Biomedical subjects

Yifan Cheng

Publications and source records attributed to Yifan Cheng.

18 recordsLinked to original sources

PubMed2006

Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly.

Clathrin-coated vesicles mediate vesicular traffic in cells. Three-dimensional image reconstructions of homogenous populations of in vitro assembled clathrin coats have yielded a molecular model for clathrin and its interactions with some of its partners. The intrinsic averaging required for those calculations has precluded detailed analysis of heterogeneous populations of clathrin-coated vesicles isolated from cells. We have therefore used cryo-electron tomography to study the lattice organization of individual clathrin-coated vesicles and the disposition of the captured vesicle with respect to the surrounding coat. We find a wide range of designs for the clathrin lattice, with different patterns of pentagonal, hexagonal, and occasionally heptagonal facets. Many coats, even smaller ones, enclose membrane vesicles, which are generally offset from the center of the clathrin shell. The electron density distribution between the coat and the underlying vesicle is not uniform, and the number of apparent contacts that anchor the clathrin lattice to the vesicle membrane is significantly less than the number of clathrin heavy chains in the assembly. We suggest that the eccentric position of the vesicle reflects the polarity of assembly, from initiation of coat formation to membrane pinching.

Animals↗

PubMed2006

Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain.

Subfragment 2 (S2), the segment that links the two myosin heads to the thick filament backbone, may serve as a swing-out adapter allowing crossbridge access to actin, as the elastic component of crossbridges and as part of a phosphorylation-regulated on-off switch for crossbridges in smooth muscle. Low-salt expansion increases interfilament spacing (from 52 nm to 67 nm) of rigor insect flight muscle fibers and exposes a tethering segment of S2 in many crossbridges. Docking an actoS1 atomic model into EM tomograms of swollen rigor fibers identifies in situ for the first time the location, length and angle assignable to a segment of S2. Correspondence analysis of 1831 38.7 nm crossbridge repeats grouped self-similar forms from which class averages could be computed. The full range of the variability in angles and lengths of exposed S2 was displayed by using class averages for atomic fittings of acto-S1, while S2 was modeled by fitting a length of coiled-coil to unaveraged individual repeats. This hybrid modeling shows that the average length of S2 tethers along the thick filament (except near the tapered ends) is approximately 10 nm, or 16% of S2's total length, with an angular range encompassing 90 degrees axially and 120 degrees azimuthally. The large range of S2 angles indicates that some rigor bridges produce positive force that must be balanced by others producing drag force. The short tethering segment clarifies constraints on the function of S2 in accommodating variable myosin head access to actin. We suggest that the short length of S2 may also favor intermolecular head-head interactions in IFM relaxed thick filaments.

Animals↗

PubMed2006

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy.

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.

Clathrin↗

PubMed2006

Activation of leukocyte beta2 integrins by conversion from bent to extended conformations.

We used negative stain electron microscopy (EM) to examine the conformational changes in the ectodomains required for activation of the leukocyte integrins alpha(X)beta(2) and alpha(L)beta(2). They transitioned between a bent conformation and two extended conformations in which the headpiece was in either a closed or an open state. Extended integrins exhibited marked flexibility at the alpha subunit genu and between integrin epidermal growth factor-like (I-EGF) domains 1 and 2. A clasp to mimic juxtamembrane association between the integrin alpha and beta subunits stabilized the bent conformation strongly for alpha(X)beta(2) and less so for alpha(L)beta(2). A small molecule allosteric antagonist induced the extended, open headpiece conformation. A Fab known to activate beta(2) integrins on leukocytes induced extension, and a Fab reporter of activation bound only after extension had been induced. The results establish an intimate relationship between extension of beta(2) integrins and their activation in immune responses and leukocyte trafficking.

Antibodies↗

PubMed2006

Three-dimensional structure of an AMPA receptor without associated stargazin/TARP proteins.

