Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]²⁺ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]²⁺ clusters, two [2Fe-2S]²⁺ clusters are converted into a [4Fe-4S]²⁺ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]²⁺ cluster from ISCA1-ISCA2 complex. A structural view of the protein–protein recognition events occurring along the [4Fe-4S]²⁺ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]²⁺ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]²⁺ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]²⁺ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]²⁺ cluster transfer.     

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

The conformational landscape of the ribosomal protein S15 and its influence on the protein interaction with 16S RNA

The interaction between the ribosomal protein S15 and its binding sites in the 16S RNA was examined from two points of view. First, the isolated protein S15 was studied by comparing NMR conformer sets, available in the PDB and recalculated using the CNS-ARIA protocol. Molecular dynamics (MD) trajectories were then recorded starting from a conformer of each set. The recalculation of the S15 NMR structure, as well as the recording of MD trajectories, reveals that several orientations of the N-terminal alpha-helix alpha1 with respect to the structure core are populated. MD trajectories of the complex between the ribosomal protein S15 and RNA were also recorded in the presence and absence of Mg(2+) ions. The Mg(2+) ions are hexacoordinated by water and RNA oxygens. The coordination spheres mainly interact with the RNA phosphodiester backbone, reducing the RNA mobility and inducing electrostatic screening. When the Mg(2+) ions are removed, the internal mobility of the RNA and of the protein increases at the interaction interface close to the RNA G-U/G-C motif as a result of a gap between the phosphate groups in the UUCG capping tetraloop and of the disruption of S15-RNA hydrogen bonds in that region. On the other hand, several S15-RNA hydrogen bonds are reinforced, and water bridges appear between the three-way junction region and S15. The network of hydrogen bonds observed in the loop between alpha1 and alpha2 is consequently reorganized. In the absence of Mg(2+), this network has the same pattern as the network observed in the isolated protein, where the helix alpha1 is mobile with respect to the protein core. The presence of Mg(2+) ions may thus play a role in stabilizing the orientation of the helix alpha1 of S15.     

Physical characteristics of ribosomal protein S4 from Escherichia coli

A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric. A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model. This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies. This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality. Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4.     

Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension

We have examined the effect of binding ribosomal protein S4 to 16 S rRNA on the susceptibility of the RNA to a variety of chemical and enzymatic probes. We have used dimethyl sulfate to probe unpaired adenines (at N-1) and cytosines (at N-3), kethoxal to probe unpaired guanines (at N-1 and N-2) and cobra venom (V1) ribonuclease as a probe of base-paired regions of 16 S rRNA. Sites of attack by the probes were identified by primer extension using synthetic oligodeoxynucleotides. Comparison of probing results for naked and S4-bound rRNA shows: Protein S4 protects a relatively compact region of the 5' domain of 16 S rRNA from chemical and enzymatic attack. This region is bounded by nucleotides 27 to 47 and 394 to 556, and has a secondary structure characterized by the junction of five helical elements. Phylogenetically conserved irregular features (bulged nucleotides, internal loops and flanking unpaired nucleotides) and helical phosphodiester bonds of four of the helices are specifically protected in the S4-RNA complex. We conclude that this is the major, and possibly sole region of contact between 16 S rRNA and S4. Many of the S4-dependent changes mimic those observed on assembly of 16 S rRNA into 30 S ribosomal subunits. Binding of S4 causes enhanced chemical reactivity coupled with protection from V1 nuclease outside the S4 junction region in the 530, 720 and 1140 loops. We interpret these results as indicative of loss of structure, and suggest that S4 binding causes disruption of adventitious pairing in these regions, possibly by stabilizing the geometry of the RNA such that these interactions are prevented from forming.     

Structural studies on the 30 S ribosomal subunit from Escherichia coli

Small-angle X-ray scattering curves computed for various 30 S subunit structures have been compared with the experimental scattering curve. The curve from the 30 S subunit is best approximated by that calculated for a 1:3.6:3.6 ellipsoidal structure. The rather prolate ellipsoidal model suggested by recent electron microscope studies gives a scattering curve considerably different from the 30 S curve, suggesting that the electron microscope model is not that found in solution. Analysis of the more extended portions of the experimental scattering curve suggests some internal structure. A model is proposed that contains RNA and protein in positions such that the calculated scattering curve shows more extensive, yet similar internal structure. Resultant constraints on the structure of the 30 S subunit in solution are given.     

Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes

C4b-binding protein (C4BP) is a large complex assembly of eight subunits that functions as an inhibitor of the complement cascade. A portion of the C4BP in serum exists as a complex with protein S. This study demonstrated that another protein, serum amyloid P component (SAP), also formed a calcium-dependent complex with C4BP. The C4BP.SAP complex was detected by several methods including light scattering intensity, gel filtration, and sucrose density gradient ultracentrifugation. This complex was of high affinity relative to serum levels of these proteins so that no dissociation was detected at 3% of serum protein concentrations. The C4BP.SAP complex was also detected in normal serum and the results suggested that there was virtually no free SAP or uncomplexed C4BP in normal serum. In addition to its complex with C4BP, SAP underwent other calcium-dependent associations such as binding to phospholipid vesicles and self-aggregation. Self-aggregation was highly cooperative with kinetics corresponding to a reaction that was 6th-order with respect to calcium and required about 1.5 mM calcium. In contrast, formation of the SAP.C4BP complex and interaction of SAP with membranes required only about 0.4 and 1.0 mM calcium, respectively. Thus, selection of the correct conditions allowed study of the SAP.C4BP interaction without interference from self-aggregation. All three of these interactions of SAP were mutually exclusive and the SAP. C4BP interaction appeared to be favored over self-aggregation or binding of SAP to phospholipids. It seems likely that the biologically dominant interaction for SAP is with C4BP. The SAP.C4BP complex interacted with protein S and these binding sites appeared to be entirely independent. Furthermore, SAP had little or no effect on the ability of C4BP to bind C4b. Finally, the entire complex of proteins (C4BP, SAP, protein S, and C4b) could associate with membranes in the presence of calcium. Membrane binding occurred through the protein S component. This rather complicated assemblage of proteins probably functions in a regulatory role for the complement cascade or other biological systems. It is possible that elevated levels of SAP or nonequivalent levels of SAP and C4BP could contribute to certain pathological conditions.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.     

ISCA1 Orchestrates ISCA2 and NFU1 in the Maturation of Human Mitochondrial [4Fe-4S] Proteins

The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2, which, instead do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, thanks to its specific interaction with the C-terminal cluster-binding domain of NFU1, drives [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from where it is assembled on the ISCA1-ISCA2 complex to NFU1, thereby resulting the [4Fe-4S] cluster available for the mitochondrial apo proteins specifically requiring NFU1 for their maturation.     

An RNA core in the 30S ribosomal subunit of Escherichia coli and its structural and functional significance.

1. Evidence is presented for the occurrence of a very stable RNA core (S4-RNA) in "native" 16S RNA that is also present in the 30S subunit of Escherichia coli. A model giving the approximate location of this RNA core in the 30S subunit is presented. 2. It is proposed (a) that this S4-RNA acts as a nucleus for the assembly of the 30S subunit, and (b) that a small class of "linkage" proteins, including S4, further facilitate the assembly of the proteins to the RNA, thereby explaining some of the "cooperative" effects that are observed during in vitro assembly. 3. Evidence for the importance of the RNA core in the functioning of the ribosome is discussed.     

Assembly of protein S and C4b-binding protein on membranes

The interaction of protein S with membranes and subsequent combination with complement C4b-binding protein (C4BP) was studied. Protein S interacted with phospholipid vesicles in a calcium-dependent manner typical of other vitamin K-dependent proteins. Association of C4BP with protein S showed no apparent selectivity for membrane-bound or solution phase protein S. When bound to the membrane, the protein complexes projected out from the vesicle surface and induced vesicle radius changes of 11.4 nm for tightly packed protein S alone and 17.5 nm for the protein S-C4BP complex. Due to a low density of the protein S-C4BP on the membrane at saturation, the actual projection of this complex out from the membrane surface would be much greater than 17.5 nm. A low saturation density suggested that the protein complex had a large two-dimensional hydrodynamic radius in the plane of the membrane that prevented tight packing of protein. In the presence of calcium, the protein-protein interaction was rapid (ka greater than or equal to 1.10(6) M-1 s-1) and had very high affinity (KD less than or equal to 10(-10) M). The dissociation rate was slow with an estimated rate constant of less than or equal to 2.10(-4) s-1 at 25 degrees C. Protein-protein interaction was much slower in the absence of calcium with an estimated association rate constant of only 2.10(4) M-1 s-1. Consequently, the protein-protein interaction was greatly enhanced by calcium. The very high affinity interaction between protein S and C4BP suggested specificity and an important function for the protein S-C4BP complex in blood. In this regard it was important that C4BP which was bound to protein S on the phospholipid surface could interact with complement protein C4b. These results suggested that protein S may serve an important role in localizing C4BP to negatively charged phospholipid. This would provide regulation of complement activation at sites where the coagulation system is activated such as on the surface of activated platelets.     

