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Biomedical subjects

Helen R Saibil

Publications and source records attributed to Helen R Saibil.

At least 19 recordsLinked to original sources
PubMed2006

Structure of an Hsp90-Cdc37-Cdk4 complex.

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.

Cell Cycle Proteins↗
PubMed2006

The calcium channel alpha2delta-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum: implications for localization and function.

The accessory alpha2delta subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel alpha1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar alpha2delta-2 subunits with alpha2delta-2 stably expressed in tsA-201 cells. We identified that alpha2delta-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel alpha1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, alpha2delta-2 is not completely proteolytically processed into alpha2-2 and delta-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which alpha2delta-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/alpha2delta-2/beta4 currents. Furthermore, alpha2delta-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of alpha2delta-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable alpha2delta-2 cell line. In contrast, alpha2delta-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant alpha2delta-2 shows reduced functionality in terms of enhancement of CaV2.1/beta4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of alpha2delta-2. Together, these results indicate that both alpha2delta-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.

Alanine↗
PubMed2006

The mechanism of pore formation by bacterial toxins.

A remarkable group of proteins challenge the notions that protein sequence determines a unique three-dimensional structure, and that membrane and soluble proteins are very distinct. The pore-forming toxins typically transform from soluble, monomeric proteins to oligomers that form transmembrane channels. Recent structural studies provide ideas about how these changes take place. The recently solved structures of the beta-pore-forming toxins LukS, epsilon-toxin and intermedilysin confirm that the pore-forming regions are initially folded up on the surfaces of the soluble precursors. To create the transmembrane pores, these regions must extend and refold into membrane-inserted beta-barrels.

Bacterial Toxins↗
PubMed2006

An expanded protein folding cage in the GroEL-gp31 complex.

Bacteriophage T4 produces a GroES analogue, gp31, which cooperates with the Escherichia coli GroEL to fold its major coat protein gp23. We have used cryo-electron microscopy and image processing to obtain three-dimensional structures of the E.coli chaperonin GroEL complexed with gp31, in the presence of both ATP and ADP. The GroEL-gp31-ADP map has a resolution of 8.2 A, which allows accurate fitting of the GroEL and gp31 crystal structures. Comparison of this fitted structure with that of the GroEL-GroES-ADP structure previously determined by cryo-electron microscopy shows that the folding cage is expanded. The enlarged volume for folding is consistent with the size of the bacteriophage coat protein gp23, which is the major substrate of GroEL-gp31 chaperonin complex. At 56 kDa, gp23 is close to the maximum size limit of a polypeptide that is thought to fit inside the GroEL-GroES folding cage.

Adenosine Diphosphate↗
PubMed2006

Elongated oligomers assemble into mammalian PrP amyloid fibrils.

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly alpha-helical state into beta-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a beta form of recombinant mouse PrP (residues 91-231). The beta-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of approximately 60 A. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-beta amyloid fold.

Amyloid↗
PubMed2006

Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.

The double-ring chaperonin GroEL and its lid-like cochaperonin GroES form asymmetric complexes that, in the ATP-bound state, mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptides and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by cryo-EM and single-particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and the substrate-binding domains, admitting ATP and new substrate protein.

Adenosine Diphosphate↗
PubMed2006

An expanded and flexible form of the vacuolar ATPase membrane sector.

The vacuolar ATPase integral membrane c-ring from Nephrops norvegicus occurs in paired complexes in a double membrane. Using cryo-electron microscopy and single particle image processing of 2D crystals, we have obtained a projection structure of the c-ring of N. norvegicus. The c-ring was found to be very flexible, most likely as a result of an expanded conformation of the c subunits. This structure may support a role for the vacuolar ATPase c-rings in membrane fusion.

Amino Acid Sequence↗
PubMed2006

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26.

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.

Binding Sites↗
PubMed2005

Dodecameric structure of the small heat shock protein Acr1 from Mycobacterium tuberculosis.

Small heat shock proteins are a ubiquitous and diverse family of stress proteins that have in common an alpha-crystallin domain. Mycobacterium tuberculosis has two small heat shock proteins, Acr1 (alpha-crystallin-related protein 1, or Hsp16.3/16-kDa antigen) and Acr2 (HrpA), both of which are highly expressed under different stress conditions. Small heat shock proteins form large oligomeric assemblies and are commonly polydisperse. Nanoelectrospray mass spectrometry showed that Acr2 formed a range of oligomers composed of dimers and tetramers, whereas Acr1 was a dodecamer. Electron microscopy of Acr2 showed a variety of particle sizes. Using three-dimensional analysis of negative stain electron microscope images, we have shown that Acr1 forms a tetrahedral assembly with 12 polypeptide chains. The atomic structure of a related alpha-crystallin domain dimer was docked into the density to build a molecular structure of the dodecameric Acr1 complex. Along with the differential regulation of these two proteins, the differences in their quaternary structures demonstrated here supports their distinct functional roles.

Amino Acid Motifs↗
PubMed2005

The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.

The Tat system mediates Sec-independent transport of folded precursor proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. Tat transport involves distinct high-molecular-weight TatA and TatBC complexes. Here we report the 3D architecture of the TatA complex from Escherichia coli obtained by single-particle electron microscopy and random conical tilt reconstruction. TatA forms ring-shaped structures of variable diameter in which the internal channels are large enough to accommodate known Tat substrate proteins. This morphology strongly supports the proposal that TatA forms the protein-conducting channel of the Tat system. One end of the channel is closed by a lid that might gate access to the channel. On the basis of previous protease accessibility measurements, the lid is likely to be located at the cytoplasmic side of the membrane. The observed variation in TatA diameter suggests a model for Tat transport in which the number of TatA protomers changes to match the size of the channel to the size of the substrate being transported. Such dynamic close packing would provide a mechanism to maintain the membrane permeability barrier during transport.

