The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated

Ab initio compressive phase retrieval

Any object on earth has two fundamental properties: it is finite, and it is made of atoms. Structural information about an object can be obtained from diffraction amplitude measurements that account for either one of these traits. Nyquist-sampling of the Fourier amplitudes is sufficient to image single particles of finite size at any resolution. Atomic resolution data is routinely used to image molecules replicated in a crystal structure. Here we report an algorithm that requires neither information, but uses the fact that an image of a natural object is compressible. Intended applications include tomographic diffractive imaging, crystallography, powder diffraction, small angle x-ray scattering and random Fourier amplitude measurements.Comment: 7 pages, 4 figures, presented at the XXI IUCr Congress, Aug. 2008, Osaka Japa

Ab-initio phasing using nanocrystal shape transforms with incomplete unit cells

abstract: X-ray free electron lasers are used in measuring diffraction patterns from nanocrystals in the 'diffract-before-destroy' mode by outrunning radiation damage. The finite-sized nanocrystals provide an opportunity to recover intensity between Bragg spots by removing the modulating function that depends on crystal shape, i.e. the transform of the crystal shape. This shape-transform dividing-out scheme for solving the phase problem has been tested using simulated examples with cubic crystals. It provides a phasing method which does not require atomic resolution data, chemical modification to the sample, or modelling based on the protein databases. It is common to find multiple structural units (e.g. molecules, in symmetry-related positions) within a single unit cell, therefore incomplete unit cells (e.g. one additional molecule) can be observed at surface layers of crystals. In this work, the effects of such incomplete unit cells on the 'dividing-out' phasing algorithm are investigated using 2D crystals within the projection approximation. It is found that the incomplete unit cells do not hinder the recovery of the scattering pattern from a single unit cell (after dividing out the shape transforms from data merged from many nanocrystals of different sizes), assuming that certain unit-cell types are preferred. The results also suggest that the dynamic range of the data is a critical issue to be resolved in order to apply the shape transform method practically

Structural investigation of the molecular mechanisms underlying titin elasticity and signaling

Titin is a giant protein that spans >1µm from the Z-disc to the M-line, forming an intrasarcomeric filament system in vertebrate striated muscle, which is not only essential for the assembly of the sarcomere, but also critical for myofibril signaling and metabolism. Furthermore, it provides the sarcomere with resting tension, elasticity and restoring forces upon stretch, ensuring the correct positioning of the actin-myosin motors during muscle function. Titin is composed of ~300 immunoglobulin (Ig) and fibronectin-III (FnIII) domains, arranged in linear tandems. They are interspersed by an auto-inhibited Ser kinase (TK) close to its C-terminus as well as several unique sequences, most prominently a differentially spliced stretch rich in PEVK residues which localizes to the I-band part of titin where its elastic properties reside. There, the PEVK segment is flanked by a long Ig tandem, which together act as serial molecular springs that determine titin elastic response. The focus of this work lay in the elucidation of the molecular mechanisms governing titin I-band elasticity and the recruitment of the M-line signalosome around TK involved in the control of myofibril turnover and the trophic state of muscle. To that effect, we have elucidated the crystal structure of a six-Ig fragment representative of the elastic Ig-tandem at 3.3Å resolution. The model reveals the molecular principles of Ig-arraying at the skeletal I-band of titin as mediated by conserved Ig-Ig transition motifs. Regular domain arrangements within this fragment point at the existence of a high-order in the fine structure of the filament, which is confirmed by EM data on a 19-mer poly-Ig segment. Our findings indicate a long-range, supra-order in the skeletal I-band of titin, where assembly of Ig domains into dynamical super-motifs is essential for the elastic function of the filament. We propose a novel model of spring mechanism for poly-Ig elasticity in titin based on a “carpenter ruler” model of skeletal I-band architecture. Furthermore, we have focused on the recruitment of the ubiquitin ligase MURF1 to the M-line signalosome through its specific interaction with titin domains A168 A170. MuRF1 contains several oligomerization motifs in succession, which indicates a possible need for tight regulation. We have therefore analyzed their influence on the oligomeric state of the protein. Our SEC-MALS data showed that the a-helical region of MuRF1 is dimeric in isolation, while in combination with the preceding B-Box domain, itself a dimerization motif, higher-order assembly is induced, which might be of physiological importance. We could also show that higher-order assembly of MuRF1 did not disrupt binding to A168-A170 in pull-down assays. Further biophysical or structural characterization of the complex of A168-A170 with MuRF1 constructs was hindered by the severely compromised solubility of the complex. Finally, we have successfully solved the crystal structure of the FnIII-Kin-Ig region of twitchin, which corresponds to titin A170-TK-M1. The N-terminal linker wraps around the kinase domain and positions the preceding FnIII domain in such a way that it blocks the autoregulatory tail in its inhibitory positon. Thus, from the structure we could conclude that stretch-activation of Twc kinase seems unlikely and instead propose phosphorylation of Y 104 as a possible activation mechanism. Our findings illustrate how the structural and functional diversity in titin’s modular architecture has evolved not only on the basis of individual domains. Rather, functionality often involves adaptation of several neighboring domains or even whole Ig tandems/super-repeats. This is reflected in variations in mechanical and dynamic properties observed in different parts of the chain and highlights the necessity of working with representative multi-domain fragments to gain a comprehensive understanding of the titin chai

