Tet(O) is an elongation factor‐like protein which confers resistance to the protein synthesis inhibitor tetracycline by promoting the release of the drug from its inhibitory site on the ribosome. ...Here we investigated the interaction of Tet(O) with the elongating ribosome and show, using dimethyl sulfate (DMS) probing and binding assays, that it interacts preferentially with the post‐translocational ribosome. Furthermore, using an XTP‐dependent mutant of Tet(O), we demonstrated that Tet(O) induces conformational rearrangements within the ribosome which can be detected by EF‐Tu, and manifested as a stimulation in the GTPase activity of this elongation factor. As such, these conformational changes probably involve the ribosomal GTPase‐associated center and, accordingly, Tet(O) alters the DMS modification pattern of the L11 region. Additionally, tetracycline binding is associated with an Ea of 58 kJ/mol. These results suggest a model where both Tet(O) and tetracycline induce a conformational change in functionally opposite directions and the Tet(O)‐induced conformation persists after it has left the ribosome; this prevents rebinding of the drug while allowing productive A‐site occupation by a ternary complex in the presence of tetracycline.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we ...present a cryo-electron microscopic reconstruction, at 16 Å resolution, of its complex with the
E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Reconstitution into proteoliposomes is a powerful method for studying calcium transport in a chemically pure membrane environment. By use of this approach, we have studied the regulation of ...Ca2+-ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Co-reconstitution of PLB and Ca2+-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca2+-ATPase (K Ca) and unexpected effects of PLB on maximal activity (Vmax). Wild-type PLB, six loss-of-function mutants (L7A, R9E, I12A, N34A, I38A, L42A), and three gain-of-function mutants (N27A, L37A, and I40A) were evaluated for their effects on K Ca and V max. With the loss-of-function mutants, their ability to shift K Ca correlated with their ability to increase V max. A total loss-of-function mutant, N34A, had no effect on K Ca of the calcium pump and produced only a marginal increase in V max. A near-wild-type mutant, I12A, significantly altered both K Ca and V max of the calcium pump. With the gain-of-function mutants, their ability to shift K Ca did not correlate with their ability to increase V max. The “super-shifting” mutants N27A, L37A, and I40A produced a large shift in K Ca of the calcium pump; however, L37A decreased V max, while N27A and I40A increased V max. For wild-type PLB, phosphorylation completely reversed the effect on K Ca, but had no effect on V max. We conclude that PLB increases V max of Ca2+-ATPase, and that the magnitude of this effect is sensitive to mutation. The mutation sensitivity of PLB Asn34 and Leu37 identifies a region of the protein that is responsible for this regulatory property.
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IJS, KILJ, NUK, PNG, UL, UM
The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for ...the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane α-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.
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IJS, KILJ, NUK, PNG, UL, UM
Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca(2+)-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport ...properties. PLB interacts with and regulates the cardiac Ca(2+)-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca(2+)-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca(2+)-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups ofEscherichia coli ...Me2SO reductase (DmsABC). In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = −120 mV 4Fe-4S cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA. This interaction is significantly modified in a DmsA-C38S mutant that contains a 3Fe-4S cluster in DmsA, suggesting that the 3Fe-4S cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = −120 mV cluster of DmsB. In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway. In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized 3Fe-4S cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum. In a double mutant, DmsAS176ABC102SC, which contains an engineered 3Fe-4S cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized 3Fe-4S cluster and the Mo(V). These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the 4Fe-4S clusters of DmsB to the Mo-bisMGD of DmsA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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