Intermolecular Complementation Research Articles

Anti-tumor activity of anthrax toxin variants that form a functional translocation pore by intermolecular complementation.

Anthrax lethal toxin is a typical A-B type protein toxin secreted by Bacillus anthracis. Lethal factor (LF) is the catalytic A-subunit, a metalloprotease having MEKs as targets. LF relies on the cell-binding B-subunit, protective antigen (PA), to gain entry into the cytosol of target cells. PA binds to cell surface toxin receptors and is activated by furin protease to form an LF-binding-competent oligomer-PA pre-pore, which converts to a functional protein-conductive pore in the acidic endocytic vesicles, allowing translocation of LF into the cytosol. During PA pre-pore-to-pore conversion, the intermolecular salt bridge interactions between Lys397 and Asp426 on adjacent PA protomers play a critical role in positioning neighboring luminal Phe427 residues to form the Phe-clamp, an essential element of the PA functional pore. This essential intermolecular interaction affords the opportunity to create pairs of PA variants that depend on intermolecular complementation to form a functional pore. We have previously generated PA variants with furin-cleavage site replaced by substrate sequences of tumor-associated proteases, such as urokinase or MMPs. Here we show that PA-U2-K397Q, a urokinase-activated PA variant with Lys397 residue replaced by glutamine, and PA-L1-D426K, a MMP-activated PA variant with Asp426 changed to lysine, do not form functional pores both in vitro or in vivo unless they are used together. Further, the mixture of PA-U2-K397Q and PA-L1-D426K displayed potent anti-tumor activity in the presence of LF. Thus, PA-U2-K397Q and PA-L1-D426K form a novel intermolecular complementation system with toxin activation relying on the presence of two distinct tumor-associated proteases, i.e., urokinase and MMPs.

An anthrax toxin variant with an improved activity in tumor targeting.

Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation.

An eccentric calpain, CAPN3/p94/calpain-3.

Calpains are Ca(2+)-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called "modulator proteases." CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases. For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na(+) for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research.

Intermolecular Complementation between Two Varicella-Zoster Virus pORF30 Terminase Domains Essential for DNA Encapsidation.

The herpesviral terminase complex is part of the intricate machinery that delivers a single viral genome into empty preformed capsids (encapsidation). The varicella-zoster virus (VZV) terminase components (pORF25, pORF30, and pORF45/42) have not been studied as extensively as those of herpes simplex virus 1 and human cytomegalovirus (HCMV). In this study, VZV bacterial artificial chromosomes (BACs) were generated with small (Δ30S), medium (Δ30M), and large (Δ30L) ORF30 internal deletions. In addition, we isolated recombinant viruses with specific alanine substitutions in the putative zinc finger motif (30-ZF3A) or in a conserved region (region IX) with predicted structural similarity to the human topoisomerase I core subdomains I and II (30-IXAla, 30-620A, and 30-622A). Recombinant viruses replicated in an ORF30-complementing cell line (ARPE30) but failed to replicate in noncomplementing ARPE19 and MeWo cells. Transmission electron microscopy of 30-IXAla-, 30-620A-, and 30-622A-infected ARPE19 cells revealed only empty VZV capsids. Southern analysis showed that cells infected with parental VZV (VZVLUC) or a repaired virus (30R) contained DNA termini, whereas cells infected with Δ30L, 30-IXAla, 30-620A, or 30-622A contained little or no processed viral DNA. These results demonstrated that pORF30, specifically amino acids 619 to 624 (region IX), was required for DNA encapsidation. A luciferase-based assay was employed to assess potential intermolecular complementation between the zinc finger domain and conserved region IX. Complementation between 30-ZF3A and 30-IXAla provided evidence that distinct pORF30 domains can function independently. The results suggest that pORF30 may exist as a multimer or participate in higher-order assemblies during viral DNA encapsidation. Antivirals with novel mechanisms of action are sought as additional therapeutic options to treat human herpesvirus infections. Proteins involved in the viral DNA encapsidation process have become promising antiviral targets. For example, letermovir is a small-molecule drug targeting HCMV terminase that is currently in phase III clinical trials. It is important to define the structural and functional characteristics of proteins that make up viral terminase complexes to identify or design additional terminase-specific compounds. The VZV ORF30 mutants described in this study represent the first VZV terminase mutants reported to date. Targeted mutations confirmed the importance of a conserved zinc finger domain found in all herpesvirus ORF30 terminase homologs but also identified a novel, highly conserved region (region IX) essential for terminase function. Homology modeling suggested that the structure of region IX is present in all human herpesviruses and thus represents a potential structurally conserved antiviral target.

