Cameron Mura

The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core

Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-Å crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP–RNA interactions.

The oligomerization and ligand‐binding properties of Sm‐like archaeal proteins (SmAPs)

Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand‐binding properties of Sm‐like archaeal proteins (SmAPs) from Pyrobaculum aerophilum (Pae) and Methanobacterium thermautotrophicum (Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide‐bonded 14‐mers or sub‐heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head‐to‐tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14‐mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 Å‐resolution structure of Mth SmAP1 bound to uridine‐5′‐monophosphate (UMP) reveals conserved ligand‐binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus (Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel‐shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single‐stranded nucleic acid‐binding activity.

Archaeal and eukaryotic homologs of Hfq A structural and evolutionary perspective on Sm function

Hfq and other Sm proteins are central in RNA metabolism, forming an evolutionarily conserved family that plays key roles in RNA processing in organisms ranging from archaea to bacteria to human. Sm-based cellular pathways vary in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in each of these pathways being mediated by an RNA-associated molecular assembly built upon Sm proteins. Though the first structures of Sm assemblies were from archaeal systems, the functions of Sm-like archaeal proteins (SmAPs) remain murky. Our ignorance about SmAP biology, particularly vis-à-vis the eukaryotic and bacterial Sm homologs, can be partly reduced by leveraging the homology between these lineages to make phylogenetic inferences about Sm functions in archaea. Nevertheless, whether SmAPs are more eukaryotic (RNP scaffold) or bacterial (RNA chaperone) in character remains unclear. Thus, the archaeal domain of life is a missing link, and an opportunity, in Sm-based RNA biology.

Structure and assembly of an augmented Sm-like archaeal protein 14-mer

To better understand the roles of Sm proteins in forming the cores of many RNA-processing ribonucleoproteins, we determined the crystal structure of an atypical Sm-like archaeal protein (SmAP3) in which the conserved Sm domain is augmented by a previously uncharacterized, mixed α/β C-terminal domain. The structure reveals an unexpected SmAP3 14-mer that is perforated by a cylindrical pore and is bound to 14 cadmium (Cd2+) ions. Individual heptamers adopt either “apical” or “equatorial” conformations that chelate Cd2+ differently. SmAP3 forms supraheptameric oligomers (SmAP3)n = 7,14,28 in solution, and assembly of the asymmetric 14-mer is modulated by differential divalent cation-binding in apical and equatorial subunits. Phylogenetic and sequence analyses substantiate SmAP3s as a unique subset of SmAPs. These results distinguish SmAP3s from other Sm proteins and provide a model for the structure and properties of Sm proteins >100 residues in length, e.g., several human Sm proteins.

