Protein Complexes in Bacteria

Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies.     

Septins: the fourth component of the cytoskeleton

Septins belong to a family of proteins that is highly conserved in eukaryotes and is increasingly recognized as a novel component of the cytoskeleton. All septins are GTP-binding proteins that form hetero-oligomeric complexes and higher-order structures, including filaments and rings. Recent studies have provided structural information about the different levels of septin organization; however, the crucial structural determinants and factors responsible for septin assembly remain unclear. Investigations on the molecular functions of septins have highlighted their roles as scaffolds for protein recruitment and as diffusion barriers for subcellular compartmentalization in numerous biological processes, including cell division and host-microorganism interactions.     

Mammalian tight junctions in the regulation of epithelial differentiation and proliferation

Tight junctions are important for the permeability properties of epithelial and endothelial barriers as they restrict diffusion along the paracellular space. Recent observations have revealed that tight junctions also function in the regulation of epithelial proliferation and differentiation. They harbour evolutionarily conserved protein complexes that regulate polarisation and junction assembly. Tight junctions also recruit signalling proteins that participate in the regulation of cell proliferation and differentiation. These signalling proteins include components that affect established signalling cascades and dual localisation proteins that can associate with junctions as well as travel to the nucleus where they regulate gene expression.     

Identification of protein complexes by comparative analysis of yeast and bacterial protein interaction data.

Mounting evidence shows that many protein complexes are conserved in evolution. Here we use conservation to find complexes that are common to the yeast S. cerevisiae and the bacteria H. pylori. Our analysis combines protein interaction data that are available for each of the two species and orthology information based on protein sequence comparison. We develop a detailed probabilistic model for protein complexes in a single species and a model for the conservation of complexes between two species. Using these models, one can recast the question of finding conserved complexes as a problem of searching for heavy subgraphs in an edge- and node-weighted graph, whose nodes are orthologous protein pairs. We tested this approach on the data currently available for yeast and bacteria and detected 11 significantly conserved complexes. Several of these complexes match very well with prior experimental knowledge on complexes in yeast only and serve for validation of our methodology. The complexes suggest new functions for a variety of uncharacterized proteins. By identifying a conserved complex whose yeast proteins function predominantly in the nuclear pore complex, we propose that the corresponding bacterial proteins function as a coherent cellular membrane transport system. We also compare our results to two alternative methods for detecting complexes and demonstrate that our methodology obtains a much higher specificity.     

Identification of conserved protein complexes based on a model of protein network evolution

MOTIVATION: Data on protein-protein interactions (PPIs) are increasing exponentially. To date, large-scale protein interaction networks are available for human and most model species. The arising challenge is to organize these networks into models of cellular machinery. As in other biological domains, a comparative approach provides a powerful basis for addressing this challenge. RESULTS: We develop a probabilistic model for protein complexes that are conserved across two species. The model describes the evolution of conserved protein complexes from an ancestral species by protein interaction attachment and detachment and gene duplication events. We apply our model to search for conserved protein complexes within the PPI networks of yeast and fly, which are the largest networks in public databases. We detect 150 conserved complexes that match well-known complexes in yeast and are coherent in their functional annotations both in yeast and in fly. In comparison with two previous approaches, our model yields higher specificity and sensitivity levels in protein complex detection. AVAILABILITY: The program is available upon request.     

Conserved positions for ribose recognition: importance of water bridging interactions among ATP,ADP and FAD-protein complexes

Analysis of the spatial arrangement of protein and water atoms that form polar interactions with ribose has been performed for a structurally non-redundant dataset of ATP, ADP and FAD-protein complexes. The 26 ligand-protein structures were separated into two groups corresponding to the most populated furanose ring conformations (N and S-domains). Four conserved positions were found for S-domain protein-ligand complexes and five for N-domain complexes. Multiple protein folds and secondary structural elements were represented at a single conserved position. The following novel points were revealed: (i) Two complementary positions sometimes combine to describe a putative atomic spatial location for a specific conserved binding spot. (ii) More than one third of the interactions scored were water-mediated. Thus, conserved spatial positions rich in water atoms are a significant feature of ribose-protein complexes.     

Replication initiation and DNA topology: The twisted life of the origin

Genomic propagation in both prokaryotes and eukaryotes is tightly regulated at the level of initiation, ensuring that the genome is accurately replicated and equally segregated to the daughter cells. Even though replication origins and the proteins that bind onto them (initiator proteins) have diverged throughout the course of evolution, the mechanism of initiation has been conserved, consisting of origin recognition, multi‐protein complex assembly, helicase activation and loading of the replicative machinery. Recruitment of the multiprotein initiation complexes onto the replication origins is constrained by the dense packing of the DNA within the nucleus and unusual structures such as knots and supercoils. In this review, we focus on the DNA topological barriers that the multi‐protein complexes have to overcome in order to access the replication origins and how the topological state of the origins changes during origin firing. Recent advances in the available methodologies to study DNA topology and their clinical significance are also discussed. J. Cell. Biochem. 110: 35–43, 2010. © 2010 Wiley‐Liss, Inc.     