The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation.

CAPN3/p94/calpain-3, a calpain protease family member predominantly expressed in skeletal muscle, possesses unusually rapid and exhaustive autolytic activity. Mutations in the human CAPN3 gene impairing its protease functions cause limb-girdle muscular dystrophy type 2A (LGMD2A); yet, the connection between CAPN3's autolytic activity and the enzyme's function in vivo remain unclear. Here, we demonstrated that CAPN3 protease activity was reconstituted by intermolecular complementation (iMOC) between its two autolytic fragments. Furthermore, the activity of full-length CAPN3 active-site mutants was surprisingly rescued through iMOC with autolytic fragments containing WT amino acid sequences. These results provide evidence that WT CAPN3 can be formed by the iMOC of two different complementary CAPN3 mutants. The finding of iMOC-mediated restoration of calpain activity indicates a novel mechanism for the genotype-phenotype links in LGMD2A.

Characterization of Chlamydomonas 102 and 104 Mutants Reveals Intermolecular Complementation in the Molybdenum Cofactor Protein CNX1E

Chlamydomonas reinhardtii is a eukaryotic, unicellular, biflagellate green alga. In Chlamydomonas nitrate assimilation depends on the activity of the enzyme nitrate reductase that requires the molybdenum cofactor (Moco). We have characterized the Chlamydomonas strains 102 (nit5nit6), 104 (nit4nit5) and 106 (nit6). These mutations lead to the exchanges V171A (nit4) and G183D (nit6) in the CNX1E protein of Moco biosynthesis that result in Moco deficiency and hence inability to grow on nitrate. CNX1E inserts molybdenum into molybdopterin (MPT) to yield Moco through the intermediate MPT-AMP. The CNX1E variant G183D is unable to bind MPT-AMP. However, the CNX1E variant V171A is able to bind MPT-AMP but is not able to hydrolyze it and insert the molybdenum to obtain Moco. We show that intermolecular complementation between the CNX1E variants V171A and G183D both in vivo and in vitro restored its activity. The molecular characterization of strain 106 led us to discover the presence of wild type and nit6 CNX1E alleles in this strain (nit6/+). Then, we found out that 106 is indeed a diploid strain. It is proposed that 102, the strain from which 106 derived, may contain a genetic alteration that produces an aberrant meiotic division of the mature zygotes.

Rescue of defective G protein-coupled receptor function in vivo by intermolecular cooperation.

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

Symmetric signalling within asymmetric dimers of the Staphylococcus aureus receptor histidine kinase AgrC

Virulence in Staphylococcus aureus is largely under control of the accessory gene regulator (agr) quorum-sensing system. The AgrC receptor histidine kinase detects its autoinducing peptide (AIP) ligand and generates an intracellular signal resulting in secretion of virulence factors. Although agr is a well-studied quorum-sensing system, little is known about the mechanism of AgrC activation. By co-immunoprecipitation analysis and intermolecular complementation of receptor mutants, we showed that AgrC forms ligand-independent dimers that undergo trans-autophosphorylation upon interaction with AIP. Remarkably, addition of specific AIPs to AgrC mutant dimers with only one functional sensor domain caused symmetric activation of either kinase domain despite the sensor asymmetry. Furthermore, mutant dimers involving one constitutive protomer demonstrated ligand-independent activity, irrespective of which protomer was kinase deficient. These results demonstrate that signalling through either individual AgrC protomer causes symmetric activation of both kinase domains. We suggest that such signalling across the dimer interface may be an important mechanism for dimeric quorum-sensing receptors to rapidly elicit a response upon signal detection.

Dss1 Interaction with Brh2 as a Regulatory Mechanism for Recombinational Repair

Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling Rad51 and is in turn regulated by Dss1. Interplay with Rad51 is conducted via the BRC element located in the N-terminal region of the protein and through an unrelated domain, CRE, at the C terminus. Mutation in either BRC or CRE severely reduces functional activity, but repair deficiency of the brh2 mutant can be complemented by expressing BRC and CRE on different molecules. This intermolecular complementation is dependent upon the presence of Dss1. Brh2 molecules associate through the region overlapping with the Dss1-interacting domain to form at least dimer-sized complexes, which in turn, can be dissociated by Dss1 to monomer. We propose that cooperation between BRC and CRE domains and the Dss1-provoked dissociation of Brh2 complexes are requisite features of Brh2's molecular mechanism.

