Mechanism Of Complex Assembly Research Articles

Architecture of a complete Bce-type antimicrobial peptide resistance module

Gram-positive bacteria synthesize and secrete antimicrobial peptides that target the essential process of peptidoglycan synthesis. These antimicrobial peptides not only regulate the dynamics of microbial communities but are also of clinical importance as exemplified by peptides such as bacitracin, vancomycin, and daptomycin. Many gram-positive species have evolved specialized antimicrobial peptide sensing and resistance machinery known as Bce modules. These modules are membrane protein complexes formed by an unusual Bce-type ABC transporter interacting with a two-component system sensor histidine kinase. In this work, we provide the first structural insight into how the membrane protein components of these modules assemble into a functional complex. A cryo-EM structure of an entire Bce module revealed an unexpected mechanism of complex assembly, and extensive structural flexibility in the sensor histidine kinase. Structures of the complex in the presence of a non-hydrolysable ATP analog reveal how nucleotide binding primes the complex for subsequent activation. Accompanying biochemical data demonstrate how the individual membrane protein components of the complex exert functional control over one another to create a tightly regulated enzymatic system.

Disulfide Trapping and Spectroscopic Studies of Bacterial Chemosensory Core Signaling Complexes: Probing Molecular Mechanisms of Complex Assembly and Receptor-Regulated On-Off Switching

The core unit of the bacterial chemosensory array is multi-protein complex comprised of 6 transmembrane chemoreceptor homodimers, 1 CheA His kinase homodimer, and 2 CheW adaptor protein monomers. We are reconstituting core units on isolated bacterial membranes, yielding individual core units and oligomers of core units ranging in size up to small hexagonal arrays. This approach generates functional, membrane-bound core complexes and allows incorporation of modified kinase and adaptor proteins possessing pairs of engineered Cys residues for disulfide trapping, or spectroscopic probes for fluorescence or EPR studies. The resulting core complexes display native receptor-stimulated kinase activities that are fully regulated by attractant binding to the receptor, or covalent modification of the receptor adaptation sites. The findings shed new light on the kinetics and order of core complex assembly, and provide insights into the molecular mechanisms underlying receptor-mediated kinase on-off switching.

Different subunits belonging to the same protein complex often exhibit discordant expression levels and evolutionary properties.

Hetero-oligomeric protein complexes are involved in many of the key processes in cells. Given that the subunits of a complex function together, it has often been expected to find that (i) they are expressed at similar levels in cells; (ii) they are simultaneously present or absent in genomes; and that (iii) the effects on fitness of deleting their genes should be similar. Such a coherence is, however, often found to be weak or absent. Multi-functionality of subunits and mechanisms of complex assembly are discussed as possible sources for the lack of coherence.

Active centrum hypothesis: The origin of chiral homogeneity and the RNA-world

I propose a hypothesis on the origin of chiral homogeneity of bio-molecules based on chiral catalysis. The first chiral active centre may have formed on the surface of complexes comprising metal ions, amino acids, other coenzymes and oligomers (short RNAs). The complexes must have been dominated by short RNAs capable of self-reproduction with ligation. Most of the first complexes may have catalysed the production of nucleotides. A basic assumption is that such complexes can be assembled from their components almost freely, in a huge variety of combinations. This assumption implies that “a few” components can constitute “a huge” number of active centre types. Moreover, an experiment is proposed to test the performance of such complexes in vitro. If the complexes were built up freely from their elements, then Darwinian evolution would operate on the assembly mechanism of complexes. For the production of complexes, first their parts had to appear by forming a proper three-dimensional structure. Three possible re-building mechanisms of the proper geometric structure of complexes are proposed. First, the integration of RNA parts of complexes was assisted presumably by a pre-intron. Second, the binding of RNA parts of a complex may give rise to a “polluted” RNA world. Third, the pairing of short RNA parts and their geometric conformation may have been supported by a pre-genetic code. Finally, an evolutionary step-by-step scenario of the origin of homochirality and a “polluted” RNA world is also introduced based on the proposed combinatorial complex chemistry. Homochirality is evolved by Darwinian selection whenever the efficiency of the reflexive autocatalysis of a dynamical combinatorial library increases with the homochirality of the active centres of reactions cascades and the homochirality of the elements of the dynamical combinatorial library. Moreover, the potential importance of phospholipid membrane is also discussed.

Structure and Function of the Pre-mRNA Splicing Machine

Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.

Complex assembly mechanism and an RNA‐binding mode of the human p14‐SF3b155 spliceosomal protein complex identified by NMR solution structure and functional analyses

The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.

