Cryo-EM Studies of Microtubules and Bacterial Nanocompartments

This dissertation focuses largely on structure-function relationship of microtubules, with a supplemental focus on bacterial nanocompartments known as encapsulins. Microtubules (MTs) are an essential component of the eukaryotic cell. MTs are crucial for intracellular trafficking, cellular division, and motility, along with many other functions within the cell. The building blocks of a MT are tubulin heterodimers, which are GTPases that self-assemble to form a hollow, cylindrical MT architecture. The diversity of MT functions is achieved in part by a curious phenomenon known as dynamic instability whereby MTs are in a constant state of flux between growth and catastrophic depolymerization. These dynamics are directly linked to the nucleotide state of the MT, whereby the interplay the GTP and GDP nucleotide states determines the propensity for growth or shrinkage. The intrinsic regulation of dynamic instability, in addition to extrinsic regulation by various MT-associated proteins (MAPs), is absolutely critical for proper cellular function. Cryo-electron microscopy (cryo-EM) was used to directly visualize these important biological assemblies in their native states. The first aspect of my work was to determine how the nucleotide state or the binding of common MAPs (EBs and Kinesins), affected the MT structure. Previous MT structures required MAP-binding to act as fiducials in order to facilitate high-resolution, and Chapter One of this dissertation shows the first high-resolution structures of undecorated MTs bound to various different nucleotides. The next aspect of my work was to examine the GTP-bound MT structure, in order to learn how certain MAPs specifically recognize the GTP state, and the conformational dynamics that occur upon GTP hydrolysis. Prior to this study, the MT field has used a GTP analog, GMPCPP, as a proxy for the GTP bound state. However, no one had actually observed MTs in the GTP state. Chapter Two in this dissertation uses mutated recombinant human tubulin that is hydrolysis-deficient to trap MTs that are truly GTP-bound. I found that these tubulin mutants are an invaluable platform for studying MTs. However, it appears that some mutants appear to create non-physiological assemblies, creating caveats for this system similar to the imperfections of nucleotide analogs. The last chapter of this dissertation is the culmination of work using cryo-EM to better understand the assembly principles for a bacterial nanocompartment known as the encapsulin. Encapsulins were first discovered in 2008, and are typically composed of an enzymatic cargo confined within a proteinaceous shell. This compartmentalization can provide the cell with protection from toxic intermediates, or help with increasing local concentration to make the reaction more efficient. The methods driving cargo encapsulation to support these diverse functions remained unknown. For one encapsulin species, I determined how the cargo enzyme was associated with the encapsulin shell, and found that encapsulin cargos do indeed use a form of symmetry-matching. In this experiment, I found that a pentameric cargo protein binds at the pentameric vertices of the encapsulin shell, thus regulating where the cargo is within the compartment as well as the stoichiometry of cargo encapsulation. Serendipitously, while purifying this encapsulin, I also realized it was a previously overlooked flavoprotein. Given the fact that the cargo enzyme is a ferritin-like protein performing redox chemistry, this flavoprotein designation is very intriguing and warrants additional experiments. Taken together this dissertation utilizes cryo-EM as a tool to probe the structural underpinnings that drive microtubule and encapsulin assembly and function

The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein.

Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.We thank Claire Thomas for help with expressing and purifying recombinant tubulin in insect cells, Abhiram Chintangal and Paul Tobias for support with computation, and Dan Toso, Jonathan Remis, and Patricia Grob for support with EM, as well as Simone Kunzelmann and Iain Taylor who helped with the determination of nucleotide content of E254N MTs by HPLC. We thank Juan Estévez-Gallego for critically reading the manuscript. B.J.L. was supported by NSF-Graduate Research Fellowship Program Grant 1106400. J.R., G.H., and T.S. were supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163), the UK Medical Research Council (FC001163), and the Wellcome Trust (FC001163). J.R. was supported by a Sir Henry Wellcome Postdoctoral Fellowship (100145/Z/12/Z). E.N. acknowledges support from the NIH (R35GM127018). T.S. acknowledges support from the European Research Council (Advanced Grant, project no. 323042). G.H., D.N., and T.S. acknowledge the support of the Spanish Ministry of Economy, Industry and Competitiveness to the CRG-EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme of the Generalitat de Catalunya. T.S. also acknowledges support from the Miller Institute for Basic Research in Science at UC Berkeley. E.N. is a Howard Hughes Medical Institute Investigator

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism.

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria