Structure Determination Of Domain Research Articles

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Tandem domain structure determination based on a systematic enumeration of conformations

Protein structure determination is undergoing a change of perspective due to the larger importance taken in biology by the disordered regions of biomolecules. In such cases, the convergence criterion is more difficult to set up and the size of the conformational space is a obstacle to exhaustive exploration. A pipeline is proposed here to exhaustively sample protein conformations using backbone angle limits obtained by nuclear magnetic resonance (NMR), and then to determine the populations of conformations. The pipeline is applied to a tandem domain of the protein whirlin. An original approach, derived from a reformulation of the Distance Geometry Problem is used to enumerate the conformations of the linker connecting the two domains. Specifically designed procedure then permit to assemble the domains to the linker conformations and to optimize the tandem domain conformations with respect to two sets of NMR measurements: residual dipolar couplings and paramagnetic resonance enhancements. The relative populations of optimized conformations are finally determined by fitting small angle X-ray scattering (SAXS) data. The most populated conformation of the tandem domain is a semi-closed one, fully closed and more extended conformations being in minority, in agreement with previous observations. The SAXS and NMR data show different influences on the determination of populations.

Small Angle X-ray Scattering Analysis of Clostridium thermocellum Cellulosome N-terminal Complexes Reveals a Highly Dynamic Structure

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the "reaching dimer" previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition.

Surface microstructure and magnetic behavior in FeSiB amorphous ribbons from magneto-optical Kerr effect

The magneto-optical Kerr effect (MOKE) completed by other surface sensitive methods as integral low-energy and conversion electron Mössbauer spectroscopy, scanning and transmission electron microscopy and by X-ray diffraction have been used with the aim to trace the surface microstructure and magnetic properties of FeSiB amorphous ribbons prepared by planar flow casting. The general composition of studied samples is Fe80SixB20−x, where x=4, 6, 8, 10at.%.It is shown that MOKE used for magnetization, hysteresis loop, and domain structure determination is highly beneficial in a detection of both surface crystallization and local ordering of atoms into magnetically different clusters of amorphous structure. Moreover, a combination of blue and red laser with different penetration depths yields unique results concerning the surface anisotropy and depth sensitivity. In the case of samples with 4, 6, and 8at.% Si MOKE detects two magnetically different phases diverging in coercivity values Hc, however, not varying with the sample composition. These phases have been identified by Mössbauer measurements as FeSi and FeB clusters. Their relationship changes with Si concentration. On the other hand, a strong increase in the surface Hc found for the sample with 10at.% Si has indicated a nanocrystallization. It was confirmed by electron microscopy, Mössbauer and X-ray diffraction results. The size of nanocrystals has varied between 200nm and 500nm.

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions

We developed and implemented an ensemble-refinement method to study dynamic biomolecular assemblies with intrinsically disordered segments. Data from small angle X-ray scattering (SAXS) experiments and from coarse-grained molecular simulations were combined by using a maximum-entropy approach. The method was applied to CHMP3 of ESCRT-III, a protein with multiple helical domains separated by flexible linkers. Based on recent SAXS data by Lata etal. (J. Mol. Biol. 378, 818, 2008), we constructed ensembles of CHMP3 at low-and high-salt concentration to characterize its closed autoinhibited state and open active state. At low salt, helix α(5) is bound to the tip of helices α(1) and α(2), in excellent agreement with a recent crystal structure. Helix α(6) remains free in solution and does not appear to be part of the autoinhibitory complex. The simulation-based ensemble refinement is general and effectively increases the resolution of SAXS beyond shape information to atomically detailed structures.

Structural basis for recognition of phosphodiester-containing lysosomal enzymes by the cation-independent mannose 6-phosphate receptor

Mannose 6-phosphate (Man-6-P)-dependent trafficking is vital for normal development. The biogenesis of lysosomes, a major cellular site of protein, carbohydrate, and lipid catabolism, depends on the 300-kDa cation-independent Man-6-P receptor (CI-MPR) that transports newly synthesized acid hydrolases from the Golgi. The CI-MPR recognizes lysosomal enzymes bearing the Man-6-P modification, which arises by the addition of GlcNAc-1-phosphate to mannose residues and subsequent removal of GlcNAc by the uncovering enzyme (UCE). The CI-MPR also recognizes lysosomal enzymes that elude UCE maturation and instead display the Man-P-GlcNAc phosphodiester. This ability of the CI-MPR to target phosphodiester-containing enzymes ensures lysosomal delivery when UCE activity is deficient. The extracellular region of the CI-MPR is comprised of 15 repetitive domains and contains three distinct Man-6-P binding sites located in domains 3, 5, and 9, with only domain 5 exhibiting a marked preference for phosphodiester-containing lysosomal enzymes. To determine how the CI-MPR recognizes phosphodiesters, the structure of domain 5 was determined by NMR spectroscopy. Although domain 5 contains only three of the four disulfide bonds found in the other seven domains whose structures have been determined to date, it adopts the same fold consisting of a flattened beta-barrel. Structure determination of domain 5 bound to N-acetylglucosaminyl 6-phosphomethylmannoside, along with mutagenesis studies, revealed the residues involved in diester recognition, including Y679. These results show the mechanism by which the CI-MPR recognizes Man-P-GlcNAc-containing ligands and provides new avenues to investigate the role of phosphodiester-containing lysosomal enzymes in the biogenesis of lysosomes.

