Jeremy Phillips

Structure–function mapping of a heptameric module in the nuclear pore complex

Integration of EM, protein–protein interaction, and phenotypic data reveals novel insights into the structure and function of the nuclear pore complex’s ∼600-kD heptameric Nup84 complex.

Integrative Structure Modeling of Macromolecular Assemblies from Proteomics Data

Proteomics techniques have been used to generate comprehensive lists of protein interactions in a number of species. However, relatively little is known about how these interactions result in functional multiprotein complexes. This gap can be bridged by combining data from proteomics experiments with data from established structure determination techniques. Correspondingly, integrative computational methods are being developed to provide descriptions of protein complexes at varying levels of accuracy and resolution, ranging from complex compositions to detailed atomic structures.

The structural dynamics of macromolecular processes

Dynamic processes involving macromolecular complexes are essential to cell function. These processes take place over a wide variety of length scales from nanometers to micrometers, and over time scales from nanoseconds to minutes. As a result, information from a variety of different experimental and computational approaches is required. We review the relevant sources of information and introduce a framework for integrating the data to produce representations of dynamic processes.

Assembly of macromolecular complexes by satisfaction of spatial restraints from electron microscopy images

To obtain a structural model of a macromolecular assembly by single-particle EM, a large number of particle images need to be collected, aligned, clustered, averaged, and finally assembled via reconstruction into a 3D density map. This process is limited by the number and quality of the particle images, the accuracy of the initial model, and the compositional and conformational heterogeneity. Here, we describe a structure determination method that avoids the reconstruction procedure. The atomic structures of the individual complex components are assembled by optimizing a match against 2D EM class-average images, an excluded volume criterion, geometric complementarity, and optional restraints from proteomics and chemical cross-linking experiments. The optimization relies on a simulated annealing Monte Carlo search and a divide-and-conquer message-passing algorithm. Using simulated and experimentally determined EM class averages for 12 and 4 protein assemblies, respectively, we show that a few class averages can indeed result in accurate models for complexes of as many as five subunits. Thus, integrative structural biology can now benefit from the relative ease with which the EM class averages are determined.

Structure of the C-terminal domain of Saccharomyces cerevisiae Nup133, a component of the nuclear pore complex

Nuclear pore complexes (NPCs), responsible for the nucleo-cytoplasmic exchange of proteins and nucleic acids, are dynamic macromolecular assemblies forming an eight-fold symmetric co-axial ring structure. Yeast (Saccharomyces cerevisiae) NPCs are made up of at least 456 polypeptide chains of {approx}30 distinct sequences. Many of these components (nucleoporins, Nups) share similar structural motifs and form stable subcomplexes. We have determined a high-resolution crystal structure of the C-terminal domain of yeast Nup133 (ScNup133), a component of the heptameric Nup84 subcomplex. Expression tests yielded ScNup133(944-1157) that produced crystals diffracting to 1.9{angstrom} resolution. ScNup133(944-1157) adopts essentially an all {alpha}-helical fold, with a short two stranded {beta}-sheet at the C-terminus. The 11 {alpha}-helices of ScNup133(944-1157) form a compact fold. In contrast, the previously determined structure of human Nup133(934-1156) bound to a fragment of human Nup107 has its constituent {alpha}-helices are arranged in two globular blocks. These differences may reflect structural divergence among homologous nucleoporins.

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ∼456 polypeptide chains contributed by ∼30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N‐terminal “FG” repeats containing a Gle2p‐binding sequence motif and a NPC targeting domain at its C‐terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882–1034 [CgNup116(882–1034)], at 1.94 Å resolution. The X‐ray structure of CgNup116(882–1034) is consistent with the molecular envelope determined in solution by small‐angle X‐ray scattering. Structural similarities of CgNup116(882–1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed. Proteins 2012;

Structure, Dynamics, Evolution and Function of a Major Scaffold Component in the Nuclear Pore Complex

The Nuclear Pore Complex, composed of proteins termed Nucleoporins (Nups), is responsible for the nucleo-cytoplasmic transport in eukaryotes. NPCs form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings.Nup192 is a major component of the NPCs inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2 to 960 [ScNup192(2-960)], which adopts an α-helical fold with three domains (i.e., D1, D2 and D3). SAXS and EM studies reveal that ScNup192(2-960) could undergo long-range transition between an “open” and “closed” conformations. We obtained a structural model of full-length ScNup192 based on EM, structure of ScNup192(2-960), and homology modeling.Evolutionary analyses using ScNup192(2-960) structure suggest that NPCs and vesicle coating complexes are descended from a common membrane-coating ancestral complex.We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.