Three-protein Complex Research Articles

Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

Tropomyosin R160A is crucial for TnI phosphorylation-induced effects on calcium sensitivity.

The thin filament, consisting of the three-protein complex troponin (TnI, TnT, and TnC) and tropomyosin (Tpm) bound to filamentous actin, acts as the main transducer that relates sarcoplasmic calcium levels to the contraction-relaxation cycles of the myocardium. In the absence of calcium, TnI biases Tpm in a position on actin that blocks myosin binding whereas calcium binding to TnC causes a propagated conformational change in Tn/Tpm that relieves this inhibition. During heart failure and adrenergic stimulation, phosphorylation of the N-terminal region of troponin I (TnI) at residues S23 and S24, has been shown to play a role in heart muscle adaptation through a decrease in calcium sensitivity. Previous modeling studies performed in thin filament models have suggested that positively charged groups on Tpm, namely R160, could interact with the phosphorylated serine residues on TnI. Here we tested the hypothesis that interactions between Tpm-R160 and phosphorylated S23/24 participate in phosphorylation-induced effects on calcium sensitivity (Pavadai et al., 2022). Tpm mutated from wild-type R160 to alanine (Tpm-R160A) and phosphomimetic TnI affecting both serine 23 and 24 (S23/24D) were generated and expressed recombinantly. Using in vitro motility (IVM) assays, we quantified the effects of these mutations alone and in combination. Regulated IVM experiments using thin filaments reconstituted with phosphomimetic S23/24D-TnI and wild-type Tpm revealed a decrease in calcium sensitivity, showing that phosphorylation is successfully mimicked by mutating serine to an aspartic acid,consistent with previous studies on human cardiac tissue. However, when TnI-S23/24D is paired with mutant R160A-Tpm, the phosphorylation-induced changes in calcium sensitivity are diminished. These results are consistent with Tpm-R160 participating in the lusitropy observed when the N-terminal of TnI is phosphorylated.

Assembly of a three-protein complex on DNA probed by nanofluidic stretching.

Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. While the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail.

Reduction of Drosophila Mitochondrial RNase P in Skeletal and Heart Muscle Causes Muscle Degeneration, Cardiomyopathy, and Heart Arrhythmia.

In this study, we examine the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. The loss of mitochondrial function can cause devastating, often multisystemic failures. When mitochondria do not provide enough energy and metabolites, the result can be skeletal muscle weakness, cardiomyopathy, and heart arrhythmias. These symptoms are complex and often difficult to interpret, making disease models useful for diagnosing disease onset and progression. Previously, we identified Drosophila orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) and found that the loss of each subunit causes lethality and decreased mitochondrial tRNA processing in vivo. Here, we use Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. We find that mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, we find that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully. The phenotypes shown here are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues.

In Vitro Reconstitution of Cinnamoyl Moiety Reveals Two Distinct Cyclases for Benzene Ring Formation.

Cinnamoyl-containing natural products (CCNPs) are a small class of bacterial metabolites with notable bioactivities. The biosynthesis of cinnamoyl moiety has been proposed to be assembled by an unusual highly reducing (HR) type II polyketide synthases (PKS). However, the biosynthetic route, especially the cyclization step for the benzene ring formation, remains unclear. In this work, we successfully reconstituted the pathway of cinnamoyl moiety in kitacinnamycin biosynthesis through a step-wise approach in vitro and demonstrated that a three-protein complex, Kcn17-Kcn18-Kcn19, can catalyze 6π-electrocyclization followed by dehydrogenation to form the benzene ring. We found that the three-protein homologues were widely distributed among 207 HR type II PKS biosynthetic gene clusters including five known CCNPs. In contrast, in the biosynthesis of youssoufene, a cinnamoyl-containing polyene, we identified that the benzene ring formation was accomplished by a distinct orphan protein. Thus, our work resolved the long-standing mystery in cinnamoyl biosynthesis and revealed two distinct enzymes that can synthesize benzene rings via polyene precursors.

Polycistronic baculovirus expression of SUGT1 enables high-yield production of recombinant leucine-rich repeat proteins and protein complexes

The SHOC2-MRAS-PPP1CA (SMP) complex is a holoenzyme that plays a vital role in the MAP kinase signaling pathway. Previous attempts to produce this challenging three-protein complex have relied on co-infection with multiple viruses and the use of affinity tags to attempt to isolate functional recombinant protein complexes. Leucine-rich repeat containing proteins have been historically challenging to express, and we hypothesized that co-expression of appropriate chaperones may be necessary for optimal production. We describe here how the SUGT1 chaperone can, in conjunction with polycistronic protein expression in baculovirus-infected insect cells, dramatically enhance production yield and quality of recombinant SHOC2, the SMP complex, and other leucine-rich repeat proteins.

Single-molecule imaging of cytoplasmic dynein in cellulo reveals the mechanism of motor activation and cargo capture

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin and a cargo adaptor. How this process of dynein activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualise and track fluorescently tagged dynein.