Escherichia coli↗
PubMed2005

Structural basis of pore formation by the bacterial toxin pneumolysin.

The bacterial toxin pneumolysin is released as a soluble monomer that kills target cells by assembling into large oligomeric rings and forming pores in cholesterol-containing membranes. Using cryo-EM and image processing, we have determined the structures of membrane-surface bound (prepore) and inserted-pore oligomer forms, providing a direct observation of the conformational transition into the pore form of a cholesterol-dependent cytolysin. In the pore structure, the domains of the monomer separate and double over into an arch, forming a wall sealing the bilayer around the pore. This transformation is accomplished by substantial refolding of two of the four protein domains along with deformation of the membrane. Extension of protein density into the bilayer supports earlier predictions that the protein inserts beta hairpins into the membrane. With an oligomer size of up to 44 subunits in the pore, this assembly creates a transmembrane channel 260 A in diameter lined by 176 beta strands.

Bacterial Outer Membrane Proteins↗
PubMed2004

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation.

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.

Adenosine Triphosphatases↗
PubMed2004

Location of auxilin within a clathrin cage.

The Dna J homologue, auxilin, acts as a co-chaperone for Hsc70 in the uncoating of clathrin-coated vesicles during endocytosis. Biochemical studies have aided understanding of the uncoating mechanism but until now there was no structural information on how auxilin interacts with the clathrin cage. Here we have determined the three-dimensional structure of a complex of auxilin with clathrin cages by cryo-electron microscopy and single particle analysis. We show that auxilin forms a discrete shell of density on the inside of the clathrin cage. Peptide competition assays confirm that a candidate clathrin box motif in auxilin, LLGLE, can bind to a clathrin construct containing the beta-propeller domain and also displace the well-characterised LLNLD clathrin box motif derived from the beta-adaptin hinge region. The means by which auxilin could both aid clathrin coat assembly and displace clathrin from AP2 during uncoating is discussed.

Amino Acid Motifs↗
PubMed2004

Recognition and separation of single particles with size variation by statistical analysis of their images.

Macromolecules may occupy conformations with structural differences that cannot be resolved biochemically. The separation of mixed molecular populations is a pressing problem in single-particle analysis. Until recently, the task of distinguishing small structural variations was intractable, but developments in cryo-electron microscopy hardware and software now make it possible to address this problem. We have developed a general strategy for recognizing and separating structures of variable size from cryo-electron micrographs of single particles. The method uses a combination of statistical analysis and projection matching to multiple models. Identification of size variations by multivariate statistical analysis was used to do an initial separation of the data and generate starting models by angular reconstitution. Refinement was performed using alternate projection matching to models and angular reconstitution of the separated subsets. The approach has been successful at intermediate resolution, taking it within range of resolving secondary structure elements of proteins. Analysis of simulated and real data sets is used to illustrate the problems encountered and possible solutions. The strategy developed was used to resolve the structures of two forms of a small heat shock protein (Hsp26) that vary slightly in diameter and subunit packing.

Computer Simulation↗
PubMed2004

Structure determination of macromolecular assemblies by single-particle analysis of cryo-electron micrographs.

A new generation of electron microscopes equipped with field emission gun electron sources and the ability to image molecules in their native environment at liquid nitrogen or helium temperatures has enabled the analysis of macromolecular structures at medium resolution (approximately 10 angstroms) and in different conformational states. The amalgamation of electron microscopy and X-ray crystallographic approaches makes it possible to solve structures in the 100-1000 angstroms size range, advancing our understanding of the function of complex assemblies. Many new structures have been solved during the past two years, including one of the smallest complexes to be determined by single-particle cryo-electron microscopy, the transferrin receptor-transferrin complex. Other notable results include the near atomic level resolution structure of the nicotinic acetylcholine receptor in helical arrays and an icosahedral virus structure with an asymmetric polymerase resolved.

Adenosine Triphosphatases↗
PubMed2003

Folding with and without encapsulation by cis- and trans-only GroEL-GroES complexes.

Although a cis mechanism of GroEL-mediated protein folding, occurring inside a hydrophilic chamber encapsulated by the co-chaperonin GroES, has been well documented, recently the GroEL-GroES-mediated folding of aconitase, a large protein (82 kDa) that could not be encapsulated, was described. This process required GroES binding to the ring opposite the polypeptide (trans) to drive release and productive folding. Here, we have evaluated this mechanism further using trans-only complexes in which GroES is closely tethered to one of the two GroEL rings, blocking polypeptide binding by that ring. In vitro, trans-only folded aconitase with kinetics identical to GroEL-GroES. Surprisingly, trans-only also folded smaller GroEL-GroES-dependent substrates, Rubisco and malate dehydrogenase, but at rates slower than the cis reaction. Remarkably, in vivo, a plasmid encoding a trans-only complex rescued a GroEL-deficient strain, but the colony size was approximately one-tenth that produced by wild-type GroEL-GroES. We conclude that a trans mechanism, involving rounds of binding to an open ring and direct release into the bulk solution, can be generally productive although, where size permits, cis encapsulation supports more efficient folding.

Chaperonin 10↗
PubMed2002

The protofilament structure of insulin amyloid fibrils.

Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-beta structure. However, there is a lack of information relating the 4.8 A beta-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-beta ribbon is composed of relatively flat beta-sheets rather than being the highly twisted, beta-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in beta-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.

Amyloid↗