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Axel T Brunger

Publications and source records attributed to Axel T Brunger.

15 recordsLinked to original sources
PubMed2003

Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations.

Vesicle fusion in eukaryotes is thought to involve the assembly of a highly conserved family of proteins termed soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a highly stable parallel four-helix bundle. We have used intermolecular single-molecule fluorescence resonance energy transfer to characterize preassembled neuronal SNARE complexes consisting of syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa on deposited lipid bilayers. Surprisingly, we found a mixture of parallel as well as antiparallel configurations involving the SNARE motifs of syntaxin and synaptobrevin as well as those of syntaxin and synaptosome-associated protein of 25 kDa. The subpopulation with the parallel four-helix bundle configuration could be greatly enriched by an additional purification step in the presence of denaturant, indicating that the parallel configuration is the energetically most favorable state. Interconversion between the configurations was not observed. From this observation, we infer the conversion rate to be <1.5 h-1. The existence of antiparallel configurations suggests a regulatory role of chaperones, such as N-ethylmaleimide-sensitive factor, or the membrane environment during SNARE complex assembly in vivo, and it could be a partial explanation for the relatively slow rates of vesicle fusion observed by reconstituted fusion experiments in vitro.

Amino Acid Motifs↗
PubMed2003

NSF and p97/VCP: similar at first, different at last.

N-Ethylmaleimide sensitive factor (NSF) and p97/valosin-containing protein (VCP) are distantly related members of the ATPases associated with a variety of cellular activities (AAA) family of proteins. While both proteins have been implied in cellular morphology changes involving membrane compartments or vesicles, more recent evidence seems to imply that NSF is primarily involved in the soluble NSF attachment receptor (SNARE)-mediated vesicle fusion by disassembling the SNARE complex whereas p97/VCP is primarily involved in the extraction of membrane proteins. These functional differences are now corroborated by major structural differences based on recent crystallographic and cryo-electron microscopy studies. This review discusses these recent findings.

Adenosine Triphosphatases↗
PubMed2003

Role of the gamma-phosphate of ATP in triggering protein folding by GroEL-GroES: function, structure and energetics.

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.

Adenosine Triphosphate↗
PubMed2003

Domain flexibility in the 1.75 A resolution structure of Pb2+-calmodulin.

Calmodulin (CaM) regulates a variety of cellular processes by interacting with a large number of proteins in a Ca(2+)-dependent manner. Conformational flexibility plays a key role in CaM function, although the full extent and detailed features of this flexibility are not fully characterized. Here, the 1.75 A resolution crystal structure of Pb(2+)-bound Paramecium tetraurelia CaM crystallized in a previously unobserved monoclinic lattice is reported. Pb(2+)-CaM is disordered in this new lattice and only a portion of each of the two molecules in the asymmetric unit can be modeled. Comparison of the structures of Ca(2+)-CaM and Pb(2+)-CaM show close agreement in the C-terminal domain but significant structural differences in the N-terminal domain. In addition, translation-libration-screw (TLS) refinement and Rosenfield difference analysis reveal inter-helical flexibility in the metal-bound N-terminal domain of the protein that is absent in the metal-bound C-terminal domain and indicates that the two structurally similar domains of CaM are dynamically distinct. These results demonstrate that TLS refinement and Rosenfield difference analysis allow detailed information about macromolecular flexibility to be extracted from X-ray diffraction data even when the crystal lattice prohibits full manifestation of this flexibility.

Animals↗
PubMed2003

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution.

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.

Adenosine Triphosphatases↗
PubMed2003

Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains.

The ATPase p97/VCP affects multiple events within the cell. These events include the alteration of both nuclear and mitotic Golgi membranes, the dislocation of ubiquitylated proteins from the endoplasmic reticulum and regulation of the NF-kappa b pathway. Here we present the crystal structure of full-length Mus musculus p97/VCP in complex with a mixture of ADP and ADP-AlF(x) at a resolution of 4.7 A. This is the first complete hexameric structure of a protein containing tandem AAA (ATPases associated with a variety of cellular activities) domains. Comparison of the crystal structure and cryo-electron microscopy (EM) reconstructions reveals large conformational changes in the helical subdomains during the hydrolysis cycle. Structural and functional data imply a communication mechanism between the AAA domains. A Zn(2+) occludes the central pore of the hexamer, suggesting that substrate does not thread through the pore of the molecule.

Adenosine Triphosphatases↗
PubMed2003

Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8 complex.