Abstract 123: Intestinal Deletion Of 3-hydroxy-3-methylglutaryl-coenzyme A Reductase Promotes Expansion Of The Resident Stem Cell Compartment

While the intestine is the critical interface between cholesterol absorption and excretion, surprisingly little is known about the role of de novo cholesterol synthesis in this organ and how it affects whole body cholesterol homeostasis. The mevalonate pathway is most well-known for the production of cholesterol, but it is also required for the production of essential non-sterol isoprenoids. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, is regulated by a three-protein complex made up of INSIG, SCAP, and SREBP2. Intestine specific knockouts of Scap and Srebp2 both result in severe enteropathy and reduced mouse survival. Here, we assessed the hypothesis that Hmgcr is required for enterocyte viability. Mice harboring floxed alleles for Hmgcr were bred with the Villin-Cre transgene to specifically knock out this enzyme in the villus and crypt epithelial cells of the small intestine (i-KO). The i-KO mice are viable through adulthood and fertile. Hmgcr is efficiently deleted based on analysis via drop digital PCR and qPCR. RNA sequencing shows reduction in all SREBP2 target genes throughout the mevalonate pathway in intestinal epithelial cells. Lipidomics confirms substantial reductions in abundance of all sterol and non-sterol isoprenoids, except 7-dehydrocholesterol and cholesterol. Cholesterol is likely maintained through reabsorption of biliary cholesterol or increased uptake from the circulation. Circulating cholesterol levels and cholesterol absorption are not altered in i-KO mice, but triglyceride absorption is increased through compensatory changes in bile acid composition and intestinal growth. At the cellular level, the intestine rapidly compensates for loss of Hmgcr via dramatic expansion of the stem cell compartment within the crypts. In conclusion, the mechanisms by which the intestine compensates for genetic loss of Hmgcr include altered triglyceride absorption, bile acid composition, increased absorptive surface area, and expansion of the resident stem cell compartment. Together these studies provide insight into the effects of HMGCR knockout in intestinal development and lipid metabolism.

Loss of Individual Mitochondrial Ribonuclease P Complex Proteins Differentially Affects Mitochondrial tRNA Processing In Vivo.

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5′-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.

Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions.

The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.

Vaccinia Virus Glycoproteins A33, A34, and B5 Form a Complex for Efficient Endoplasmic Reticulum to trans-Golgi Network Transport.

Orthopoxviruses produce two, antigenically distinct, infectious enveloped virions termed intracellular mature virions and extracellular virions. Extracellular virions are required for cell-to-cell spread and pathogenesis. Specific to the extracellular virion membrane, glycoproteins A33, A34, and B5 are highly conserved among orthopoxviruses and have roles during extracellular virion formation and subsequent infection. B5 is dependent on an interaction with either A33 or A34 for localization to the site of intracellular envelopment and incorporation into the envelope of released extracellular virions. In this report we show that an interaction between A33 and A34 can be detected in infected cells. Furthermore, we show that a three-protein complex between A33, A34, and B5 forms in the endoplasmic reticulum (ER) that disassociates post ER export. Finally, immunofluorescence reveals that coexpression of all three glycoproteins results in their localization to a juxtanuclear region that is presumably the site of intracellular envelopment. These results demonstrate the existence of two previously unidentified interactions: one between A33 and A34 and another simultaneous interaction between all three of the glycoproteins. Furthermore, these results indicate that interactions among A33, A34, and B5 are vital for proper intracellular trafficking and subcellular localization.IMPORTANCE The secondary intracellular envelopment of poxviruses at the trans-Golgi network to release infectious extracellular virus (EV) is essential for their spread and pathogenesis. Viral glycoproteins A33, A34, and B5 are critical for the efficient production of infectious EV and interactions among these proteins are important for their localization and incorporation into the outer extracellular virion membrane. We have uncovered a novel interaction between glycoproteins A33 and A34. Furthermore, we show that B5 can interact with the A33-A34 complex. Our analysis indicates that the three-protein complex has a role in ER exit and proper localization of the three glycoproteins to the intracellular site of wrapping. These results show that a complex set of interactions occur in the secretory pathway of infected cells to ensure proper glycoprotein trafficking and envelope content, which is important for the release of infectious poxvirus virions.

Articles of Significant Interest in This Issue

Three glycoproteins of vaccinia virus must localize to the intracellular envelopment site at the trans-Golgi network (TGN) to ensure proper glycoprotein content and infectious virus production. Monticelli et al. (e02155-19) discovered a set of novel glycoprotein interactions and the formation of a three-protein complex that results in efficient localization of all three glycoproteins at the TGN where envelopment occurs. These findings shed light on the events that mediate intracellular wrapping of orthopoxviruses and other enveloped viruses.

HLA-B-associated transcript 3 (Bat3) stabilizes and activates p53 in a HAUSP-dependent manner.

