Frank Alber

The molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC’s 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of ‘columns’. These findings provide clues to the evolutionary origins of the NPC.

Determining the architectures of macromolecular assemblies

To understand the workings of a living cell, we need to know the architectures of its macromolecular assemblies. Here we show how proteomic data can be used to determine such structures. The process involves the collection of sufficient and diverse high-quality data, translation of these data into spatial restraints, and an optimization that uses the restraints to generate an ensemble of structures consistent with the data. Analysis of the ensemble produces a detailed architectural map of the assembly. We developed our approach on a challenging model system, the nuclear pore complex (NPC). The NPC acts as a dynamic barrier, controlling access to and from the nucleus, and in yeast is a 50 MDa assembly of 456 proteins. The resulting structure, presented in an accompanying paper, reveals the configuration of the proteins in the NPC, providing insights into its evolution and architectural principles. The present approach should be applicable to many other macromolecular assemblies.

Genome architectures revealed by tethered chromosome conformation capture and population-based modeling

We describe tethered conformation capture (TCC), a method for genome-wide mapping of chromatin interactions. By performing ligations on solid substrates rather than in solution, TCC substantially enhances the signal-to-noise ratio, thereby facilitating a detailed analysis of interactions within and between chromosomes. We identified a group of regions in each chromosome in human cells that account for the majority of interchromosomal interactions. These regions are marked by high transcriptional activity, suggesting that their interactions are mediated by transcriptional machinery. Each of these regions interacts with numerous other such regions throughout the genome in an indiscriminate fashion, partly driven by the accessibility of the partners. As a different combination of interactions is likely present in different cells, we developed a computational method to translate the TCC data into physical chromatin contacts in a population of three-dimensional genome structures. Statistical analysis of the resulting population demonstrates that the indiscriminate properties of interchromosomal interactions are consistent with the well-known architectural features of the human genome.

Components of Coated Vesicles and Nuclear Pore Complexes Share a Common Molecular Architecture

Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107–160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a β-propeller fold, an α-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107–160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes.

Integrating diverse data for structure determination of macromolecular assemblies.

To understand the cell, we need to determine the macromolecular assembly structures, which may consist of tens to hundreds of components. First, we review the varied experimental data that characterize the assemblies at several levels of resolution. We then describe computational methods for generating the structures using these data. To maximize completeness, resolution, accuracy, precision, and efficiency of the structure determination, a computational approach is required that uses spatial information from a variety of experimental methods. We propose such an approach, defined by its three main components: a hierarchical representation of the assembly, a scoring function consisting of spatial restraints derived from experimental data, and an optimization method that generates structures consistent with the data. This approach is illustrated by determining the configuration of the 456 proteins in the nuclear pore complex (NPC) from bakers yeast. With these tools, we are poised to integrate structural information gathered at multiple levels of the biological hierarchy--from atoms to cells--into a common framework.

Physical tethering and volume exclusion determine higher-order genome organization in budding yeast

In this paper we show that tethering of heterochromatic regions to nuclear landmarks and random encounters of chromosomes in the confined nuclear volume are sufficient to explain the higher-order organization of the budding yeast genome. We have quantitatively characterized the contact patterns and nuclear territories that emerge when chromosomes are allowed to behave as constrained but otherwise randomly configured flexible polymer chains in the nucleus. Remarkably, this constrained random encounter model explains in a statistical manner the experimental hallmarks of the S. cerevisiae genome organization, including (1) the folding patterns of individual chromosomes; (2) the highly enriched interactions between specific chromatin regions and chromosomes; (3) the emergence, shape, and position of gene territories; (4) the mean distances between pairs of telomeres; and (5) even the co-location of functionally related gene loci, including early replication start sites and tRNA genes. Therefore, most aspects of the yeast genome organization can be explained without calling on biochemically mediated chromatin interactions. Such interactions may modulate the pre-existing propensity for co-localization but seem not to be the cause for the observed higher-order organization. The fact that geometrical constraints alone yield a highly organized genome structure, on which different functional elements are specifically distributed, has strong implications for the folding principles of the genome and the evolution of its function.

Substrate Diversity of Herpes Simplex Virus Thymidine Kinase IMPACT OF THE KINEMATICS OF THE ENZYME

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.

An analysis of the helix-to-strand transition between peptides with identical sequence

An analysis of peptide segments with identical sequence but that differ significantly in structure was performed over non‐redundant databases of protein structures. We focus on those peptides, which fold into an α‐helix in one protein but a β‐strand in another. While the study shows that many such structurally ambivalent peptides contain amino acids with a strong helical preference collocated with amino acids with a strong strand preference, the results overwhelmingly indicate that the peptides environment ultimately dictates its structure. Furthermore, the first naturally occurring structurally ambivalent nonapeptide from evolutionary unrelated proteins is described, highlighting the intrinsic plasticity of peptide sequences. We even find seven proteins that show structural ambivalence under different conditions. Finally, a computer algorithm has been implemented to identify regions in a given sequence where secondary structure prediction programs are likely to make serious mispredictions. Proteins 2000;41:248–256.

Structural modeling of protein interactions by analogy: application to PSD-95.

We describe comparative patch analysis for modeling the structures of multidomain proteins and protein complexes, and apply it to the PSD-95 protein. Comparative patch analysis is a hybrid of comparative modeling based on a template complex and protein docking, with a greater applicability than comparative modeling and a higher accuracy than docking. It relies on structurally defined interactions of each of the complex components, or their homologs, with any other protein, irrespective of its fold. For each component, its known binding modes with other proteins of any fold are collected and expanded by the known binding modes of its homologs. These modes are then used to restrain conventional molecular docking, resulting in a set of binary domain complexes that are subsequently ranked by geometric complementarity and a statistical potential. The method is evaluated by predicting 20 binary complexes of known structure. It is able to correctly identify the binding mode in 70% of the benchmark complexes compared with 30% for protein docking. We applied comparative patch analysis to model the complex of the third PSD-95, DLG, and ZO-1 (PDZ) domain and the SH3-GK domains in the PSD-95 protein, whose structure is unknown. In the first predicted configuration of the domains, PDZ interacts with SH3, leaving both the GMP-binding site of guanylate kinase (GK) and the C-terminus binding cleft of PDZ accessible, while in the second configuration PDZ interacts with GK, burying both binding sites. We suggest that the two alternate configurations correspond to the different functional forms of PSD-95 and provide a possible structural description for the experimentally observed cooperative folding transitions in PSD-95 and its homologs. More generally, we expect that comparative patch analysis will provide useful spatial restraints for the structural characterization of an increasing number of binary and higher-order protein complexes.

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization

Significance We provide a method for population-based structure modeling of whole diploid genomes using Hi-C data. The method considers the stochastic nature of chromosome structures, which allows a detailed analysis of the dynamic landscape of genome organizations. We predict and experimentally validate the presence of chromosome-specific higher-order centromere clusters, which can play a key role in the spatial organization of the human genome, specifically influencing the overall chromosome positioning, as well as the preference of specific chromosome conformations. Our approach generate predictive structural models of diploid genomes from Hi-C data, which can provide insights into the guiding principles of 3D genome organizations. Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.