Assembly of chaperonin complexes

Chaperonins are a subclass of molecular chaperones that assist both the folding of newly synthesized proteins and the maintenance of proteins in a folded state during periods of stress. The best studied members of this family are the type I chaperonins, occurring in bacteria and evolutionarily derived organelles. Type II chaperonins occur in archaea and the eukaryotic cytosol. An intriguing question pertains to the mechanism by which chaperonins themselves are folded and assembled into functional oligomers. The available evidence for the assembly/disassembly of type I and II chaperonins points to a process that is highly cooperative and suggests a prominent role for nucleotides. Interestingly, the intracellular assembly of type I chaperonins appears to be a chaperone-dependent process itself and requires functional preformed chaperonin complexes.     

Assembly of Polycomb complexes and silencing mechanisms

Polycomb complexes assemble at their target sites and silence neighboring genes when these are not actively transcribed. The action of these complexes and of Trithorax complexes bound to the Polycomb Response Element establish alternative silent or derepressed states that are remembered through cell division and maintained for the rest of development. Recent results that may help explain the properties of these states are reviewed.     

Assembly and function of RNA silencing complexes

In the RNA-interference pathway, double-stranded RNA induces sequence-specific mRNA degradation through the action of the RNA-induced silencing complex (RISC). Recent work has provided our first glimpses of the RISC-assembly pathway and uncovered the biochemical roles of critical RISC components. These advances have taken our mechanistic understanding of RNA interference to a new level and promise to improve our ability to exploit this biological process for use in experimental biology and medicine.     

Assembly and self-association of oxytricha telomeric nucleoprotein complexes

Two types of specific telomeric protein-DNA complex are reconstituted upon incubation of purified Oxytricha telomere protein with (T4G4)4, an oligodeoxynucleotide of telomeric sequence. The complexes differ in electrophoretic mobility, in protein-DNA contacts, and in the rate of DNA exchange. The patterns of protein-DNA interaction determined by modification interference suggest a model in which the protein can bind either to the two T4G4 repeats at the 3' end or to two internal repeats; in the latter case, it can make a different set of contacts with the terminal repeat to form the more stable complex. Native telomeric chromatin isolated from Oxytricha contains both types of complexes. The reconstituted monomeric complexes associate to give a high molecular weight form that has an altered chemical footprint. Such interactions may mediate the association of chromosomal telomeres in vivo.     

Assembly and disassembly kinetics of anthrax toxin complexes

Proteolytic activation of the protective antigen (PA) component of anthrax toxin allows it to self-associate into a ring-shaped homoheptamer, [PA(63)](7), which can bind the enzymatic components lethal factor (LF) and edema factor (EF). [PA(63)](7) is a pore-precursor (prepore), and under the low-pH conditions of the endosome, it forms a transmembrane pore that allows LF and EF to enter the cytosol. PA was labeled with donor and acceptor fluorescent dyes, and F?rster resonance energy transfer was used to measure the assembly and disassembly kinetics of the prepore complex in solution. The dissociation rate constant for [PA(63)](7) was 1 x 10(-)(6) s(-)(1) (t(1/2) approximately 7 days). In contrast, a ternary complex containing the PA-binding domain of LF (LF(N)) bound to a PA(63) dimer composed of two nonoligomerizing mutants dissociated rapidly (t(1/2) approximately 1 min). Thus, the substantial decrease in the rate of disassembly of [PA(63)](7) relative to the ternary complex is due to the cooperative interactions among neighboring subunits in the heptameric ring. Low concentrations of LF(N) promoted assembly of the prepore from proteolytically activated PA, whereas high concentrations inhibited assembly of both the prepore and the ternary complex. A self-assembly scheme of anthrax toxin complexes is proposed.     

Assembly of membrane-bound respiratory complexes by the Tat protein-transport system

The Tat protein-export system serves to translocate folded proteins, often containing redox cofactors, across the bacterial inner membrane. Substrate proteins are directed to the Tat apparatus by distinctive N-terminal signal peptides containing a consensus SRRxFLK 'twin-arginine' motif. Here we review recent studies of the Tat system with particular emphasis on the assembly of membrane-bound respiratory complexes. We discuss the connection between Tat targeting and topological organisation of the complexes and consider the role of chaperone proteins in cofactor insertion and Tat targeting. The crystal structure of Escherichia coli formate dehydrogenase-N demonstrates that some Tat substrates are integral membrane proteins. Sequence analysis suggests that one-quarter of all traffic on the E. coli Tat pathway is inner-membrane proteins.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.     

