feed-forward loop between HnoB and HnoD response regulators with phosphodiesterase domains and phosphorylation-mediated activation intricately regulated c-di-GMP levels. In vivo phenotypic characterization established a direct link between NO signaling and increased biofilm formation. Cellular adhesion in biofilms may provide a general protection mechanism for bacteria against high concentrations of reactive and damaging NO. HnoC functions as transcription factor controlling expression of the signaling components in the network. Mechanistic studies of HnoC revealed an unprecedented regulation mechanism, involving phosphorylation-induced dissociation of the response regulator tetramer. The transcriptional feedback loop created by HnoC further regulates the dynamics of the H-NOX signaling network in response to NO stimuli.

One of the phosphotransfer targets of the H-NOX-associated histidine kinase in the signaling network in S. oneidensis and V. cholerae contains a HD-GYP domain: a predicted but poorly characterized phosphodiesterase domain for cyclic-di-GMP hydrolysis. The HnoD HD-GYP domains contain degenerate residues that cause them to be catalytically inactive. To understand the functional consequence of the degeneracy and to gain general insights on the catalytic mechanism of HD-GYP domains, two catalytically active HD-GYP enzymes were characterized. The enzymes contained a binuclear iron center, and reconstitution experiments demonstrated that a heterovalent Fe(III)-Fe(II) cluster is likely required for catalysis. The absence of the metal-coordination site in HnoD eliminates any phosphodiesterase activity.

The prevalence of orphan H-NOX/histidine kinase pairs highlights the necessity for improved methods to map connectivities in two-component signaling. The identity of cognate response regulator(s) is needed to define the biological function of the signaling system. Analogous to approaches in eukaryotic kinases, protein engineering was applied to histidine kinases to permit the use of unnatural ATP analogs as substrates, which are unreactive with other enzymes. Analog-sensitive alleles of two model histidine kinases were developed, and the phosphotransfer transfer reactions to their respective response regulators were optimized. Additionally, a panel of kinase inhibitors was screened for specificity against the analog-sensitive alleles. The analog-sensitive histidine kinases could become useful for in situ identification of phosphotransfer partners of orphan histidine kinases.

ABSTRACT

Structure and Mechanism of Fungal Polysaccharide Monooxygenases

by

Elise Arielle Span

Doctor of Philosophy of Biophysics

University of California, Berkeley

Professor Michael A. Marletta, Chair

Polysaccharide monooxygenases (PMOs) are a newly discovered and growing superfamily of secreted copper catalysts found in nature. Initial PMO discoveries were made in 2011 describing the role of these enzymes in the cellulolytic machinery of fungi. Since then, additional families predominantly from fungi and bacteria have been characterized as having oxidative activity towards a variety of polysaccharide substrates. Chapter 1 introduces these enzymes and describes their origins, structure, function, as well as what can be surmised about their mechanism.

Chapter 2 describes a study that probes PMO mechanism in a system utilizing insoluble (and non-quantifiable) cellulose as a substrate. A combination of in vitro assays and electron paramagnetic resonance studies of a panel of single amino acid variants illustrates the function of the secondary coordination sphere in O2 activation by a cellulose-active PMO. This study bears directly on the recent controversy over the co-substrate of PMOs: although under some conditions PMOs can utilize peroxide, there is clearly a pathway for O2 utilization.

An example of discoveries that are on the horizon in the PMO field is described in Chapter 3. Genetic experiments and biochemical characterization of a homologously expressed PMO from Neurospora crassa shed light on a new family of PMOs that represents a significant departure from those studied previously. These enzymes are necessary for hyphal homing and fusion in filamentous fungi.

Nitric oxide (NO) signaling in mammals occurs through activation of the soluble isoform of guanylate cyclase (sGC), which results in diverse physiological processes such as blood vessel dilation and neurotransmission. sGC is a heterodimeric heme protein that has evolved to be a specific sensor for NO. Although sGC contains the same histidyl-ligated porphyrin as the globins, it has no measureable affinity for oxygen and does not oxidize in air. These unusual features facilitate selective and robust activation by NO in aerobic cellular contexts. Questions regarding the mechanism of ligand discrimination in sGC led to the identification of sGC heme domain homologues in many different organisms where these proteins function as NO and/or oxygen sensors for gas-mediated signaling pathways. The members of the protein family have been subsequently termed Heme Nitric oxide/OXygen binding (H-NOX) domains due to their divergent ligand-binding properties. Functional characterization of H-NOX domains from prokaryotes has provided important clues about the structural features that control ligand discrimination across the H-NOX family. However, additional fundamental questions remain about the influence of protein structure on heme chemistry.