A quantitative analysis of interfacial amino acid conservation in protein-protein hetero complexes

A long-standing question in molecular biology is whether interfaces of protein-protein complexes are more conserved than the rest of the protein surfaces. Although it has been reported that conservation can be used as an indicator for predicting interaction sites on proteins, there are recent reports stating that the interface regions are only slightly more conserved than the rest of the protein surfaces, with conservation signals not being statistically significant enough for predicting protein-protein binding sites. In order to properly address these controversial reports we have studied a set of 28 well resolved hetero complex structures of proteins that consists of transient and non-transient complexes. The surface positions were classified into four conservation classes and the conservation index of the surface positions was quantitatively analyzed. The results indicate that the surface density of highly conserved positions is significantly higher in the protein-protein interface regions compared with the other regions of the protein surface. However, the average conservation index of the patches in the interface region is not significantly higher compared with other surface regions of the protein structures. This finding demonstrates that the number of conserved residue positions is a more appropriate indicator for predicting protein-protein binding sites than the average conservation index in the interacting region. We have further validated our findings on a set of 59 benchmark complex structures. Furthermore, an analysis of 19 complexes of antigen-antibody interactions shows that there is no conservation of amino acid positions in the interacting regions of these complexes, as expected, with the variable region of the immunoglobulins interacting mostly with the antigens. Interestingly, antigen interacting regions also have a higher number of non-conserved residue positions in the interacting region than the rest of the protein surface.     

YidC--an evolutionary conserved device for the assembly of energy-transducing membrane protein complexes

Members of the YidC/Oxa1/Alb3 membrane protein family are multifunctional mediators of membrane protein integration, folding and assembly into large complexes. Their evolutionary conserved and physiologically important role appears to relate to the assembly of major energy-transducing membrane protein complexes.     

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.     

Single-molecule analysis of DNA-protein complexes using nanopores

We present a method for rapid measurement of DNA-protein interactions using voltage-driven threading of single DNA molecules through a protein nanopore. Electrical force applied to individual ssDNA-exonuclease I complexes pulls the two molecules apart, while ion current probes the dissociation rate of the complex. Nanopore force spectroscopy (NFS) reveals energy barriers affecting complex dissociation. This method can be applied to other nucleic acid-protein complexes, using protein or solid-state nanopore devices.     

Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive

The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of cell growth. In budding yeast, TOR is found in structurally and functionally distinct protein complexes: TORC1 and TORC2. A mammalian counterpart of TORC1 (mTORC1) has been described, but it is not known whether TORC2 is conserved in mammals. Here, we report that a mammalian counterpart of TORC2 (mTORC2) also exists. mTORC2 contains mTOR, mLST8 and mAVO3, but not raptor. Like yeast TORC2, mTORC2 is rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton. mTORC2 is not upstream of the mTORC1 effector S6K. Thus, two distinct TOR complexes constitute a primordial signalling network conserved in eukaryotic evolution to control the fundamental process of cell growth.     

Some assembly required: yeast septins provide the instruction manual

Septins are a family of conserved proteins that form hetero-oligomeric complexes that assemble into filaments. The filaments can be organized into linear arrays, coils, rings and gauzes. They serve as membrane-associated scaffolds and as barriers to demarcate local compartments, especially for the establishment of the septation site for cytokinesis. Studies in budding and fission yeast have revealed many of the protein-protein interactions that govern the formation of multi-septin complexes. GTP binding and phosphorylation direct the polymerization of filaments that is required for septin-collar assembly in budding yeast, whereas a homolog of anillin instructs timely formation of the ring of septin filaments at the medial cortex in fission yeast. These insights should aid understanding of the organization and function of the diverse septin structures in animal cells.     

Gamma-tubulins and their functions

gamma-Tubulin is a ubiquitous phylogenetically conserved member of tubulin superfamily. In comparison with alpha beta-tubulin dimers, it is a low abundance protein present within the cells in both various types of microtubule-organizing centers and cytoplasmic protein complexes. gamma-Tubulin small complexes are subunits of the gamma-tubulin ring complex, which is involved in microtubule nucleation and capping of the minus ends of microtubules. In the past years important findings have advanced the understanding of the structure and function of gamma-tubulin ring complexes. Recent evidences suggest that the functions of gamma-tubulin extend beyond microtubule nucleation.     

Conservation of telomere protein complexes: shuffling through evolution

The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species-specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology.     

Viral strategies for intracellular trafficking: motors and microtubules

To overcome barriers to diffusion, many viruses utilize the microtubule-associated molecular motor cytoplasmic dynein 1 to drive transport towards the nucleus of a target cell. Cytoplasmic dynein 1 generates movement towards the minus end of microtubules located at the microtubule organizing centre (MTOC), a structure that is typically in close proximity to the nucleus. Physiological cargoes for cytoplasmic dynein include membranous organelles, protein complexes and aggregates of misfolded protein. In this review, we discuss the study of microtubule-based translocation of viruses and raise questions about the mechanisms for association with and then dissociation from cytoplasmic dynein with a goal of understanding whether viruses are seen by the intracellular trafficking machinery as functional protein complexes or misfolded protein aggregates.     

Overcoming natural replication barriers: differential helicase requirements

DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.