The crystal structure of IB␣ in complex with the transcription factor, nuclear factor -B (NF-B) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high-affinity binding. To characterize the folded state of IB␣ (67-317) when it is not in complex with NF-B, we have carried out circular dichroism (CD) spectroscopy, 8-anilino-1-napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS-binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. IB␣ unfolds with a T m of 42°C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IB␣ are discussed.

SUMMARY Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO3−. The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into 3-dimensional collagen matrix.

SUMMARYIκBα is an ankyrin repeat protein that inhibits NF-κB transcriptional activity by sequestering NF-κB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IκBα ankyrin repeat domain to NF-κB(p50/p65) using surface plasmon (SPR) resonance and isothermal titration calorimetry (ITC). SPR data showed that the IκBα and NF-κB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K D.obs of approximately 40 pM at 37 °C. As reported previously, the amino-terminal/DNA binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high affinity binding. This was surprising given the small size of the binding interface formed by this part of the p65. The NF-κB(p50/p65) heterodimer and p65 homodimer bound IκBα with almost indistinguishable thermodynamics except that the NF-κB p65 homodimer was characterized by a more favorable ΔH obs relative to the NF-κB (p50p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (ΔC P,obs ), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be readily accounted for by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can enable the target protein to fold free from interference. Using both naturally-occurring and artificial polypeptides we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have low sequence complexity and high net charge, but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently-used fusions such as maltose-binding-protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physio-chemical properties they did not perturb the structure, conformational stability nor function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically-disordered fusions, and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

One advantage of detecting amide H/2 H exchange by mass spectrometry instead of NMR is that the more rapidly exchanging surface amides are still detectable. In this study, we present quench-flow amide H/ 2 H exchange experiments to probe how rapidly the surfaces of two different proteins exchange. We compared the amide H/ 2 H exchange behavior of thrombin, a globular protein, and IB␣, a nonglobular protein, to explore any differences in the determinants of amide H/ 2 H exchange rates for each class of protein. The rates of exchange of only a few of the surface amides were as rapid as the "intrinsic" exchange rates measured for amides in unstructured peptides. Most of the surface amides exchanged at a slower rate, despite the fact that they were not seen to be hydrogen bonded to another protein group in the crystal structure. To elucidate the influence of the surface environment on amide H/ 2 H exchange, we compared exchange data with the number of amides participating in hydrogen bonds with other protein groups and with the solvent accessible surface area. The best correlation with amide H/ 2 H exchange was found with the total solvent accessible surface area, including side chains. In the case of the globular protein, the correlation was modest, whereas it was well correlated for the nonglobular protein. The nonglobular protein also showed a correlation between amide exchange and hydrogen bonding. These data suggest that other factors, such as complex dynamic behavior and surface burial, may alter the expected exchange rates in globular proteins more than in nonglobular proteins where all of the residues are near the

The solvent accessibility of thrombin in its substrate-free and substrate-bound forms has been compared by amide hydrogen/deuterium (H/(2)H) exchange. The optimized inhibitor peptide dPhe-Pro-Arg chloromethyl ketone (PPACK) was used to simulate the substrate-bound form of thrombin. These studies were motivated by the lack of observed changes in the active site of thrombin in the crystal structure of the thrombin-thrombomodulin complex. This result appeared to contradict amide exchange studies on the thrombin-thrombomodulin complex that suggested subtle changes occur in the active site loops upon thrombomodulin binding. Our results show that two active site loops, residues 214-222 and residues 126-132, undergo decreases in solvent accessibility due to steric contacts with PPACK substrate. However, we also observe two regions outside the active site undergoing solvent protection upon substrate binding. The first region corresponds to anion binding exosite 1, and the second is a beta-strand-containing loop which runs through the core of the molecule and contains Trp141 which makes critical contacts with anion binding exosite 1. These results indicate two pathways of allosteric change that connect the active site to the distal anion binding exosite 1.

Human melanomas show oncogenic B-Raf mutations which activate the B-Raf/MKK/ERK cascade. We screened microarrays to identify cellular targets of this pathway, and found that genes upregulated by B-Raf/MKK/ERK showed highest association with cell cycle regulators, whereas genes downregulated were most highly associated with axon guidance genes, including plexin-semaphorin family members. Plexin B1 was strongly inhibited by MAP kinase signaling in melanoma cells and melanocytes. In primary melanoma cells, plexin B1 blocked tumorigenesis as measured by growth of colonies in soft agar, spheroids in extracellular matrix, and xenograft tumors. Tumor suppression depended on residues in the C-terminal domain of plexin B1 which mediate receptor GAP activity, and also correlated with AKT inhibition. Interestingly, the inhibitory response to plexin B1 was reduced or absent in cells from a matched metastatic tumor, suggesting that changes occur in metastatic cells which bypass the tumor suppressor mechanisms. Plexin B1 also inhibited cell migration, but this was seen in metastatic cells and not in matched primary cells. Thus, plexin B1 has tumor suppressor function in early-stage cells, while suppressing migration in late-stage cells. Our findings suggest that B-Raf/MKK/ERK provides a permissive environment for melanoma genesis by modulating plexin B1.

The M 4 receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M 4 allosteric functional screen. Although unsuitable as a therapeutic due to M 2 receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 59-[g- triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M 4 receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M 2 receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M 2 active-state structure recently solved (Kruse et al., 2013), a structure that provides crucial insights into the mechanisms of orthosteric activation and allosteric modulation of muscarinic receptors.