Construction of Intramolecular Luciferase Complementation Probe for Detecting Specific RNA

Intermolecular enzyme complementation assay is a useful method for detecting protein-protein interactions. Specifically, bioluminescent signals produced from reconstructed split luciferase fragments are powerful tools for in vivo analysis because the bioluminescent signals have been visualized both in cultured cells and living animals. However, they are limited for detection and evaluation of biological events relevant to intermolecular protein-protein interactions. In this study, we constructed an intramolecular luciferase complementation probe for detecting target biomolecules other than protein-protein interactions. It consists of peptide-inserted firefly luciferase (PI-FLuc) containing a short peptide between internally divided firefly luciferase. The inserted short peptide triggers FLuc complementation or discomplementation and subsequent reactivation or inactivation of FLuc activity through its induced fit conformational changes. We chose RNA binding arginine rich motif (ARM) peptides, Rev and/or Tat, for model peptide insertion, and expressed constructed PI-FLuc probe variants using a wheat germ cell-free protein synthesis system. They showed FLuc activity changes, reactivation, or inactivation after binding to their specific RNA targets. Furthermore, to expand the versatility of the PI-FLuc RNA detection system, we designed split-RNA probes built to reform the ARM peptide binding site in the presence of arbitrarily selected target-RNA. As a result, the target RNA was homogeneously detected by FLuc luminescent signals mediated by a cooperative function of the PI-FLuc and split-RNA probe sets.

Substitutions in the N‐terminal alpha helical spine of Neisseria gonorrhoeae pilin affect Type IV pilus assembly, dynamics and associated functions

Type IV pili (Tfp) are multifunctional surface appendages expressed by many Gram negative species of medical, environmental and industrial importance. The N-terminally localized, so called alpha-helical spine is the most conserved structural feature of pilin subunits in these organelles. Prevailing models of pilus assembly and structure invariably implicate its importance to membrane trafficking, organelle structure and related functions. Nonetheless, relatively few studies have examined the effects of missense substitutions within this domain. Using Neisseria gonorrhoeae as a model system, we constructed mutants with single and multiple amino acid substitutions localized to this region of the pilin subunit PilE and characterized them with regard to pilin stability, organelle expression and associated phenotypes. The consequences of simultaneous expression of the mutant and wild-type PilE forms were also examined. The findings document for the first time in a defined genetic background the phenomenon of pilin intermolecular complementation in which assembly defective pilin can be rescued into purifiable Tfp by coexpression of wild-type PilE. The results further demonstrate that pilin subunit composition can impact on organelle dynamics mediated by the PilT retraction protein via a process that appears to monitor the efficacy of subunit-subunit interactions. In addition to confirming and extending the evidence for PilE multimerization as an essential component for competence for natural genetic transformation, this work paves the way for detailed studies of Tfp subunit-subunit interactions including self-recognition within the membrane and packing within the pilus polymer.

Intermolecular complementation achieves high-specificity tumor targeting by anthrax toxin

Anthrax toxin protective antigen (PrAg) forms a heptamer in which the binding site for lethal factor (LF) spans two adjacent monomers. This suggested that high cell-type specificity in tumor targeting could be obtained using monomers that generate functional LF-binding sites only through intermolecular complementation. We created PrAg mutants with mutations affecting different LF-binding subsites and containing either urokinase plasminogen activator (uPA) or matrix metalloproteinase (MMP) cleavage sites. Individually, these PrAg mutants had low toxicity as a result of impaired LF binding, but when administered together to uPA- and MMP-expressing tumor cells, they assembled into functional LF-binding heteroheptamers. The mixture of two complementing PrAg variants had greatly reduced toxicity in mice and was highly effective in the treatment of aggressive transplanted tumors of diverse origin. These results show that anthrax toxin, and by implication other multimeric toxins, offer excellent opportunities to introduce multiple-specificity determinants and thereby achieve high therapeutic indices.

Heterodimeric interactions among the 1-amino-cyclopropane-1-carboxylate synthase polypeptides encoded by the Arabidopsis gene family.