Most excitatory synaptic transmissions in the central nervous system are mediated by the neurotransmitter glutamate. Binding of glutamate released from the presynaptic membrane causes glutamate receptors in the postsynaptic membrane to open, which results in a transient depolarization of the postsynaptic membrane. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) subtype of glutamate receptors is responsible for the majority of excitatory postsynaptic currents and is thought to play a central role in synaptic plasticity. Because modulation of glutamate receptors is believed to be involved in the basic mechanism underlying information storage in the brain, the molecular architecture of native AMPA receptors (AMPA-Rs) is of great interest. Previously, we have shown that AMPA-Rs purified from the brain are tightly associated with members of the stargazin/TARP (transmembrane AMPA receptor regulatory protein) family of membrane proteins [Nakagawa et al., Nature 433 (2005), pp. 545-549]. Here, we present a three-dimensional (3D) density map of the hetero-tetrameric AMPA-R without associated stargazin/TARP proteins as determined by cryo-negative stain single-particle electron microscopy. In the absence of stargazin/TARP proteins, the density representing the transmembrane region of the AMPA-R particles is substantially smaller, corroborating our previous analysis that was based solely on projection images.

Animals↗

PubMed2005

ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins.

The archaeal ATPase complex PAN, the homolog of the eukaryotic 26S proteasome-regulatory ATPases, was shown to associate transiently with the 20S proteasome upon binding of ATP or ATPgammaS, but not ADP. By electron microscopy (EM), PAN appears as a two-ring structure, capping the 20S, and resembles two densities in the 19S complex. The N termini of the archaeal 20S alpha subunits were found to function as a gate that prevents entry of seven-residue peptides but allows entry of tetrapeptides. Upon association with the 20S particle, PAN stimulates gate opening. Although degradation of globular proteins requires ATP hydrolysis, the PAN-20S complex with ATPgammaS translocates and degrades unfolded and denatured proteins. Rabbit 26S proteasomes also degrade these unfolded proteins upon ATP binding, without hydrolysis. Thus, although unfolding requires energy from ATP hydrolysis, ATP binding alone supports ATPase-20S association, gate opening, and translocation of unfolded substrates into the proteasome, which can occur by facilitated diffusion through the ATPase.

Adenosine Triphosphatases↗

PubMed2005

Lipid-protein interactions in double-layered two-dimensional AQP0 crystals.

Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.

Animals↗

PubMed2005

Single particle reconstructions of the transferrin-transferrin receptor complex obtained with different specimen preparation techniques.

The outcome of three-dimensional (3D) reconstructions in single particle electron microscopy (EM) depends on a number of parameters. We have used the well-characterized structure of the transferrin (Tf)-transferrin receptor (TfR) complex to study how specimen preparation techniques influence the outcome of single particle EM reconstructions. The Tf-TfR complex is small (290kDa) and of low symmetry (2-fold). Angular reconstitution from images of vitrified specimens does not reliably converge on the correct structure. Random conical tilt reconstructions from negatively stained specimens are reliable, but show variable degrees of artifacts depending on the negative staining protocol. Alignment of class averages from vitrified specimens to a 3D negative stain reference model using FREALIGN largely eliminated artifacts in the resulting 3D maps, but not completely. Our results stress the need for critical evaluation of structures determined by single particle EM.

Carbon↗

PubMed2005

Single particle reconstruction of the human apo-transferrin-transferrin receptor complex.

Most organisms depend on iron as a co-factor for proteins catalyzing redox reactions. Iron is, however, a difficult element for cells to deal with, as it is insoluble in its ferric (Fe3+) form and potentially toxic in its ferrous (Fe2+) form. Thus, in vertebrates iron is transported through the circulation bound to transferrin (Tf) and delivered to cells through an endocytotic cycle involving the transferrin receptor (TfR). We have previously presented a model for the Tf-TfR complex in its iron-bearing form, the diferric transferrin (dTf)-TfR complex [Cheng, Y., Zak, O., Aisen, P., Harrison, S.C., Walz, T., 2004. Structure of the human transferrin receptor-transferrin complex. Cell 116, 565-576]. We have now calculated a single particle reconstruction for the complex in its iron-free form, the apo-transferrin (apoTf)-TfR complex. The same density map was obtained by aligning raw particle images or class averages of the vitrified apoTf-TfR complex to reference models derived from the structures of the dTf-TfR or apoTf-TfR complex. We were unable to improve the resolution of the apoTf-TfR density map beyond 16A, most likely because of significant structural variability of Tf in its iron-free state. The density map does, however, support the model for the apoTf-TfR we previously proposed based on the dTf-TfR complex structure, and it suggests that receptor-bound apoTf prefers to adopt an open conformation.

Apoproteins↗

PubMed2005

Structure and different conformational states of native AMPA receptor complexes.