Small-angle scattering studies of macromolecular solutions

In recent years, major progress has been achieved in developing novel approaches to interpret small-angle scattering data from solutions of biological macromolecules in terms of three-dimensional models. These advanced methods include: ab initio low-resolution shape and domain structure determination; modelling of quaternary structure by rigid-body refinement; simultaneous analysis of multiple scattering patterns, e.g. from contrast variation in neutron scattering to study multicomponent complexes; validation of high-resolution models; and addition of missing loops and domains. The new techniques will be presented and practical applications of the methods are illustrated by recent examples. The use of additional information from other methods, joint applications of X-ray and neutron scattering, and the possibilities for assessing and validating the models constructed based on small-angle scattering data will be discussed.

New methods for domain structure determination of proteins from solution scattering data

New approaches for domain structure determination of macromolecules from X-ray solution scattering are presented. An ab initio method for building structural models of proteins from the scattering data implemented in the computer program GASBOR employs simulated annealing to find a chain-like spatial distribution of dummy residues (DR), which fits the experimental scattering pattern up to a resolution of 0.5 nm. This method substantially improves the resolution and reliability of models derived from the scattering data. A modification of GASBOR fitting distance distribution data in real space substantially (by a factor of 3 to 7) speeds up the calculation of DR models. The DR approach is further extended to reconstruct missing domains in multisubunit proteins and fusion proteins and to find probable configurations of missing loops in crystallographic models. Four computer programs have been written to provide appropriate tools for different situations in which a high or low resolution structural model lacks a loop or domain. These methods permit solution scattering analysis to usefully complement the results obtained by high-resolution methods like X-ray crystallography and nuclear magnetic resonance spectroscopy. The efficiency of the presented approaches is illustrated by their application for structure analysis of several proteins, with known and unknown crystal structure, from experimental scattering data.

Homologous repair of DNA damage and tumorigenesis: the BRCA connection.

Homologous recombination has been recognized in recent years to be an important DNA repair pathway in mammalian cells, for such damage as chromosomal double-strand breaks. Cells mutated for the genes involved in the hereditary breast and ovarian cancer susceptibility syndromes, i.e. BRCA1 and BRCA2, show defects in DNA repair by homologous recombination, implicating this repair pathway in protecting individuals against tumorigenesis. This review summarizes recent advances in our understanding of BRCA1 and BRCA2 in DNA repair, as well as insight into these proteins gleaned from structure determination of domains of these proteins and the broader evolutionary conservation than previously appreciated.

Thermal stability of pTRMs created from different magnetic states

The apparent successful application of the Thellier’s method of paleointensity or paleofield direction determinations on multi-domain (MD) and pseudo-single-domain (PSD) grains suggests the validity of the Thellier’s laws of independence and additivity of partial thermoremanent magnetizations (pTRMs). However, it is well known that these laws are violated for PSD and especially for MD samples, leading to a noticeable inequality of blocking ( T b) and unblocking ( T ub) temperatures. Thus, a preliminary determination of domain structure (DS) of a sample is desirable. The thermomagnetic criterion of DS is based on a direct check of the equality of T b and T ub by measuring the parameter A a( T 1, T 2)=intensity of the tail[pTRM a( T 1, T r)] normalized to the original pTRM a( T 1, T r), both measured at T r. Here T r is room temperature and pTRM a( T 1, T r) is a pTRM acquired when the upper temperature T 1 of pTRM acquisition is reached by cooling a sample in zero field from the Curie temperature T C. A detailed study of properties of this pTRM and its tail was performed in our previous paper [J. Geophys. Res. 105 (2000) 767]. However, the application of the thermomagnetic criterion for pre-selection of the rock samples requires the undesirable heating of samples to T C. To avoid the heating, one can try to impart a pTRM AF( T 1, T r) from the AF-demagnetized state and use the tail of this pTRM as a more suitable test of DS. The advantage of this approach is that the measurement of the tail[pTRM AF( T 1, T r)] requires a preliminary heating of a sample to a moderate temperature T 1 only (say, T 1=300°C). It is shown that the tail[pTRM AF(300, T r)] is usually much shorter than that of pTRM a(300, T r) and of smaller magnitudes by a factor of about 3. Thus, this test is suitable mainly for the pre-selection of MD samples having relatively large tails of pTRM AF(300, T r). For PSD grains, the tails of pTRM AF(300, T r) are often too small to distinguish them from the single-domain (SD) ones. A detailed study of properties of different pTRMs created from AF-demagnetized, thermodemagnetized and zero-field cooled states has been undertaken. Physically the dependence of values of both pTRMs and their tails on the initial magnetic state of a sample is explained by the phenomenon of metastability.