Molecular Determinants of the Yeast Arc1p–Aminoacyl-tRNA Synthetase Complex Assembly

Eukaryotic aminoacyl-tRNA synthetases are usually organized into high-molecular-weight complexes, the structure and function of which are poorly understood. We have previously described a yeast complex containing two aminoacyl-tRNA synthetases, methionyl-tRNA synthetase and glutamyl-tRNA synthetase, and one noncatalytic protein, Arc1p, which can stimulate the catalytic efficiency of the two synthetases. To understand the complex assembly mechanism and its relevance to the function of its components, we have generated specific mutations in residues predicted by a recent structural model to be located at the interaction interfaces of the N-terminal domains of all three proteins. Recombinant wild-type or mutant forms of the proteins, as well as the isolated N-terminal domains of the two synthetases, were overexpressed in bacteria, purified and used for complex formation in vitro and for determination of binding affinities using surface plasmon resonance. Moreover, mutant proteins were expressed as PtA or green fluorescent protein fusion polypeptides in yeast strains lacking the endogenous proteins in order to monitor in vivo complex assembly and their subcellular localization. Our results show that the assembly of the Arc1p–synthetase complex is mediated exclusively by the N-terminal domains of the synthetases and that the two enzymes bind to largely independent sites on Arc1p. Analysis of single-amino-acid substitutions identified residues that are directly involved in the formation of the complex in yeast cells and suggested that complex assembly is mediated predominantly by van der Waals and hydrophobic interactions, rather than by electrostatic forces. Furthermore, mutations that abolish the interaction of methionyl-tRNA synthetase with Arc1p cause entry of the enzyme into the nucleus, proving that complex association regulates its subcellular distribution. The relevance of these findings to the evolution and function of the multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases is discussed.

Endoplasmic reticulum retention of the γ‐secretase complex component Pen2 by Rer1

gamma-Secretase is involved in the production of amyloid beta-peptide, which is the principal component of amyloid plaques in the brains of patients with Alzheimer disease. gamma-Secretase is a complex composed of presenilin (PS), nicastrin, anterior pharynx-defective phenotype 1 (Aph1) and PS enhancer 2 (Pen2). We previously proposed a mechanism of complex assembly by which unassembled subunits are retained in the endoplasmic reticulum (ER) and only the fully assembled complex is exported from the ER. We have now identified Retention in endoplasmic reticulum 1 (Rer1) as a protein that is involved in the retention/retrieval of unassembled Pen2 to the ER. Direct binding of unassembled Pen2 to Rer1 is mediated by the first transmembrane domain of Pen2, and a conserved asparagine in this domain is required. Downregulation of Rer1 leads to increased surface localization of Pen2, whereas overexpression of Rer1 stabilizes unassembled Pen2. To our knowledge, Rer1 is the first identified interaction partner of mammalian transmembrane-based retention/retrieval signals.

A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimentation Velocity Analysis

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes.

Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

Formation of Novel TRPC Channels by Complex Subunit Interactions in Embryonic Brain

Mammalian short TRP channels (TRPCs) are putative receptor- and store-operated cation channels that play a fundamental role in the regulation of cellular Ca2+ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo- and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerol-sensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5DN). Co-expression of TRPC5DN suppressed currents through TRPC5- and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.

Macromolecular Assemblage of Aminoacyl-tRNA Synthetases: Quantitative Analysis of Protein-Protein Interactions and Mechanism of Complex Assembly

The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches. This macromolecular assemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the seven monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 proteins. The scaffold p38 protein was expressed in Escherichia coli and purified to homogeneity as a His-tagged protein. The different components of the complex were shown to associate in vitro with p38 immobilized on Ni2+-coated plates. Interactions between peripheral enzymes and p38 are referred to as central interactions, as opposed to lateral interactions between peripheral enzymes. Kinetic parameters of the interactions were determined by the means of a biosensor-based approach. The two dimeric proteins LysRS and AspRS were found to tightly bind to p38, with a Kd value of 0.3 and 4.7nM, respectively. These interactions involved the catalytic core of the enzymes. By contrast, binding of ArgRS or GlnRS to p38 was much weaker (>5μM). ArgRS and p43, two peripheral components, were shown to interact with moderate affinity (Kd=93nM). Since all the components of the complex are tightly associated within this particle, lateral interactions were believed to contribute to the stabilization of this assemblage. Using an in vitro binding assay, concomitant association of several components of the complex on immobilized p38 could be demonstrated, and revealed the involvement of synergistic effects for association of weakly interacting proteins. Taking into account the possible synergy between central and lateral contributions, a sub-complex containing p38, p43, ArgRS and GlnRS was reconstituted in vitro. These data provide compelling evidence for an ordered and concerted mechanism of complex assembly.

Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells.

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.