RNA-protein cross-linking in Eschericia coli 30S ribosomal subunits: a method for the direct analysis of the RNA regions involved in the cross-links.

A prerequisite for topographical studies on ribosomal subunits involving RNA-protein cross-linking is that the cross-linking sites on the RNA should be determined. Methodology is presented which offers a solution to this problem, using as a test system 30S subunits in which protein S7 has been cross-linked to the 16S RNA by ultraviolet irradiation. The method is based on a gel separation system in the presence of a non-ionic detergent. When a ribonucleoprotein fragment containing RNA-protein cross-links is applied to this system, non-cross-linked protein is removed, and simultaneously the cross-linked RNA-protein complex is separated from non-cross-linked RNA. Oligonucleotide analysis of the S7-RNA complex isolated in this manner showed it to consist of a region of RNA from sections P-A of the 16S RNA. A single characteristic oligonucleotide was absent from this region, and it was tentatively concluded that this missing oligonucleotide contains the actual site of cross-linking.     

Identification of a protein at the ribosomal donor-site by affinity labeling

p-nitrophenylcarbamyl-methionyl-tRNA_f^Met is shown to act as an analogue of fMet-tRNA_f^Met in initiation complex formation. It binds to E. coli ribosomes in the presence of initiation factors and R 17-RNA as messenger. Covalent bond formation occurs in the complex between the Met-tRNA_f^Met derivative and protein of the 50 S ribosomal subunit. The protein labeled predominantly in the reaction has been identified as L 27 indicating that this protein is located at the donor-site of the ribosome.     

Localisation of part of the binding sites of 30S ribosomal proteins S4 and S20 in a small uninterrupted fragment of 16S RNA.

Analyses of the T1 ribonuclease-alkaline phosphatase fingerprint of a continuous fragment of the 16S rRNA, 170-230 nucleotides long, isolated from the products of autodigestion of 30S ribosome subunits show that it contains a sequence near the 5'-phosphate terminus of intact 16S rRNA and corresponds to segment H'-M of this molecule as defined by Ehresmann et al [29]. Incubation of this fragment with total 30S ribosomal proteins under reconstitution conditions leads to the formation of a complex containing proteins S4, S20, and one or both of proteins S16 and S17. The stoichiometry of these proteins in the complex is discussed.     

Role of p70S6K1-mediated Phosphorylation of eIF4B and PDCD4 Proteins in the Regulation of Protein Synthesis

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5′-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.     

Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells

Apoptosis is characterized by a lack of inflammatory reaction in surrounding tissues, suggesting local control of complement activation. During the initial stage of apoptosis, cells expose negatively charged phospholipid phosphatidylserine on their surfaces. The vitamin K-dependent protein S has a high affinity for this type of phospholipid. In human plasma, 60-70% of protein S circulates in complex with C4b-binding protein (C4BP). The reason why protein S and C4BP form a high-affinity complex in plasma is not known. However, C4BP is an important regulator of the classical pathway of the complement system where it acts as a cofactor in degradation of complement protein C4b. Using Jurkat cells as a model system for apoptosis, we now show protein S to bind to apoptotic cells. We further demonstrate protein S-mediated binding of C4BP to apoptotic cells. Binding of the C4BP-protein S complex to apoptotic cells was calcium-dependent and could be blocked with Abs directed against the phospholipid-binding domain in protein S. Annexin V, which binds to exposed phosphatidylserine on the apoptotic cell surface, could inhibit the binding of protein S. The C4BP that was bound via protein S to the apoptotic cells was able to interact with the complement protein C4b, supporting a physiological role of the C4BP/protein S complex in regulation of complement on the surface of apoptotic cells.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.