Our research focuses on two fundamental questions in cell biology: How do cells interact with each other and with their environment? How do cells obtain the nutrients essential for their survival? We focus on integral membrane proteins because they play a crucial role in mediating information transfer between cells and between compartments within cells and because they are involved in vital processes such as energy conversion, sensing, fusion, and nutrient and solute uptake. More than 20 percent of the genes in the human genome encode membrane-traversing proteins. Thus it is critical to study structures of membrane proteins to unravel important biological and physiological processes. Our model system is the eye lens, whose cells are rich in junction-forming membrane proteins and transporters needed for nutrient uptake.
My laboratory is interested in revealing the structural and molecular mechanisms by which membrane proteins mediate these cross-membrane processes with high specificity and efficiency. Using high-resolution cryo-electron microscopy (cryo-EM), together with other biophysical and biochemical approaches, we are focusing on membrane channels, transporters, and macromolecular machines that form biological pores.
We use a multidisciplinary approach, including cryo-EM, x-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy for determining protein structure and characterizing membrane protein complex formation and binding stoichiometries. To assay protein function, we use biophysical techniques with reconstituted proteoliposomes and investigate protein structure under a variety of physiologically important regulatory states.
My previous work with Thomas Walz (HHMI, Harvard Medical School) led to a number of research contributions to the fields of electron microscopy, membrane biology, water channel physiology, and structural biology. I studied aquaporin-0 (AQP0), the only water channel having two distinct functions: it forms a pore for water permeation, and it creates adhesive junctions in the eye lens. I crystallized AQP0 in two dimensions (2D) for electron crystallography and collected a data set to 1.9-Å resolution—the highest resolution achieved using this technique, and the highest resolution achieved for any mammalian membrane channel. I developed procedures for molecular replacement and refinement—the first successful implementation of x-ray crystallographic techniques to an electron crystallographic study; this set the stage for using electron crystallography to determine protein structure rapidly.
[[nid:3985]]Our structure—the first atomic resolution structure for an adhesive junction—highlighted how AQP0 membrane channels can mediate the formation of cell-cell membrane junctions. Our study also revealed that the AQP0 water channels close when the protein forms membrane junctions. This allowed us to investigate aspects of water channel regulation at high resolution and furthered our understanding of how these ubiquitous channels function. A striking feature of these 2D crystals is the presence of a continuous lipid bilayer surrounding AQP0. Our membrane model is the first, and only, experimentally determined structure of a eukaryotic membrane surrounding a mammalian protein. The structure allowed us to begin to understand how membrane protein functions may depend on the ability of these proteins to organize the lipid environment around them.
We have now extended our water channel studies to include channel structure in general and channel regulation in particular. AQP0 is regulated by at least three bona fide mechanisms: pH, calmodulin, and phosphorylation.
We are studying the structural consequences of AQP0's pH regulation. AQP0's permeability doubles when extracellular pH drops from 7.2 to 6.5 (a physiologically significant event in the eye lens). Two-dimensional crystals are biological membranes in which water channels form functional pores. These crystals are robust enough to allow us to change buffer pH without affecting crystal order, making it possible to use the same 2D crystals to determine AQP0's structure under different conditions. We anticipate that at pH 6.5 the channel will be in a high-permeability state; at pH 7.2, the permeability of AQP0 halves and we expect the channel to be in a low-permeability state.
AQP0's permeability is regulated by calmodulin (CaM). Calmodulin is a bilobed Ca2+-binding protein that functions as a ubiquitous secondary messenger in several Ca2+-signaling pathways. CaM modulates the activity of many channels and transporters, including communicating channels, water channels, and voltage-gated cardiac Ca2+ channels. CaM is therefore arguably the most important signaling molecule in the cell, yet no structure of any channel/transporter in complex with CaM has been determined.
