SrmB Rescues Trapped Ribosome Assembly Intermediates.

RNA helicases play various roles in ribosome biogenesis depending on the ribosome assembly pathway and stress state of the cell. However, it is unclear how most RNA helicases interact with ribosome assembly intermediates or participate in other cell processes to regulate ribosome assembly. SrmB is a DEAD-box helicase that acts early in the ribosome assembly process, although very little is known about its mechanism of action. Here, we use a combined quantitative mass spectrometry/cryo-electron microscopy approach to detail the protein inventory, rRNA modification state, and structures of 40S ribosomal intermediates that form upon SrmB deletion. We show that the binding site of SrmB is unperturbed by SrmB deletion, but the peptidyl transferase center, the uL7/12 stalk, and 30S contact sites all show severe assembly defects. Taking into account existing data on SrmB function and the experiments presented here, we propose several mechanisms by which SrmB could guide assembling particles from kinetic traps to competent subunits during the 50S ribosome assembly process.

Long-range effects and functional consequences of stabilizing mutations in the ankyrin repeat domain of IκBα.

Protein domains containing three or more ankyrin repeats (ARs) are ubiquitous in all phyla. Sequence alignments previously identified certain conserved positions, which have been shown to stabilize AR domains and promote their folding. Consensus mutations [Y254L/T257A (YLTA) and C186P/A220P (CPAP)] stabilize the naturally occuring AR domain of human IκBα to denaturation; however, only the YLTA mutations stabilize the protein to proteasomal degradation. We present results from NMR experiments designed to probe the roles of these consensus mutations in IκBα. According to residual dipolar coupling analysis, the gross structures of the AR domains of both mutants appear to be similar to the wild type (WT). Comparison of chemical shifts of mutant and WT proteins reveals that the YLTA and CPAP consensus mutations cause unexpected long-range effects throughout the AR domains. Backbone dynamics experiments reveal that the YLTA mutations in the sixth AR order the C-terminal PEST sequence on the picosecond-to-nanosecond timescale, compared to either the WT or the CPAP mutant IκBαs. This property is likely the mechanism by which the half-life of YLTA IκBα is extended in vivo.

The RelA nuclear localization signal folds upon binding to IκBα.

The nuclear localization signal (NLS) polypeptide of RelA, the canonical nuclear factor-κB family member, is responsible for regulating the nuclear localization of RelA-containing nuclear factor-κB dimers. The RelA NLS polypeptide also plays a crucial role in mediating the high affinity and specificity of the interaction of RelA-containing dimers with the inhibitor IκBα, forming two helical motifs according to the published X-ray crystal structure. In order to define the nature of the interaction between the RelA NLS and IκBα under solution conditions, we conducted NMR and isothermal titration calorimetry studies using a truncated form of IκBα containing residues 67-206 and a peptide spanning residues 293-321 of RelA. The NLS peptide, although largely unfolded, has a weak tendency toward helical structure when free in solution. Upon addition of the labeled peptide to unlabeled IκBα, the resonance dispersion in the NMR spectrum is significantly greater, providing definitive evidence that the RelA NLS polypeptide folds upon binding IκBα. Isothermal titration calorimetry studies of single-point mutants reveal that residue F309, which is located in the middle of the more C-terminal of the two helices (helix 4) in the IκBα-bound RelA NLS polypeptide, is critical for the binding of the RelA NLS polypeptide to IκBα. These results help to explain the role of helix 4 in mediating the high affinity of RelA for IκBα.

Enhanced conformational space sampling improves the prediction of chemical shifts in proteins.

A biased-potential molecular dynamics simulation method, accelerated molecular dynamics (AMD), was combined with the chemical shift prediction algorithm SHIFTX to calculate (1)H(N), (15)N, (13)Calpha, (13)Cbeta, and (13)C' chemical shifts of the ankyrin repeat protein IkappaBalpha (residues 67-206), the primary inhibitor of nuclear factor kappa-B (NF-kappaB). Free-energy-weighted molecular ensembles were generated over a range of acceleration levels, affording systematic enhancement of the conformational space sampling of the protein. We have found that the predicted chemical shifts, particularly for the (15)N, (13)Calpha, and (13)Cbeta nuclei, improve substantially with enhanced conformational space sampling up to an optimal acceleration level. Significant improvement in the predicted chemical shift data coincides with those regions of the protein that exhibit backbone dynamics on longer time scales. Interestingly, the optimal acceleration level for reproduction of the chemical shift data has previously been shown to best reproduce the experimental residual dipolar coupling (RDC) data for this system, as both chemical shift data and RDCs report on an ensemble and time average in the millisecond range.

Functional dynamics of the folded ankyrin repeats of I kappa B alpha revealed by nuclear magnetic resonance.

Inhibition of nuclear factor kappaB (NF-kappaB) is mainly accomplished by IkappaB alpha, which consists of a signal response sequence at the N-terminus, a six-ankyrin repeat domain (ARD) that binds NF-kappaB, and a C-terminal PEST sequence. Previous studies with the ARD revealed that the fifth and sixth repeats are only partially folded in the absence of NF-kappaB. Here we report NMR studies of a truncated version of IkappaB alpha, containing only the first four ankyrin repeats, IkappaB alpha(67-206). This four-repeat segment is well-structured in the free state, enabling full resonance assignments to be made. H-D exchange, backbone dynamics, and residual dipolar coupling (RDC) experiments reveal regions of flexibility. In addition, regions consistent with the presence of micro- to millisecond motions occur periodically throughout the repeat structure. Comparison of the RDCs with the crystal structure gave only moderate agreement, but an ensemble of structures generated by accelerated molecular dynamics gave much better agreement with the measured RDCs. The regions showing flexibility correspond to those implicated in entropic compensation for the loss of flexibility in ankyrin repeats 5 and 6 upon binding to NF-kappaB. The regions showing micro- to millisecond motions in the free protein are the ends of the beta-hairpins that directly interact with NF-kappaB in the complex.

Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples.

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.

Pre-folding IkappaBalpha alters control of NF-kappaB signaling.

Transcription complex components frequently show coupled folding and binding but the functional significance of this mode of molecular recognition is unclear. IkappaBalpha binds to and inhibits the transcriptional activity of NF-kappaB via its ankyrin repeat (AR) domain. The beta-hairpins in ARs 5-6 in IkappaBalpha are weakly-folded in the free protein, and their folding is coupled to NF-kappaB binding. Here, we show that introduction of two stabilizing mutations in IkappaBalpha AR 6 causes ARs 5-6 to fold cooperatively to a conformation similar to that in NF-kappaB-bound IkappaBalpha. Free IkappaBalpha is degraded by a proteasome-dependent but ubiquitin-independent mechanism, and this process is slower for the pre-folded mutants both in vitro and in cells. Interestingly, the pre-folded mutants bind NF-kappaB more weakly, as shown by both surface plasmon resonance and isothermal titration calorimetry in vitro and immunoprecipitation experiments from cells. One consequence of the weaker binding is that resting cells containing these mutants show incomplete inhibition of NF-kappaB activation; they have significant amounts of nuclear NF-kappaB. Additionally, the weaker binding combined with the slower rate of degradation of the free protein results in reduced levels of nuclear NF-kappaB upon stimulation. These data demonstrate clearly that the coupled folding and binding of IkappaBalpha is critical for its precise control of NF-kappaB transcriptional activity.

Stabilizing IkappaBalpha by "consensus" design.

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.