The sec6/8 complex or exocyst is an octameric protein complex that functions during cell polarization by regulating the site of exocytic vesicle docking to the plasma membrane, in concert with small GTP-binding proteins. The Sec5 subunit of the mammalian sec6/8 complex binds Ral in a GTP-dependent manner. Here we report the crystal structure of the complex between the Ral-binding domain of Sec5 and RalA bound to a non-hydrolyzable GTP analog (GppNHp) at 2.1 A resolution, providing the first structural insights into the mechanism and specificity of sec6/8 regulation. The Sec5 Ral-binding domain folds into an immunoglobulin-like beta-sandwich structure, which represents a novel fold for an effector of a GTP-binding protein. The interface between the two proteins involves a continuous antiparallel beta-sheet, similar to that found in other effector/G-protein complexes, such as Ras and Rap1A. Specific interactions unique to the RalA.Sec5 complex include Sec5 Thr11 and Arg27, and RalA Glu38, which we show are required for complex formation by isothermal titration calorimetry. Comparison of the structures of GppNHp- and GDP-bound RalA suggests a nucleotide-dependent switch mechanism for Sec5 binding.

Amino Acid Sequence↗
PubMed2002

Mutational analysis of synaptobrevin transmembrane domain oligomerization.

Synaptobrevin 2 is thought to facilitate fusion of synaptic vesicles with the presynaptic membrane through formation of a soluble NSF attachment protein receptor complex (SNARE) with syntaxin 1a and a synaptosomal associated protein of 25 kDa (SNAP-25). Previous reports have described a homodimer of synaptobrevin that is dependent on the transmembrane domain. However, these reports disagree about the magnitude of dimerization, which makes it difficult to assess the biological relevance of this interaction. We used SDS-PAGE and the TOXCAT genetic assay to reexamine the homodimerization of the synaptobrevin transmembrane domain in detergents and the Escherichia coli inner membrane, respectively. To gauge the magnitude of synaptobrevin homodimerization, we used the well-characterized glycophorin A homodimer as a positive standard. In contrast to previous studies, we found synaptobrevin homodimerization in E. coli is very weak when compared to glycophorin A. Recombinant synaptobrevin forms a small amount of dimer and higher order oligomers in detergents that are highly dependent on solublization conditions. We estimate a dissociation constant of 10 mM for synaptobrevin dimerization in detergent. Thus, the dimerization of synaptobrevin in membranes is very weak, questioning any possible functional role for this association in vivo.

Amino Acid Sequence↗
PubMed2002

High resolution structure, stability, and synaptotagmin binding of a truncated neuronal SNARE complex.

The structure of a truncated SNARE complex has been solved to 1.4-A resolution revealing a stabilizing salt bridge, sites of hydration, and conformational variability of the ionic central layer that were not observed in a previously published structure at 2.4-A resolution (Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347-353). The truncated complex lacks residues involved in phospholipid binding and denatures at a lower temperature than longer complexes as assessed by SDS and circular dichroism thermal melts. The truncated SNARE complex is monomeric, and it retains binding to synaptotagmin I.

Amino Acid Sequence↗
PubMed2002

The ENTH domain.

The epsin NH2-terminal homology (ENTH) domain is a membrane interacting module composed by a superhelix of alpha-helices. It is present at the NH2-terminus of proteins that often contain consensus sequences for binding to clathrin coat components and their accessory factors, and therefore function as endocytic adaptors. ENTH domain containing proteins have additional roles in signaling and actin regulation and may have yet other actions in the nucleus. The ENTH domain is structurally similar to the VHS domain. These domains define two families of adaptor proteins which function in membrane traffic and whose interaction with membranes is regulated, in part, by phosphoinositides.

Amino Acid Sequence↗
PubMed2002

Molecular dynamics applied to X-ray structure refinement.

Simulated annealing, in the form of temperature-controlled molecular dynamics, has been successfully applied to macromolecular X-ray structure optimization. The theory and practice of the method are reviewed, and some recent improvements are described.

Biopolymers↗
PubMed2002

Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle.

p97 (also called VCP), a member of the AAA ATPase family, is involved in several cellular processes, including membrane fusion and extraction of proteins from the endoplasmic reticulum for cytoplasmic degradation. We have studied the conformational changes that p97 undergoes during the ATPase cycle by cryo-EM and single-particle analysis. Three-dimensional maps show that the two AAA domains, D1 and D2, as well as the N-domains, experience conformational changes during ATP binding, ATP hydrolysis, P(i) release and ADP release. The N-domain is flexible in most nucleotide states except after ATP hydrolysis. The rings formed by D1 and D2 rotate with respect to each other, and the size of their axial openings fluctuates. Taken together, our results depict the movements that this and possibly other AAA ATPases can undergo during an ATPase cycle.

Adenosine Triphosphatases↗
PubMed2002

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin.

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.

Amino Acid Sequence↗