Extensive studies on the globins, as model heme proteins, have established the functional importance of heme protein topological features in modulating gas diffusion to and from the heme site. In globins, deep gas pockets around the porphyrin transiently capture and release oxygen, tuning oxygen affinity for transport, delivery, and storage in diverse physiological environments. Although mechanisms of gas binding in H-NOX proteins have been a subject of intense investigation, there is little knowledge regarding the functional role of protein structure in modulating gas diffusion. Using X-ray crystallography with xenon and kinetic measurements, a tunnel network that extends between the solvent and interior heme site was mapped in a prokaryotic H-NOX domain. Hindering gas diffusion through the tunnels has important consequences on diatomic gas affinity. This suggests that protein tunnels in H-NOX proteins may play functional roles in tuning gas-mediated signaling.

Unlike isolated H-NOX domains, mammalian sGC is a structurally more complex, multi-domain protein. The ability of the sGC heme to resist oxidation in air is unique among histidyl-ligated heme proteins and essential for maximal NO-induced activation. Speculations have been made about how the sGC heme resists oxidation, but no study has systemically addressed the structural and electronic factors that contribute to this critical property. To probe the accessibility of oxygen to the heme site, we sought to substitute the native heme of sGC with an unnatural porphyrin displaying emission that is quenched by oxygen. Using an expression-based methodology, a phosphorescent Ru porphyrin was incorporated in sGC constructs of varying lengths. Emission quenching results suggest that oxygen diffusion to the full-length sGC heme site is significantly hindered compared to smaller sGC constructs. Limited oxygen accessibility, combined with heme electronic factors, appear to serve as important evolutionary solutions in sGC to protect the heme cofactor under aerobic conditions.

Originally to probe sGC function, the strategies used to generate oxygen-sensing proteins have been applied to create a new family of H-NOX-based molecular sensors. Substitution of the native heme in bacterial H-NOX proteins with unnatural porphyrins has proven to be a promising strategy to develop stable protein agents for biological imaging applications. Heme proteins are highly tunable frameworks that coordinate porphyrins with high fidelity and specificity. These properties have been exploited to design heme protein-based sensors with tailored functionalities for enhancing porphyrin bioavailability. Initial work on making optical oxygen sensors has evolved into recent efforts to generate H-NOX-based MRI contrast agents with high relaxivities for future use in deep-tissue imaging. Thus, through substitution of the native heme group with unnatural porphyrins, new sensing properties have been built into the H-NOX family.

Nitric oxide (NO) is an important physiological mediator of vasodilation, platelet aggregation, and neurotransmission. An NO signal regulates these processes by signal transduction through the enzyme soluble guanylate cyclase (sGC), and dysfunction in this pathway manifests in human disease. NO activates sGC several hundred fold to produce the second messenger cGMP, and the mechanism of activation was previously thought to result exclusively from NO binding to the sGC heme. However, recent studies have shown that heme-bound NO only partially activates sGC and additional NO binding to a nonheme site is required for maximal NO activation. Therefore, understanding the nature the nonheme-NO coordination is crucial to understanding the mechanism of enzyme activation.

Thiol oxidation of sGC cysteines results in the loss of enzyme activity, therefore, the role of cysteines in NO-stimulated sGC activity was investigated. We found that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking the non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway.

Heme-oxidation also results in the loss enzyme activity and may play a role in the NO-desensitization of sGC in cardiovascular disease. In vitro, the sGC heme is typically found in the ferrous [Fe(II)] state, which is activated several hundred fold by NO. Comparatively, the ferric [Fe(III)] form of sGC is only weakly activated by NO, but the mechanism of this blunted response is not well understood. Specifically, sGC undergoes a reaction with NO termed reductive nitrosylation, where the sGC heme is reduced to the ferrous [Fe(II)] state; but despite the recovery of the initial heme state, NO-stimulated activity does not recover. We found that the reduction of the sGC heme occurs via a coupled oxidation of an sGC cysteine to a nitrosothiol, and that the S-nitrosation of this key cysteine inhibits the enzymatic response to NO. The data from chapter 2 suggests the mechanism of inhibition may be the oxidation of a cysteine that constitutes the nonheme NO binding site. Thus, thiol-oxidation is at the root of the NO-desensitization of heme-oxidized sGC and the implications to human health and disease are discussed.