The pyridoxal phosphate-dependent enzyme, 1-aminocyclopropane-1-carboxylate synthase (ACS; EC 4.4.1.14), catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, ACS11) and one nonfunctional (ACS1) homodimers. Because the enzyme is a homodimer with shared active sites, the question arises whether the various polypeptides can form functional heterodimers. Intermolecular complementation experiments in Escherichia coli by coexpressing the K278A and Y92A mutants of different polypeptides show that all of them have the capacity to heterodimerize. However, functional heterodimers are formed only among gene family members that belong to one or the other of the two phylogenetic branches. ACS7 is an exception to this rule, which forms functional heterodimers with some members of both branches when it provides the wt K278 residue. ACS1, the nonfunctional polypeptide as a homodimer, can also form functional heterodimers with members of its phylogenetic branch when its partners provide the wt K278 residue. The ACS gene family products can potentially form 45 homo- and heterodimers of which 25 are functional. Bimolecular fluorescence complementation and biochemical coaffinity purification assays show that the inactivity of certain heterodimers is not due to the absence of heterodimerization but rather to structural restraint(s) that prevents the shared active sites from being functional. We propose that functional heterodimerization enhances the isozyme diversity of the ACS gene family and provides physiological versatility by being able to operate in a broad gradient of S-adenosylmethionine concentration in various cells/tissues during plant growth and development. Nonfunctional heterodimerization may also play a regulatory role during the plant life cycle.

Domains of the TMV movement protein involved in subcellular localization.

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9-11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49-51 may be involved in co-alignment of the protein with microtubules. The region around residues 88-101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.

Independently cloned halves of cytomegalovirus assemblin, An and Ac, can restore proteolytic activity to assemblin mutants by intermolecular complementation.

Herpesviruses encode an essential serine proteinase called assemblin that is responsible for cleaving the precursor assembly protein during the process of capsid formation. In cytomegalovirus (CMV), assemblin undergoes autoproteolysis at an internal (I) site located near the middle of the molecule. I-site cleavage converts the enzyme to an active two-chain form consisting of the subunits An and Ac. We have recently shown that the recombinant An and Ac subunits can spontaneously associate within eukaryotic cells to yield active two-chain proteinase. This finding indicates that the subunits are able to independently assume their correct functional conformations and led us to test whether they are capable of intermolecular complementation. This was done by coexpressing inactive mutant (point, deletion, and insertion) forms of assemblin together with the wild-type subunit (either An or Ac) corresponding to the domain of assemblin that was mutated. Results of these experiments showed that both An and Ac are able to rescue the enzymatic activity of assemblin mutants. I-site cleavage of the mutated assemblin occurred during complementation but was not absolutely required, as shown by effective complementation of inactive assemblins with noncleavable I sites. We have also shown that intermolecular complementation can rescue the activity of an inactive mutant full-length proteinase precursor and can occur between different species of CMV (e.g., human CMV subunit can rescue activity of mutant simian CMV assemblin). These results indicate that assemblin is able to form active multimeric structures that may be of functional importance.

Intermolecular complementation of the kinase activity of CheA.

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis in Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase-defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer. The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphorylation requires the presence of both carboxy-terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.

Intermolecular complementation between two defective mutant signal-transducing receptors of Escherichia coli.

Taz1 is a hybrid signal-transducing membrane receptor between Tar, an aspartate chemoreceptor, and EnvZ, an osmosensor of Escherichia coli that is able to induce ompC expression by phosphorylating OmpR (a transcriptional activator) in response to aspartate. When the Taz1 His-277, the proposed autophosphorylation site in the cytoplasmic EnvZ domain, was replaced with a valine residue, the mutant Taz1 was unable to induce ompC expression. Similarly, when approximately two-thirds of the EnvZ domain was deleted, Taz1 was nonfunctional. However, when these two defective Taz1 proteins were coexpressed in a cell, ompC was constitutively expressed. Coinciding with this result, two mutant Taz1 molecules were able to intermolecularly complement each other to restore the OmpR kinase activity but not phosphatase activity in vitro. The identical result was also obtained with EnvZ. The present results suggest that the autophosphorylation of Taz1 and EnvZ is an intermolecular phosphorylation reaction, requiring formation of a dimer (or oligomer), and that ligand-dependent ompC expression requires not only kinase but also phosphatase activity.

Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA

The yeast mitochondrial group II intron bI1 is self-splicing in vitro. We have introduced a deletion of hairpin C1 within the structural domain 1 that abolishes catalytic activity of the intron in the normal splicing reaction in cis, but does less severely affect a reaction in trans, the reopening of ligated exons. Since exon reopening is supposed to correspond to a reverse 3′cleavage this suggests that the deletion specifically blocks the first reaction step. The intron regains its activity to self-splice in cis by intermolecular complementation with a small RNA harbouring sequences lacking in the mutant intron. These results demonstrate the feasibility to reconstitute a functionally active structure of the truncated intron by intermolecular complementation in vitro. Furthermore, the data support the hypothesis that group II introns are predecessors of nuclear pre-mRNA introns and that the small nuclear RNAs of the spliceosome arose by segregation from the original intron.