Using indirect protein-protein interactions for protein complex prediction

Protein complexes are fundamental for understanding principles of cellular organizations. As the sizes of protein-protein interaction (PPI) networks are increasing, accurate and fast protein complex prediction from these PPI networks can serve as a guide for biological experiments to discover novel protein complexes. However, it is not easy to predict protein complexes from PPI networks, especially in situations where the PPI network is noisy and still incomplete. Here, we study the use of indirect interactions between level-2 neighbors (level-2 interactions) for protein complex prediction. We know from previous work that proteins which do not interact but share interaction partners (level-2 neighbors) often share biological functions. We have proposed a method in which all direct and indirect interactions are first weighted using topological weight (FS-Weight), which estimates the strength of functional association. Interactions with low weight are removed from the network, while level-2 interactions with high weight are introduced into the interaction network. Existing clustering algorithms can then be applied to this modified network. We have also proposed a novel algorithm that searches for cliques in the modified network, and merge cliques to form clusters using a "partial clique merging" method. Experiments show that (1) the use of indirect interactions and topological weight to augment protein-protein interactions can be used to improve the precision of clusters predicted by various existing clustering algorithms; and (2) our complex-finding algorithm performs very well on interaction networks modified in this way. Since no other information except the original PPI network is used, our approach would be very useful for protein complex prediction, especially for prediction of novel protein complexes.     

Inhibitory effects of RraA and RraB on RNAse E-related enzymes imply conserved functions in the regulated enzymatic cleavage of RNA

RraA and RraB are recently discovered protein inhibitors of RNAse E, which forms a large protein complex termed the degradosome that catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli . Here, we report that these E. coli protein inhibitors physically interact with RNAse ES, a Streptomyces coelicolor functional ortholog of RNAse E, and inhibit its action in vivo as well as in vitro ; however, unlike their ability to differentially modulate E. coli RNAse E action in a substrate-dependent manner by altering the composition of the degradosome, both proteins appear to have a general inhibitory effect on the ribonucleolytic activity of RNAse ES, which does not interact with E. coli polynucleotide phosphorylase, a major component of the degradosome. Our findings suggest that these regulators of RNAse activity have a conserved intrinsic property enabling them to directly act on RNAse E-related enzymes and inhibit their general ribonucleolytic activity.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.     

DEAD box RhlB RNA helicase physically associates with exoribonuclease PNPase to degrade double-stranded RNA independent of the degradosome-assembling region of RNase E

The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.     

Identification of ZipA,a signal recognition particle-dependent protein from Neisseria gonorrhoeae

A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in Escherichia coli was used to screen a Neisseria gonorrhoeae plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related N. meningitidis. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in N. gonorrhoeae since it was not possible to generate null mutations in the gene. Although appearing unique to Neisseria at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene zipA of E. coli. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an E. coli conditional zipA mutant and therefore encodes a functional ZipA homolog in N. gonorrhoeae. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria.     

A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli.

Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.     

Isolation and characterization of a putative multidrug resistance pump from Vibrio cholerae

Multidrug-resistant strains of Vibrio cholerae (the causative agent of the diarrhoeal disease cholera) have recently been described. In an attempt to identify a homologue of the Escherichia coli TolC in V . cholerae , we isolated a DNA fragment (pVC) that enabled an E . coli tolC mutant to grow in the presence of 0.05% deoxycholate (DOC). However, other TolC defects were not complemented. Nucleotide sequence analysis of this fragment revealed the presence of two open reading frames (ORF1 and ORF2) separated by 9 bp and encoding 42.4 and 55.8 kDa proteins respectively. The translational products of these two ORFs correlated closely with the molecular weights of the predicted proteins. The deduced amino acid sequences of ORF1 and ORF2 showed a high degree of similarity with conserved regions of the E . coli efflux pump proteins, EmrA and EmrB. The presence of pVC2 within the E . coli efflux pump mutants defective in either the emrAB or the acrAB genes provided the mutants with resistance against several antibiotics. A V . cholerae isogenic mutant defective in ORF2 was constructed by gene replacement. Characterization of this mutant has shown it to be more sensitive to CCCP, PMA, PCP, nalidixic acid and DOC than the parent strain. These results suggest that ORF1 and ORF2 constitute an operon encoding two components of a putative multidrug resistance pump in V . cholerae . In addition, the presence of both structural and functional similarities between VceAB and EmrAB suggests that VceAB is a homologue of EmrAB.     

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.     

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide.

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.     

An rpoE-like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep-sea bacterium Photobacterium sp. strain SS9

Many deep-sea bacteria have evolved specialized adaptations for life at cold temperatures and high pressures. A locus required for both psychro- and baro- adaptation in the psychrophilic, moderate barophile, Photobacterium species strain SS9 was identified among SS9 transposon mutants. DNA sequence analysis of this locus identified four complete open reading frames (ORFs), which appear to comprise an operon, and a fifth incomplete ORF. All transposon insertions isolated are in ORF3. Extensive sequence similarity exists between the translation products of ORFs 1–3 and a collection of gene products proposed to include alternative RNA polymerase sigma factors and modifiers of sigma factors activity involved in extracytoplasmic sensing and regulation. Based on the similarity between ORF1 and Escherichia coli rpoE , we have tentatively designated this locus the rpoE locus. SS9 rpoE locus ORF3 insertion mutants showed altered abundances of numerous outer membrane proteins and were both baro- and psychro- sensitive. ORF3 mutant revertants that displayed enhanced high-pressure growth also displayed concomitant enhanced low-temperature growth. Most of these revertants possessed DNA rearrangements at the site of the transposon insertion, further demonstrating the importance of the rpoE locus to high-pressure and cold-temperature growth. Complementation analyses indicated that ORF3 functions in OMP synthesis regulation while ORF4 is required for baro- and psychro-adaptation.     