We adopted a multidisciplinary approach to structurally characterize the interaction between AQP0 and CaM by NMR, x-ray crystallography, and electron crystallography. Using NMR, we derived a structural model of the AQP0-CaM complex and showed that CaM may act as a channel inhibitor, by capping the water pores of two monomers within the AQP0 tetramer.
Our results suggest that CaM induces cross-cooperativity between monomers within the AQP0 tetramer, and describe for the first time the cooperativity within water channels. For a long time it was unclear why AQPs assemble into tetramers, because each monomer is functionally independent within the tetramer. Our research suggests that the tetramerization is a necessary scaffold for the binding of regulatory proteins such as CaM.
We have expressed, purified, and crystallized the closest bacterial homolog of the human facilitated GLUT—the Escherichia coli H+/galactose symporter (GalP). Surprisingly, the projection structure of GalP indicates that the transporter forms trimers in membranes—an oligomerization that is both novel and unexplained. Our functional studies on GalP 2D crystals verify that the membrane-embedded transporter is able to bind substrate. It is possible that trimerization is necessary for protein stability and function, and we are investigating this.
We use electrophysiology and patch-clamping techniques to study the function of channels and transporters. We use the Xenopus oocyte expression system, but we also record channel function from highly ordered 2D crystals for a direct correlation between structure and function of target proteins as they are embedded within a biological membrane.
The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin from Vibrio cholerae and heat-labile enterotoxin from enterotoxigenic Escherichia coli. The major outer membrane T2SS protein is the secretin GspD. Using cryo-EM and single-particle reconstruction techniques, we are investigating the structure and function of this cholera toxin secretion channel. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution revealed a dodecameric structure reminiscent of a barrel, with a large channel at its center that appears to contain a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus we provide a structural basis for a possible secretion mechanism of the T2SS. We are working to improve the resolution of our maps.
In electron crystallography, membrane protein structure is determined from 2D 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 provide experimental phases directly to high resolution. We recently developed a new approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We used the strengths of electron crystallography to rapidly obtain accurate experimental-phase information from low-resolution images and to obtain accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α-helix fragments and were 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, we determined structures of three membrane proteins rapidly and accurately to atomic resolution without high-resolution image data. We are working to fully automate this method.
Tamir Gonen Lab Head
Megan Filbin Postdoctoral Associate
Brent Nannenga Postdoctoral Associate
Stephen Reichow Research Staff
Maen Sarhan Research Staff
Dan Shi Senior Scientist
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.
A number of atomic-resolution structures of membrane proteins (better than 3Å resolution) have been determined recently by electron crystallography. While this technique was established more than 40 years ago, it is still in its infancy with regard to the two-dimensional (2D) crystallization, data collection, data analysis, and protein structure determination. In terms of data collection, electron crystallography encompasses both image acquisition and electron diffraction data collection. Other chapters in this volume outline protocols for image collection and analysis. This chapter, however, outlines detailed protocols for data collection by electron diffraction. These include microscope setup, electron diffraction data collection, and troubleshooting.
Voltage sensor ring in a native structure of a membrane-embedded potassium channel.Proceedings of the National Academy of Sciences of the United States of America 2013
L. Shi, H. Zheng, H. Zheng, B. A. Borkowski, D. Shi, T. Gonen, and Q. Jiang Proceedings of the National Academy of Sciences of the United States of America, (2013)
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.
Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 2013
B. L. Nannenga, M. G. Iadanza, B. S. Vollmar, and T. Gonen Current protocols in protein science / editorial board, John E. Coligan ... [et al.], Chapter 17:Unit17.15 (2013)
Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.
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.
The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders.
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.
A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a calmodulin (CaM)-binding domain of AQP0. The additional negative charge introduced by phosphoserine 235 perturbs electrostatic interactions between AQP0 and CaM to favour water influx through the channel. In isolated mouse lenses, displacement of PKA from the AKAP2-AQP0 channel complex promotes cortical cataracts as characterized by severe opacities and cellular damage. Thus, anchored PKA modulation of AQP0 is a homeostatic mechanism that must be physically intact to preserve lens transparency.