The p53 pathway is a highly complex signaling network including several key regulators. HAUSP is a critical component of the p53 pathway acting as a deubiquitinase for both p53 and its key repressor Mdm2. Here, we identified a novel HAUSP-interacting protein, HLA-B-associated transcript 3 (Bat3) and found it to be capable of inducing p53 stabilization and activation via a HAUSP-dependent mechanism, resulting in cell growth inhibition. Surprisingly, the deubiquitylating enzymatic activity of HAUSP was not required for this phenomenon. Co-immunoprecipitation showed that p53 coexisted in a complex with Bat3 and HAUSP in vivo, and HAUSP may serve as a binding mediator to enhance the interaction between p53 and Bat3. Further studies revealed that formation of this three-protein complex interfered with the binding of p53 to its proteasome receptor S5a and promoted the accumulation of p53 in nucleus. Notably, Mdm2 protein abundance is also regulated by Bat3 in the presence of HAUSP. Overexpression of Bat3 and HAUSP increases Mdm2 protein levels without influencing the p53–Mdm2 interaction and Mdm2-mediated p53 ubiquitination, indicating that Bat3–HAUSP-mediated protein stabilization is not specific to p53 and different mechanisms may be involved in Bat3-mediated regulation of p53–Mdm2 pathway. Together, our study unravels a novel mechanism by which p53 is stabilized and activated by HAUSP-mediated interaction with Bat3 and implies that Bat3 might function as a tumor suppressor through the stabilization of p53.

Interplay between substrate recognition, 5' end tRNA processing and methylation activity of human mitochondrial RNase P.

Human mitochondrial ribonuclease P (mtRNase P) is an essential three-protein complex that catalyzes the 5′ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). Mitochondrial RNase P Protein 3 (MRPP3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl-methionine (SAM)-dependent methyltransferase MRPP1/2 subcomplex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here, we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the TRMT10C (MRPP1) cofactor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process, and methylate human mitochondrial pre-tRNAs.

Blocking single-stranded transferred DNA conversion to double-stranded intermediates by overexpression of yeast DNA REPLICATION FACTOR A.

Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the β-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that double-stranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process.

The 3′ Overhangs at Tetrahymena thermophila Telomeres Are Packaged by Four Proteins, Pot1a, Tpt1, Pat1, and Pat2

Although studies with the ciliate Tetrahymena thermophila have played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement of Tetrahymena telomere proteins has not yet been identified. Previously, we demonstrated that in Tetrahymena, the telomeric 3' overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associated Tetrahymena 2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.

Temperature-dependent instability of the cTnI subunit in NIST SRM2921 characterized by tryptic peptide mapping

In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC–MS/MS. From a Mascot database search in combination with “semiTrypsin” tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of “unexpected” peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a “single-step” MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed.

A ribosome-associating factor chaperones tail-anchored membrane proteins

Hundreds of proteins are post-translationally inserted into the endoplasmic reticulum (ER) membrane by a single carboxy-terminal transmembrane domain (TMD)1. During targeting through the cytosol, the hydrophobic TMD of these tail-anchored (TA) proteins requires constant chaperoning to prevent aggregation or inappropriate interactions. A central component of this targeting system is TRC40, a conserved cytosolic factor that recognizes the TMD of TA proteins and delivers them to the ER for insertion2-4. The mechanism that permits TRC40 to effectively find and capture its TA protein cargos in a highly crowded cytosol is unknown. Here, we identify a conserved three-protein complex composed of Bat3, TRC35, and Ubl4A that facilitates TA protein capture by TRC40. This Bat3 complex is recruited to ribosomes synthesizing membrane proteins, interacts with the TMDs of newly released TA proteins, and transfers them to TRC40 for targeting. Depletion of the Bat3 complex allows non-TRC40 factors to compete for TA proteins, explaining their mislocalization in the analogous yeast deletion strains5-7. Thus, the Bat3 complex acts as a TMD selective chaperone that effectively channels TA proteins to the TRC40 insertion pathway.

Chemomechanical Regulation of SNARE Proteins Studied with Molecular Dynamics Simulations

SNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress.

An Unusual Pattern of CytR and CRP Binding Energetics at Escherichia coli cddP Suggests a Unique Blend of Class I and Class II Mediated Activation

Two transcription factors, CRP and CytR, mediate positive and negative control of nine cistrons involved in nucleoside catabolism and recycling in Escherichia coli. The ability of multiple transcription factors to combine in different ways to confer differential gene regulation is of significant interest in both prokaryotic and eukaryotic gene regulation. Analysis of cooperative interactions between CytR and CRP at the deoP2 and udpP promoters has implicated the importance of promoter architecture in controlling repression and induction. These studies have also identified competition between CytR and CRP as an additional contributor to differential regulation. The pattern and energetics of CytR and CRP interactions at the cdd promoter, the most strongly activated of the CytR-regulated promoters, have been delineated using DNase I footprinting. Surprisingly, CRP has greater affinity for the promoter proximal site at cddP, CRP1, than for the distal site, CRP2, in contrast to promoters studied previously. This difference is a major contributor to unusually high CRP-mediated activation of cddP. Additionally, while cytidine binding to CytR nearly eliminates the pairwise interactions between CytR and CRP bound at CRP1, it has little effect on pairwise cooperativity between CytR and CRP bound at CRP2 or as a consequence on the overall cooperativity of the three-protein complex in which CRP is bound to both sites. The effect of cytidine binding on cooperativity differs between the three promoters studied thus far. We propose that the different patterns of interaction reflect the spacing between CytR half-sites and the location of the CytR operator in relation to the two CRP sites.