An Introduction to Biomolecular Graphics

One need only compare the number of three-dimensional molecular illustrations in the first (1990) and third (2004) editions of Voet & Voet’s Biochemistry in order to appreciate this field’s profound communicative value in modern biological sciences – ranging from medicine, physiology, and cell biology, to pharmaceutical chemistry and drug design, to structural and computational biology. The cliché about a picture being worth a thousand words is quite poignant here: The information ‘content’ of an effectively-constructed piece of molecular graphics can be immense. Because biological function arises from structure, it is difficult to overemphasize the utility of visualization and graphics in molding our current understanding of the molecular nature of biological systems. Nevertheless, creating effective molecular graphics is not easy – neither conceptually, nor in terms of effort required. The present collection of Rules is meant as a guide for those embarking upon their first molecular illustrations; it most closely parallels the previous collections devoted to publishing papers [1], making oral presentations [2], and creating good posters [3].Biological function arises from detailed molecular structure, making it difficult to overemphasize the role of structural visualization and biomolecular graphics in shaping our current understanding of the molecular nature of biological systems. Indeed, one need only compare the number of three-dimensional (3D) structures illustrated in the first (1990) and fourth (2010) editions of Voet & Voets Biochemistry in order to appreciate the profound communicative value of molecular graphics in modern biosciences, ranging from medicine and physiology to drug design and computational biology. Faced with a deluge of structural genomics results over the past decade, the cliche about a picture being worth a thousand words is quite poignant: The information “content” of carefully constructed molecular graphics can be immense. Because computer-based molecular visualization (MolVis) is such an effective means for exploring and analyzing structural data, this guide introduces the science and art of biomolecular graphics, both in principle and as practiced in structural and computational biology. This guide is built around a series of practical case-studies, emphasizing the creation of biomolecular graphics for publication figures and animations. Intended primarily for those embarking upon their first illustrations, intermediate-level examples are also provided in order to facilitate the transition from novice user to advanced practitioner. For enhanced pedagogical value, the exact methods used to create each figure are provided to the reader in the form of heavily annotated computer scripts. Because the PyMOL [1] software package was used to create these illustrations, all materials (images, animations, scripts, etc.) have been made freely available as a dedicated section of the PyMOL wiki site (http://pymolwiki.org/PLoS). Additional background material on MolVis, including a detailed review of the underlying principles (Box 1), is provided as supporting information (Text S1). Further information can be found in the recent treatment by Bottomley and Helmerhorst [2], and in several reviews covering either small-molecule [3] or macromolecular visualization [4]–[6]. Box 1. MolVis Concepts and Terminology Raster, vector: Two different ways to structure images, either as combinations of simple geometric objects such as points, lines, curves (vector graphics), or as a discrete 2D array of colored pixels (raster/bitmap). Vector graphics are arbitrarily scalable, whereas the fixed array of pixels in a bitmap leads to graininess (“pixelization”) upon zooming-in of raster graphics; see Box S1 in Text S1 and ref. [2] for further information. Graphics primitives: Low-level geometric entities that are readily described in mathematical terms (lines, spheres, tetrahedra, etc.), and from which any complex shape, such as protein surfaces, can be constructed via solid geometry. Scenes are built from primitives, along with associated lighting, shading, and texturing properties; thus, primitives are how a scene is discretized for computer representation and manipulation. As an example, increasing PyMOLs “sphere_quality” beyond the default value of 1 yields smoother spheres (more triangles), while decreasing to 0 exposes the individual triangular primitives used to render spheres. Scene geometry, matrices: Several matrices are used to transform a molecular scene (atomic coordinate-based) into the image (pixel-based) shown on the actual 2D display. Along with all the primitives that represent molecular properties (atoms, bonds, surfaces, etc.), many other scene data must also be carried through these transforms, including materials, colors, lighting, shading, clipping, and depth (z) buffer data—in other words, all the attributes that define a scene. In being mapped onto the viewing plane, a scene can be rendered in either a perspective (skewed viewing matrix) or orthoscopic (orthonormal viewing matrix) projection mode; the PyMOL settings “orthoscopic” and “field_of_view” adjust this behavior, and the viewing matrix can be retrieved/modified via the “get_view” / “set_view” pair of commands. Clipping planes: The boundaries of a scene define a rectangular pyramid, with an apex at the camera(/eye), and the faces defined by top/bottom and right/left pairs of planes. In addition, far/near clipping planes can be defined behind/in front of a region of interest in this rectangular pyramid. Clipping plane geometry and behavior is adjustable; for instance, the PyMOL command “clip slab, 20” sets the slab thickness to 20 A. Ray tracing: A method to render photorealistic images by simulating the path of light rays through a scene, incorporating effects such as light sources, opacity, textures, atmospheric fog, and shading models. Ray tracing is computationally expensive for complex scenes, and more “realistic” (higher resolution) images require a greater density of light rays per pixel of the final image. Keyframes: Reference markers, either in time (animations) or in space (interpolations), that serve as the end-points that bracket an interpolation stage. For instance, in a sequence of frames consisting of structural snapshots S1→···→S2 ······ Sn, S1 and S2 define the first pair of keyframes. Linearly interpolating the gaps between S1 and S2 is essentially a form of data-smoothening. Most movie-making functionalities incorporate the keyframe concept. Anti-aliasing: A feature/setting in most MolVis programs (“antialias” in PyMOL) that greatly improves image quality by diminishing the jagged distortions (“aliasing”) of curves and diagonal lines that compose the geometric primitives of a scene.