Ionotropic glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Their modulation is believed to affect learning and memory, and their dysfunction has been implicated in the pathogenesis of neurological and psychiatric diseases. Despite a wealth of functional data, little is known about the intact, three-dimensional structure of these ligand-gated ion channels. Here, we present the structure of native AMPA receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AMPA-Rs) purified from rat brain, as determined by single-particle electron microscopy. Unlike the homotetrameric recombinant GluR2 (ref. 3), the native heterotetrameric AMPA-R adopted various conformations, which reflect primarily a variable separation of the two dimeric extracellular amino-terminal domains. Members of the stargazin/TARP family of transmembrane proteins co-purified with AMPA-Rs and contributed to the density representing the transmembrane region of the complex. Glutamate and cyclothiazide markedly altered the conformational equilibrium of the channel complex, suggesting that desensitization is related to separation of the N-terminal domains. These data provide a glimpse of the conformational changes of an important ligand-gated ion channel of the brain.

Animals↗

PubMed2004

Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating.

Clathrin-coated pits invaginate from specific membrane compartments and pinch off as coated vesicles. These vesicles then uncoat rapidly once released. The Hsc70 molecular chaperone effects the uncoating reaction, and is guided to appropriate locations on clathrin lattices by the J-domain-containing co-chaperone molecule auxilin. This raises the question of how a local event such as ATP hydrolysis by Hsc70 can catalyse a global disassembly. Here, we have used electron cryomicroscopy to determine 12-A-resolution structures of in-vitro-assembled clathrin coats in association with a carboxy-terminal fragment of auxilin that contains both the clathrin-binding region and the J domain. We have located the auxilin fragment by computing differences between these structures and those lacking auxilin (described in an accompanying paper). Auxilin binds within the clathrin lattice near contacts between an inward-projecting C-terminal helical tripod and the crossing of two 'ankle' segments; it also contacts the terminal domain of yet another clathrin 'leg'. It therefore recruits Hsc70 to the neighbourhood of a set of critical interactions. Auxilin binding produces a local change in heavy-chain contacts, creating a detectable global distortion of the clathrin coat. We propose a mechanism by which local destabilization of the lattice promotes general uncoating.

Animals↗

PubMed2004

Molecular model for a complete clathrin lattice from electron cryomicroscopy.

Clathrin-coated vesicles are important vehicles of membrane traffic in cells. We report the structure of a clathrin lattice at subnanometre resolution, obtained from electron cryomicroscopy of coats assembled in vitro. We trace most of the 1,675-residue clathrin heavy chain by fitting known crystal structures of two segments, and homology models of the rest, into the electron microscopy density map. We also define the position of the central helical segment of the light chain. A helical tripod, the carboxy-terminal parts of three heavy chains, projects inward from the vertex of each three-legged clathrin triskelion, linking that vertex to 'ankles' of triskelions centred two vertices away. Analysis of coats with distinct diameters shows an invariant pattern of contacts in the neighbourhood of each vertex, with more variable interactions along the extended parts of the triskelion 'legs'. These invariant local interactions appear to stabilize the lattice, allowing assembly and uncoating to be controlled by events at a few specific sites.

Animals↗

PubMed2004

Aquaporin-0 membrane junctions form upon proteolytic cleavage.

Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.

Amino Acid Sequence↗

PubMed2004

Aquaporin-0 membrane junctions reveal the structure of a closed water pore.

The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.

Animals↗

PubMed2004

Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy.

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens.

Journal Article↗

PubMed2004

Structure of the human transferrin receptor-transferrin complex.

Iron, insoluble as free Fe(3+) and toxic as free Fe(2+), is distributed through the body as Fe(3+) bound to transferrin (Tf) for delivery to cells by endocytosis of its complex with transferrin receptor (TfR). Although much is understood of the transferrin endocytotic cycle, little has been uncovered of the molecular details underlying the formation of the receptor-transferrin complex. Using cryo-electron microscopy, we have produced a density map of the TfR-Tf complex at subnanometer resolution. An atomic model, obtained by fitting crystal structures of diferric Tf and the receptor ectodomain into the map, shows that the Tf N-lobe is sandwiched between the membrane and the TfR ectodomain and that the C-lobe abuts the receptor helical domain. When Tf binds receptor, its N-lobe moves by about 9 A with respect to its C-lobe. The structure of TfR-Tf complex helps account for known differences in the iron-release properties of free and receptor bound Tf.

Animals↗

PubMed2004

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase.

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.

Amino Acid Sequence↗

PubMed2003

The crystal structure of the bifunctional primase-helicase of bacteriophage T7.

Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.

Bacteriophage T7↗