Assembly of oligomeric death domain complexes during Toll receptor signaling

The Drosophila Toll receptor is activated by the endogenous protein ligand Sp?tzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.     

Spotlight: Assembly of protein complexes by integrating graph clustering methods

As is generally assumed, clusters in protein–protein interaction (PPI) networks perform specific, crucial functions in biological systems. Various network community detection methods have been developed to exploit PPI networks in order to identify protein complexes and functional modules. Due to the potential role of various regulatory modes in biological networks, a single method may just apply a single graph property and neglect communities highlighted by other network properties.     

Assembly and signaling of CRLR and RAMP1 complexes assessed by BRET

Biochemical and functional evidence suggest that the calcitonin receptor-like receptor (CRLR) interacts with receptor activity-modifying protein-1 (RAMP1) to generate a calcitonin gene-related peptide (CGRP) receptor. Using bioluminescence resonance energy transfer (BRET), we investigated the oligomeric assembly of the CRLR-RAMP1 signaling complex in living cells. As for their wild-type counterparts, fusion proteins linking CRLR and RAMP1 to the energy donor Renilla luciferase (Rluc) and energy acceptor green fluorescent protein (GFP) reach the cell surface only upon coexpression of CRLR and RAMP1. Radioligand binding and cAMP production assays also confirmed that the fusion proteins retained normal functional properties. BRET titration experiments revealed that CRLR and RAMP1 associate selectively to form heterodimers. This association was preserved for a mutated RAMP1 that cannot reach the cell surface, even in the presence of CRLR, indicating that the deficient targeting resulted from the altered conformation of the complex rather than a lack of heterodimerization. BRET analysis also showed that, in addition to associate with one another, both CRLR and RAMP1 can form homodimers. The homodimerization of the coreceptor was further confirmed by the ability of RAMP1 to prevent cell surface targeting of a truncated RAMP1 that normally exhibits receptor-independent plasma membrane delivery. Although the role of such dimerization remains unknown, BRET experiments clearly demonstrated that CRLR can engage signaling partners, such as G proteins and beta-arrestin, following CGRP stimulation, only in the presence of RAMP1. In addition to shed new light on the CRLR-RAMP1 signaling complex, the BRET assays developed herein offer new biosensors for probing CGRP receptor activity.     

Assembly of membrane-bound protein complexes: detection and analysis by single molecule diffusion

Protein complexes assembled on membrane surfaces regulate a wide array of signaling pathways and cell processes. Thus, a molecular understanding of the membrane surface diffusion and regulatory events leading to the assembly of active membrane complexes is crucial to signaling biology and medicine. Here we present a novel single molecule diffusion analysis designed to detect complex formation on supported lipid bilayers. The usefulness of the method is illustrated by detection of an engineered, heterodimeric complex in which two membrane-bound pleckstrin homology (PH) domains associate stably, but reversibly, upon Ca(2+)-triggered binding of calmodulin (CaM) to a target peptide from myosin light chain kinase (MLCKp). Specifically, when a monomeric, fluorescent PH-CaM domain fusion protein diffusing on a supported bilayer binds a dark MLCKp-PH domain fusion protein, the heterodimeric complex is observed to diffuse nearly 2-fold more slowly than the monomer because both of its twin PH domains can simultaneously bind to the viscous bilayer. In a mixed population of monomers and heterodimers, the single molecule diffusion analysis resolves, identifies and quantitates the rapidly diffusing monomers and slowly diffusing heterodimers. The affinity of the CaM-MLCKp interaction is measured by titrating dark MLCKp-PH construct into the system, while monitoring the changing ratio of monomers and heterodimers, yielding a saturating binding curve. Strikingly, the apparent affinity of the CaM-MLCKp complex is ~10(2)-fold greater in the membrane system than in solution, apparently due to both faster complex association and slower complex dissociation on the membrane surface. More broadly, the present findings suggest that single molecule diffusion measurements on supported bilayers will provide an important tool for analyzing the 2D diffusion and assembly reactions governing the formation of diverse membrane-bound complexes, including key complexes from critical signaling pathways. The approach may also prove useful in pharmaceutical screening for compounds that inhibit membrane complex assembly or stability.     