Initiating Heavy-atom Based Phasing by Multi-Dimensional Molecular Replacement

To obtain an electron-density map from a macromolecular crystal the phase-problem needs to be solved, which often involves the use of heavy-atom derivative crystals and concomitantly the determination of the heavy atom substructure. This is customarily done by direct methods or Patterson-based approaches, which however may fail when only poorly diffracting derivative crystals are available, as often the case for e.g. membrane proteins. Here we present an approach for heavy atom site identification based on a Molecular Replacement Parameter Matrix (MRPM) search. It involves an n-dimensional search to test a wide spectrum of molecular replacement parameters, such as clusters of different conformations. The result is scored by the ability to identify heavy-atom positions, from anomalous difference Fourier maps, that allow meaningful phases to be determined. The strategy was successfully applied in the determination of a membrane protein structure, the CopA Cu+-ATPase, when other methods had failed to resolve the heavy atom substructure. MRPM is particularly suited for proteins undergoing large conformational changes where multiple search models should be generated, and it enables the identification of weak but correct molecular replacement solutions with maximum contrast to prime experimental phasing efforts.Comment: 19 pages total, main paper: 6 pages (2 figures), supplementary material: 13 pages (2 figures, 9 tabels

FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution

Development of crystallographic methods for phasing highly modulated macromolecular structures

[eng] Pathologies that result in highly modulated intensities in macromolecular crystal structures pose a challenge for structure solution. To address this issue two studies have been performed: a theoretical study of one of these pathologies, translational non- crystallographic symmetry (tNCS), and a practical study of paradigms of highly modulated macromolecular structures, coiled-coils. tNCS is a structural situation in which multiple, independent copies of a molecular assembly are found in similar orientations in the crystallographic asymmetric unit. Structure solution is problematic because the intensity modulations caused by tNCS cause the intensity distribution to differ from a Wilson distribution. If the tNCS is properly detected and characterized, expected intensity factors for each reflection that model the modulations observed in the data can be refined against a likelihood function to account for the statistical effects of tNCS. In this study, a curated database of 80482 protein structures from the PDB was analysed to investigate how tNCS manifests in the Patterson function. These studies informed the algorithm for detection of tNCS, which includes a method for detecting the tNCS order in any commensurate modulation. In the context of automated structure solution pipelines, the algorithm generates a ranked list of possible tNCS associations in the asymmetric unit, which can be explored to efficiently maximize the probability of structure solution. Coiled-coils are ubiquitous protein folding motifs present in a wide range of proteins that consist of two or more α-helices wrapped around each other to form a supercoil. Despite the apparent simplicity of their architecture, solution by molecular replacement is challenging due to the helical irregularities found in these domains, tendency to form fibers, large dimensions in their typically anisometric asymmetric units, low-resolution and anisotropic diffraction. In addition, the internal symmetry of the helices and their alignment in preferential directions gives rise to systematic overlap of Patterson vectors, a Patterson map that indicates tNCS is present, and intensity modulations similar to those in true tNCS. In this study, we have explored fragment phasing on a pool of 150 coiled-coils with ARCIMBOLDO_LITE, an ab initio phasing approach that combines fragment location with Phaser and density modification and autotracing with SHELXE. The results have been used to identify limits and bottlenecks in coiled-coil phasing that have been addressed in a specific mode for solving coiled-coils, allowing the solution of 95% of the test set and four previously unknown structures, and extending the resolution limit from 2.5 Å to 3.0 Å