Comparative characterization of release factor RF-3 genes of Escherichia coli, Salmonella typhimurium, and Dichelobacter nodosus.

The termination of protein synthesis in bacteria requires two codon-specific release factors, RF-1 and RF-2. A gene for a third factor, RF-3, that stimulates the RF-1 and RF-2 activities has been isolated from the gram-negative bacteria Escherichia coli and Dichelobacter nodosus. In this work, we isolated the RF-3 gene from Salmonella typhimurium and compared the three encoded RF-3 proteins by immunoblotting and intergeneric complementation and suppression. A murine polyclonal antibody against E. coli RF-3 reacted with both S. typhimurium and D. nodosus RF-3 proteins. The heterologous RF-3 genes complemented a null RF-3 mutation of E. coli regardless of having different sequence identities at the protein level. Additionally, multicopy expression of either of these RF-3 genes suppressed temperature-sensitive RF-2 mutations of E. coli and S. typhimurium by restoring adequate peptide chain release. These findings strongly suggest that the RF-3 proteins of these gram-negative bacteria share common structural and functional domains necessary for RF-3 activity and support the notion that RF-3 interacts functionally and/or physically with RF-2 during translation termination.     

Targeting and insertion of heterologous membrane proteins in E. coli

Membrane targeting and insertion of the archaeal Halobacter halobium proton pump bacterioopsin (Bop) and the human melanocortin 4 receptor (MC(4)R) were studied in vitro, using E. coli components for protein synthesis, membrane targeting and insertion. These heterologous proteins are targeted to E. coli membranes in a signal recognition particle (SRP) dependent manner and inserted into the membrane co-translationally. Furthermore, we demonstrate that nascent chains of Bop and MC(4)R first interact with SecY and then with YidC as they move through the translocon. Our results suggest that the initial stages of membrane targeting and insertion of heterologous proteins in E. coli proceed by the pathway used for native E. coli membrane proteins. No significant pausing of protein elongation was observed in the presence of E. coli SRP, in line with the suggestion that translational arrest requires an Alu domain, which is absent in SRP from E. coli.     

Interactions that drive Sec-dependent bacterial protein transport

Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported via other pathways. While targeting signals for each route have common overall characteristics, individual signal peptides vary greatly in their amino acid sequences. How do these diverse signals interact specifically with the proteins that comprise the appropriate transport machinery and, at the same time, avoid targeting to an alternate route? The recent publication of the crystal structures of components of the Sec transport machinery now allows a more thorough consideration of the interactions of signal sequences with these components.     

Comparative studies of penicillin-binding proteins in Pseudomonas aeruginosa and Escherichia coli.

Penicillin-binding proteins in Pseudomonas aeruginosa were compared with those of Escherichia coli. These in P. aeruginosa were found exclusively in the cytoplasmic membrane fraction (fraction soluble in sodium N-lauroyl sarcosinate). Sodium dodecyl sulfate/acrylamide gel electrophoresis of the proteins bound to [14C]penicillin G resulted in the separation of six major bands and several minor bands. The proteins in these bands are referred to as proteins 1A, 1B, 2, 3, 4 and 5 in order of increasing electrophoretical mobility. The electrophoretic mobilities and other properties of penicillin-binding proteins in P. aeruginosa and E. coli were compared and correlated. Fundamentally they seem to be very similar in the two bacteria, but proteins 1A and 1B in P. aeruginosa seem to correspond respectively to proteins 1B and 1A in E. coli, and protein 6 seems to be missing or present in only small amount in P. aeruginosa. In addition, the affinities of currently developed beta-lactam antibiotics to each protein of P. aeruginosa and E. coli were examined in relation to the morphological changes of the cells induced by these antibiotics and their antibacterial potencies. Mecillinam showed high affinity to only protein 2 in both P. aeruginosa and E. coli. At a minimal inhibitory concentration, it converted cells of both P. aeruginosa and E. coli from rods to spherical cells, although its minimal inhibitory concentration was much higher for P. aeruginosa than for E. coli.     

The tol gene products and the import of macronmolecules into Escherichia coli

Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single-stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer-membrane receptors and of the bacteriophage of the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed 'tolerant' (tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.     

Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response and DNA synthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not a functional oxidoreductase. A variety of proteins in E. coli interact with YbbN, including multiple ribosomal protein subunits and a strong interaction with GroEL. YbbN acts as a mild inhibitor of GroESL chaperonin function and ATPase activity, suggesting that it is a negative regulator of the GroESL system. Combined with previous observations that YbbN enhances the DnaK-DnaJ-GrpE chaperone system, we propose that YbbN coordinately regulates the activities of these two prokaryotic chaperones, thereby helping to direct client protein traffic initially to DnaK. Therefore, YbbN may play a role in integrating the activities of different chaperone pathways in E. coli and related bacteria.     

Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.