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.
Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.
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.
Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains (VSDs) and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the VSDs control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the VSDs.
The Ndc80 kinetochore complex directly modulates microtubule dynamics.Proceedings of the National Academy of Sciences of the United States of America 2012
N. T. Umbreit, D. R. Gestaut, J. F. Tien, B. S. Vollmar, T. Gonen, C. L. Asbury, and T. N. Davis Proceedings of the National Academy of Sciences of the United States of America, (2012)
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.
Synaptosomes are intact, isolated nerve terminals that contain the necessary machinery to recycle synaptic vesicles via endocytosis and exocytosis upon stimulation. Here we use this property of synaptosomes to load quantum dots into synaptic vesicles. Vesicles are then isolated from the synaptosomes, providing a method to probe isolated, individual synaptic vesicles where each vesicle contains a single, encapsulated nanoparticle. This technique provided an encapsulation efficiency of ~16%, that is, ~16% of the vesicles contained a single quantum dot while the remaining vesicles were empty. The ability to load single nanoparticles into synaptic vesicles opens new opportunity for employing various nanoparticle-based sensors to study the dynamics of vesicular transporters.
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.
The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB5 heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.
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.
Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.
N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity.Proceedings of the National Academy of Sciences of the United States of America 2011
S. Jehle, B. S. Vollmar, B. Bardiaux, K. K. Dove, P. Rajagopal, T. Gonen, H. Oschkinat, and R. E. Klevit Proceedings of the National Academy of Sciences of the United States of America, 108:6409-14 (2011)
The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.
Prior Publications (9 of 35)
Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B.The Journal of Cell Biology 2010
J. F. Tien, N. T. Umbreit, D. R. Gestaut, A. D. Franck, J. Cooper, L. Wordeman, T. Gonen, C. L. Asbury, and T. N. Davis The Journal of Cell Biology, 189:713-23 (2010)
The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.
An engineered DNA-binding protein self-assembles metallic nanostructures.Chembiochem: A European Journal of Chemical Biology 2010
R. Hall Sedlak, M. Hnilova, E. Gachelet, L. Przybyla, D. Dranow, T. Gonen, M. Sarikaya, C. Tamerler, and B. Traxler Chembiochem: A European Journal of Chemical Biology, 11:2108-12 (2010)
The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the 'secretin' GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.
Glucose is a primary source of energy for human cells. Glucose transporters form specialized membrane channels for the transport of sugars into and out of cells. Galactose permease (GalP) is the closest bacterial homolog of human facilitated glucose transporters. Here, we report the functional reconstitution and 2D crystallization of GalP. Single particle electron microscopy analysis of purified GalP shows that the protein assembles as an oligomer with three distinct densities. Reconstitution assays yield 2D GalP crystals that exhibit a hexagonal array having p3 symmetry. The projection structure of GalP at 18 A resolution shows that the protein is trimeric. Each monomer in the trimer forms its own channel, but an additional cavity (10 approximately 15 A in diameter) is apparent at the 3-fold axis of the oligomer. We show that the crystalline GalP is able to selectively bind substrate, suggesting that the trimeric form is biologically active.
Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system.mBio 2010
S. Sanowar, P. Singh, R. A. Pfuetzner, I. André, H. Zheng, T. Spreter, N. C J. Strynadka, T. Gonen, D. Baker, D. R. Goodlett, and S. I. Miller mBio, 1 (2010)
The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.
Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR.Structure (London, England : 1993) 2010
G. Wisedchaisri, D. M. Dranow, T. J. Lie, J. B. Bonanno, Y. Patskovsky, S. A. Ozyurt, M. J. Sauder, S. C. Almo, S. R. Wasserman, S. K. Burley, J. A. Leigh, and T. Gonen Structure (London, England : 1993), 18:1512-21 (2010)
Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.
Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein's architecture where they can affect the function, structure, quaternary assembly, and the stability of the membrane protein.