Assembly and transport properties of invariant chain trimers and HLA-DR-invariant chain complexes.

The MHC class II-associated invariant chain behaves as a resident endoplasmic reticulum protein in the absence of class II molecules. In humans, two predominant forms exist; one, p35, differs from the other, p33, by an N-terminal cytoplasmic extension of 16 amino acids that contains a strong endoplasmic reticulum-retention signal. Here we show that one mechanism for retention of p33 is its association with p35 in mixed invariant chain trimers. However, even for p33 homotrimers transport from the endoplasmic reticulum is inefficient. In an MHC class II-positive B cell line, the formation of invariant chain trimers is rapid and is the first intermediate in the assembly of a nine-chain alpha beta-invariant chain complex. With time, three higher molecular weight complexes are progressively formed. These correspond to an invariant chain trimer with one alpha beta dimer, two alpha beta dimers, and three alpha beta dimers, respectively. No free alpha beta dimers are detectable early in biosynthesis. However, beginning at 2 h of chase, alpha beta dimers begin to appear concomitant with the disappearance of the completely assembled alpha beta-invariant chain complex. This conversion is virtually complete by 4 h, and presumably reflects the proteolytic degradation of the invariant chain component of the alpha beta-invariant chain complex and the generation of endosomal alpha beta dimers capable of binding antigenic peptides.     

Assembly of anthrax toxin pore: Lethal‐factor complexes into lipid nanodiscs

We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N‐terminal domain of anthrax lethal factor (LF_N) into lipid nanodiscs and analyzed the resulting complexes by negative‐stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (～43%). Three‐dimensional analysis of negatively stained single particles revealed the LF_N‐PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF_N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo‐EM LF_N‐PA nanodisc structures and use of advanced automated particle selection methods.     

Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.

The in vitro assembly process for forming nucleoprotein complexes containing linear retrovirus-like DNA and integrase (IN) was investigated. Solution conditions that allowed avian myeloblastosis virus IN to efficiently pair two separate linear DNA fragments (each 487 bp in length) containing 3' OH recessed long terminal repeat termini were established. Pairing of the viral termini by IN during preincubation on ice permitted these nucleoprotein complexes to catalyze the concerted insertion of the two termini into a circular DNA target (full-site reaction), mimicking the in vivo reaction. The three major solution determinants were high concentrations of NaCl (0.33 M), 1,4-dioxane, and polyethylene glycol. The aprotic solvent dioxane (15%) was significantly better (sixfold) than 15% dimethyl sulfoxide for forming complexes capable of full-site rather than half-site integration events. Half-site reactions by IN involved the insertion of a single donor terminus into circular pGEM. Although NaCl was essential for the efficient promotion of the concerted integration reaction, dioxane was necessary to prevent half-site reactions from occurring at high NaCl concentrations. Under optimal solution conditions, the concerted integration reaction was directly proportional to a sixfold range of IN. The complexes appeared not to turn over, and few half-site donor-donor molecules were produced. In the presence of 0.15 or 0.35 M NaCl, dioxane prevented efficient 3' OH trimming of a blunt-ended donor by IN, suggesting that the complexes formed by IN with blunt-ended donors were different from those formed with donors containing 3' OH recessed termini for strand transfer. The results suggest that IN alone was capable of protein-protein and protein-DNA interactions that efficiently promote the in vitro concerted integration reaction.     

Assembly of protein-RNA complexes using natural RNA and mutant forms of an RNA cytosine methyltransferase

This work reveals that mutant forms of RNA methyltransferases that form 5-methylcytosine (m5C) have characteristics that may make them useful for biomacromolecular assembly. The experiments utilized bacterially expressed Trm4p, a tRNA methyltransferase cloned from Saccharomyces cerevisiae. Like DNA m5C methyltransferases, Trm4p mediates methylation using a covalent intermediate, which would allow Trm4p to be trapped as a stable protein-RNA complex when the substrate RNA contains a modified cytosine base such as 5-fluorocytosine. However, mutant forms of Trm4p are identified that fail to release RNA resulting in the formation of denaturant stable methyltransferase-RNA complexes that contain only natural nucleotides. The ability to form stable complexes with natural RNA gives these mutant forms of Trm4p greater potential versatility for biomacromolecule construction applications than the wild-type Trm4p enzyme or DNA methyltransferases for which the trapping of the covalent intermediate requires the presence of a nucleotide analogue at the site of modification.