Gonen Lab

Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.

Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular.

Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.

Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion.

The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.

Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.