Interplay of the Bacterial Ribosomal A-Site, S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

The conformational properties of the aminoacyl-tRNA binding site (A-site), and its surroundings in the Escherichia coli 30S ribosomal subunit, are of great relevance in designing antibacterial agents. The 30S subunit A-site is near ribosomal protein S12, which neighbors helices h27 and H69; this latter helix, of the 50S subunit, is a functionally important component of an intersubunit bridge. Experimental work has shown that specific point mutations in S12 (K42A, R53A) yield hyper-accurate ribosomes, which in turn confers resistance to the antibiotic ‘paromomycin’ (even when this aminoglycoside is bound to the A-site). Suspecting that these effects can be elucidated in terms of the local atomic interactions and detailed dynamics of this region of the bacterial ribosome, we have used molecular dynamics simulations to explore the motion of a fragment of the E. coli ribosome, including the A-site. We found that the ribosomal regions surrounding the A-site modify the conformational space of the flexible A-site adenines 1492/93. Specifically, we found that A-site mobility is affected by stacking interactions between adenines A1493 and A1913, and by contacts between A1492 and a flexible side-chain (K43) from the S12 protein. In addition, our simulations reveal possible indirect pathways by which the R53A and K42A mutations in S12 are coupled to the dynamical properties of the A-site. Our work extends what is known about the atomistic dynamics of the A-site, and suggests possible links between the biological effects of hyper-accurate mutations in the S12 protein and conformational properties of the ribosome; the implications for S12 dynamics help elucidate how the miscoding effects of paromomycin may be evaded in antibiotic-resistant mutants of the bacterial ribosome.

RapA, the SWI/SNF subunit of Escherichia coli RNA polymerase, promotes the release of nascent RNA from transcription complexes.

RapA, a prokaryotic member of the SWI/SNF protein superfamily, is an integral part of the RNA polymerase transcription complex. RapAs function and catalytic mechanism have been linked to nucleic acid remodeling. In this work, we show that mutations in the interface between RapAs SWI/SNF and double-stranded nucleic acid-binding domains significantly alter ATP hydrolysis in purified RapA. The effects of individual mutations on ATP hydrolysis loosely correlated with RapAs nucleic acid remodeling activity, indicating that the interaction between these domains may be important for the RapA-mediated remodeling of nonproductive transcription complexes. In this study, we introduced a model system for in vitro transcription of a full-length Escherichia coli gene (slyD). To study the function of RapA, we fractionated and identified in vitro transcription reaction intermediates in the presence or absence of RapA. These experiments demonstrated that RapA contributes to the formation of free RNA species during in vitro transcription. This work further refines our models for RapA function in vivo and establishes a new role in RNA management for a representative of the SWI/SNF protein superfamily.

Abstractions, algorithms and data structures for structural bioinformatics in PyCogent

To facilitate flexible and efficient structural bioinformatics analyses, new functionality for three-dimensional structure processing and analysis has been introduced into PyCogent - a popular feature-rich framework for sequence-based bioinformatics, but one which has lacked equally powerful tools for handling stuctural/coordinate-based data. Extensible Python modules have been developed, which provide object-oriented abstractions (based on a hierarchical representation of macromolecules), efficient data structures (e.g.kD-trees), fast implementations of common algorithms (e.g. surface-area calculations), read/write support for Protein Data Bank-related file formats and wrappers for external command-line applications (e.g. Stride). Integration of this code into PyCogent is symbiotic, allowing sequence-based work to benefit from structure-derived data and, reciprocally, enabling structural studies to leverage PyCogents versatile tools for phylogenetic and evolutionary analyses.