Aspects of phase retrieval in x-ray crystallography

X-ray crystallography is the primary technique for imaging the structures, or the positions of the atoms, of molecules. Knowledge of the geometrical atomic structures of molecules is key information in physics, chemistry, biology, geology and many other areas of science and technology. Structures are determinants of the properties of molecular systems. In the case of biology, knowledge of the structures of biological molecules provides essential information that allows us to understand the biological functionality of biomolecules and biomolecular systems. This knowledge is used to understand the fundamental molecular basis of biological function and processes, disease processes, and is also important in rational, or structure-based, drug design. X-ray crystallography involves irradiating a crystal specimen of the molecule under study with a beam of X-rays, and measuring the resulting pattern of diffracted X-rays. The data consisting of measured diffraction patterns is then inverted computationally to produce an image of the molecule. This is often referred to as computational imaging or computational microscopy. If both the phase and amplitude of the diffracted X- ray could be measured, then inversion of the data to produce the image would be straightforward. However, in practice, one can measure only the amplitude, but not the phase, of the diffracted X-rays. This results in the famous so-called “phase problem” in crystallography. A method of determining the phases must be devised before the structure can be calculated. The phase problem in crystallography has been studied for over one hundred years, and a number of clever methods have been devised for determining the phases in order for structures to be calculated. However, the phase problem is still an active area of research as current phasing techniques have significant limitations, and also because of the emergence of new kinds of instrumentation, specimens, and diffraction experiments. This thesis is concerned with the phase problem and phase retrieval algorithms for biological (macromolecular) crystallography that have arisen, in part, through the recent introduction of a new kind of X-ray source called an X-ray free-electron laser, and through new kinds of specimens that can be used with these sources. The thesis is divided into six chapters. The first chapter provides background information on diffraction imaging, X-ray crystallography, the phase problem, phase retrieval algorithms, and X-ray free-electron lasers and serial femtosecond crystallography. Original material is contained in Chapters 2 through 5. Concluding remarks are made in Chapter 6. Chapter 2 is concerned with properties of the phase problem for 3D crystals. New relationships are derived that more carefully formalise uniqueness for this problem, the problem for the case of an unknown molecular support is studied in detail and the theoretical results are supported by simulations, and the effects of crystallographic and noncrystallographic symmetry are elucidated. Chapters 3 and 4 form the first main part of the thesis and consider the phase problem for 2D crystals, a new kind of specimen that has been investigated with X-ray free-electron lasers. The two chapters are presented as two published journal papers for which the candidate is the primary author. In Chapter 3, the fundamental uniqueness properties of the phase problem for 2D crystals are derived, the nature of the solution set is elucidated, and the effects of various kinds of a priori information are evaluated by simulation. Chapter 4 follows up the results in Chapter 3, using simulations to investigate practical aspects of ab initio phase retrieval for 2D crystals using minimal molecular envelope information, and considering the characteristics of data available from X-ray free-electron laser sources. Chapter 5 forms the second main part of the thesis and develops a new kind of ab initio phasing technique called ab initio molecular replacement phasing. This method uses diffraction data from the same molecule crystallised in two or more crystal forms. Uniqueness of the solution for such a dataset is evaluated, and a suitable phase re- trieval algorithm is developed and tested by simulation using a small protein of known structure. Chapter 6 contains a brief summary of the outcomes of the thesis and suggestions for future research

Phasing Two-Dimensional Crystal Diffraction Pattern with Iterative Projection Algorithms

abstract: Phase problem has been long-standing in x-ray diffractive imaging. It is originated from the fact that only the amplitude of the scattered wave can be recorded by the detector, losing the phase information. The measurement of amplitude alone is insufficient to solve the structure. Therefore, phase retrieval is essential to structure determination with X-ray diffractive imaging. So far, many experimental as well as algorithmic approaches have been developed to address the phase problem. The experimental phasing methods, such as MAD, SAD etc, exploit the phase relation in vector space. They usually demand a lot of efforts to prepare the samples and require much more data. On the other hand, iterative phasing algorithms make use of the prior knowledge and various constraints in real and reciprocal space. In this thesis, new approaches to the problem of direct digital phasing of X-ray diffraction patterns from two-dimensional organic crystals were presented. The phase problem for Bragg diffraction from two-dimensional (2D) crystalline monolayer in transmission may be solved by imposing a compact support that sets the density to zero outside the monolayer. By iterating between the measured stucture factor magnitudes along reciprocal space rods (starting with random phases) and a density of the correct sign, the complex scattered amplitudes may be found (J. Struct Biol 144, 209 (2003)). However this one-dimensional support function fails to link the rod phases correctly unless a low-resolution real-space map is also available. Minimum prior information required for successful three-dimensional (3D) structure retrieval from a 2D crystal XFEL diffraction dataset were investigated, when using the HIO algorithm. This method provides an alternative way to phase 2D crystal dataset, with less dependence on the high quality model used in the molecular replacement method.Dissertation/ThesisDoctoral Dissertation Physics 201