Determination of Domain Structure of Proteins from X-Ray Solution Scattering

An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.

Determination of domain structure and abundance of epitaxial Pb(Zr, Ti)O3 thin films grown on MgO(001) by rf magnetron sputtering

Epitaxial Pb(ZrxTi1−x)O3 (x = 0.0−0.32) ferroelectric thin films of 500 nm thickness were grown on MgO(001) single crystal substrates by in situ rf magnetron sputtering, and evolution of their domain structures is characterized by employing various x-ray diffraction techniques. X-ray θ-2θ scan showed the films were grown highly c-axis oriented with a tetragonal perovskite structure. 90° domain configuration was investigated using the x-ray rocking curve analysis for PZT 100 peaks in two different Φphi; angles. The rocking curve analysis showed that the degree of c-axis orientation and the crystalline quality of the films were improved continuously with increasing Zr concentration. The c-domain abundance as a function of Zr concentration was quantified using the x-ray rocking curves of PZT 001 and 100, taking account of structural factors and Lorentz-polarization factors. High temperature x-ray technique was also employed to quantify the domain structure as a function of temperature during cooling after reheating the samples to 650 °C. During the cooling process, c-domain abundance was found to increase continuously while the crystalline quality of the films was deteriorated below the Curie temperature. The results led us to conclude that the transformation strain of the film at and below the Curie temperature plays a significant role in the final domain structure and abundance of epitaxial PZT thin films.

Wavelength dependent neutron depolarization in some metallic glasses

Ferromagnetic metallic glass ribbons Fe 34.6Ni 36.5Mo 6B 22.9, (Fe 50Ni 50) 94.9B 5.1, Fe 78Co 8.1B 13.9 and Fe 56.2Co 7Ni 15.6 Si 4B 17.2 were studied by magneto-optical methods (Kerr effect, magnetic colloid fluid technique) sensitive to the surface in combination with bulk domain structure determination by means of the neutron depolarization technique. The dependence of polarization of a polychromatic beam of polarized neutrons transmitted through the samples on the neutron wavelength was analysed by fitting to the data domain structure models consistent with the surface domain patterns; it enabled to estimate the mean domain size as well as the mean magnetic induction, its fluctuations, and orientation with respect to applied magnetic field.

Wavelength dependent neutron depolarization in soft magnetic amorphous ribbons

Results from bulk domain structure determination by analysing the wavelength dependent depolarization of a polychromatic beam of polarized neutrons transmitted through soft magnetic ribbons are presented and discussed. Information on mean magnetization, static magnetization fluctuations, and magnetic anisotropy was obtained for amorphous ferromagnetic ribbons of composition Fe78Co8.1B13.9 and Fe56.2Co7Ni15.6Si4B17.2 taken as examples. Under favourable circumstances even simple models of the domain structure are found satisfactory as in the case of carefully demagnetized samples. However, more complex modes are shown to be reasonable in describing the change in bulk magnetization state probed by the neutrons.

Determination of domain structure in igneous rocks by alternating field and other methods

AF (alternating field) demagnetization, ARM (anhysteretic remanent magnetization) and strong-field hysteresis properties of a large collection of mostly continental igneous rocks are reported here. The collection included rocks whose magnetic carriers were believed from previous work to be of one of three types: MD (multidomain); SD/PSD (single-domain/pseudo-single-domain); or a bimodal mixture of MD grains (e.g., discrete opaques) and SD/PSD material (e.g., silicate inclusions). Two series of subaerial basalts with a full range of deuteric oxidation classes included examples of all three classes of behaviour. SD/PSD rocks have relatively hard inflected AF decay curves (decay rate initially increasing, then decreasing), MD rocks have soft, exponential-like decay curves, and bimodal rocks have a combination of these characteristics. Relative hardnesses of normalized decay curves of remanences acquired in weak, intermediate and strong fields (the Lowrie-Fuller test) are also distinctively different for the three classes, and the results support the theory developed in an accompanying paper [1] that Lowrie-Fuller characteristics are an expression of the shapes of decay curves. The Lowrie-Fuller test, although its result can be expressed as a numerical parameter, is not capable of fine-scale classification of domain structure or grain size. The shape of the ARM induction curve does have a quasi-continuous variation with grain size, however. The parameter χ ar/J rs (initial anhysteretic susceptibility normalized to saturation remanence), which is easily measured with standard paleomagnetic instrumentation, is potentially useful for magnetic granulometry, although χ ar itself was not diagnostic of grain size.

Theoretical determination of domain structure at transitions from twinned phase : Application to the tetragonal-orthorhombic transition of KNb03

Abstract The theoretical determination of the initial domain structure and the phase boundary orientation inducing a wanted domain structure in the new phase seems to be a study of worthwhile interest. The W. L. R. theory {1} is not suitable for complex domain structure in each phase because mean distortions of the new phase cannot be expressed in terms of the older one. So, an other algorithm is proposed to solve this problem.