Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.     

Involvement of the TPR2 subdomain movement in the activities of ?29 DNA polymerase

The polymerization domain of ϕ29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we have constructed a ϕ29 DNA polymerase mutant able to form a disulfide bond between the apexes of TPR2 and thumb to limit the mobility of TPR2. Biochemical analysis of the mutant led us to conclude that TPR2 moves away from the thumb to allow the DNA polymerase to replicate circular ssDNA. Despite the fact that no TPR2 motion is needed to allow the polymerase to use the terminal protein (TP) as primer during the initiation of ϕ29 TP–DNA replication, the disulfide bond prevents the DNA polymerase from entering the elongation phase, suggesting that TPR2 movements are necessary to allow the TP priming domain to move out from the polymerase during transition from initiation to elongation. Furthermore, the TPR2-thumb bond does not affect the equilibrium between the polymerization and exonuclease activities, leading us to propose a primer-terminus transference model between both active sites.     

The Solution Structure of the C-Terminal Ig-like Domain of the Bacteriophage λ Tail Tube Protein

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV_C), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV_C and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV_C by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV_C structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV_C that may be important in mediating the function of this domain.     

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ? Subunit

Escherichia coli ATP synthase (F₀F₁) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F₁-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F₁ rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer¹⁰⁸ in loop 2 and ϵTyr¹¹⁴ in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F₁ rotation through interaction with other subunits.     

N-Terminal Domain of Nuclear IL-1α Shows Structural Similarity to the C-Terminal Domain of Snf1 and Binds to the HAT/Core Module of the SAGA Complex

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

Cleavage of Bacteriophage λ cI Repressor Involves the RecA C-Terminal Domain

The SOS response to DNA damage in Escherichia coli involves at least 43 genes, all under the control of the LexA repressor. Activation of these genes occurs when the LexA repressor cleaves itself, a reaction catalyzed by an active, extended RecA filament formed on DNA. It has been shown that the LexA repressor binds within the deep groove of this nucleoprotein filament, and presumably, cleavage occurs in this groove. Bacteriophages, such as λ, have repressors (cI) that are structural homologs of LexA and also undergo self-cleavage when SOS is induced. It has been puzzling that some mutations in RecA that affect the cleavage of repressors are in the C-terminal domain (CTD) far from the groove where cleavage is thought to occur. In addition, it has been shown that the rate of cleavage of cI by RecA is dependent upon both the substrate on which RecA is polymerized and the ATP analog used. Electron microscopy and three-dimensional reconstructions show that the conformation and dynamics of RecA's CTD are also modulated by the polynucleotide substrate and ATP analog. Under conditions where the repressor cleavage rates are the highest, cI is coordinated within the groove by contacts with RecA's CTD. These observations provide a framework for understanding previous genetic and biochemical observations.     

Structure–Function Analysis of the C-Terminal Domain of the Type VI Secretion TssB Tail Sheath Subunit

The type VI secretion system (T6SS) is a specialized macromolecular complex dedicated to the delivery of protein effectors into both eukaryotic and bacterial cells. The general mechanism of action of the T6SS is similar to the injection of DNA by contractile bacteriophages. The cytoplasmic portion of the T6SS is evolutionarily, structurally and functionally related to the phage tail complex. It is composed of an inner tube made of stacked Hcp hexameric rings, engulfed within a sheath and built on a baseplate. This sheath undergoes cycles of extension and contraction, and the current model proposes that the sheath contraction propels the inner tube toward the target cell for effector delivery. The sheath comprises two subunits: TssB and TssC that polymerize under an extended conformation. Here, we show that isolated TssB forms trimers, and we report the crystal structure of a C-terminal fragment of TssB. This fragment comprises a long helix followed by a helical hairpin that presents surface-exposed charged residues. Site-directed mutagenesis coupled to functional assay further showed that these charges are required for proper assembly of the sheath. Positioning of these residues in the extended T6SS sheath structure suggests that they may mediate contacts with the baseplate.     

Optically Detected Structural Change in the N-Terminal Region of?the?Voltage-Sensor Domain

The voltage-sensor domain (VSD) is a functional module that undergoes structural transitions in response to membrane potential changes and regulates its effectors, thereby playing a crucial role in amplifying and decoding membrane electrical signals. Ion-conductive pore and phosphoinositide phosphatase are the downstream effectors of voltage-gated channels and the voltage-sensing phosphatase, respectively. It is known that upon transition, the VSD generally acts on the region C-terminal to S4. However, whether the VSD also induces any structural changes in the N-terminal region of S1 has not been addressed directly. Here, we report the existence of such an N-terminal effect. We used two distinct optical reporters—one based on the Förster resonance energy transfer between a pair of fluorescent proteins, and the other based on fluorophore-labeled HaloTag—and studied the behavior of these reporters placed at the N-terminal end of the monomeric VSD derived from voltage-sensing phosphatase. We found that both of these reporters were affected by the VSD transition, generating voltage-dependent fluorescence readouts. We also observed that whereas the voltage dependencies of the N- and C-terminal effects appear to be tightly coupled, the local structural rearrangements reflect the way in which the VSD is loaded, demonstrating the flexible nature of the VSD.     

Activation of the B Cell Receptor Leads to Increased Membrane Proximity of the Igα Cytoplasmic Domain

Binding of antigen to the B cell receptor (BCR) induces conformational changes in BCR''s cytoplasmic domains that are concomitant with phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs). Recently, reversible folding of the CD3ε and ξ chain ITAMs into the plasma membrane has been suggested to regulate T cell receptor signaling. Here we show that the Igα and Igβ cytoplasmic domains of the BCR do not associate with plasma membrane in resting B cells. However, antigen binding and ITAM phosphorylation specifically increased membrane proximity of Igα, but not Igβ. Thus, BCR activation is accompanied by asymmetric conformational changes, possibly promoting the binding of Igα and Igβ to differently localized signaling complexes.     

Spatial Organization of the Chicken α-Globin Gene Domain in Cells of Different Origins

Hybridization with an oligonucleotide array was used to map the regions of DNA anchorage to the nuclear matrix. Matrix-associated DNA served as a hybridization probe. To obtain the oligonucleotide array, 60-mer oligonucleotides regularly distributed throughout the genome region of interest at 2-kb intervals were immobilized on a nylon filter. The organization of DNA into loop domains was studied in a 100-kb region of chicken chromosome 16, including the α -globin gene cluster. A 40-kb DNA loop, which was fixed to the nuclear matrix and harbored all α-globin genes, was observed in erythroid cells. One of its anchorage regions colocalized with matrix associated region (MAR) and an insulator found previously in the 5′ region of the chicken α-globin gene domain. The spatial (domain-loop) organization of the α-globin gene cluster in lymphoid cells proved to be strikingly different from that in erythroid cells.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with k_cat values of 1.9, 0.6, and 0.32 min⁻¹, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded K_d values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg²⁺ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased k_cat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.     

Open-Channel Structures of the Human Glycine Receptor α1 Full-Length Transmembrane Domain

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