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Diffusion measurements by pulsed-field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle x-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed-field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed-field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modeling and for force field benchmarking and parameterization. Diffusion measurements by pulsed-field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle x-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed-field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed-field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modeling and for force field benchmarking and parameterization.

All aerobic organisms require the cofactor heme to survive, but its synthesis requires formation of a potentially toxic intermediate protoporphyrin IX (PPIX). Little is known about the extent of PPIX’s cellular interactions. Here, we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics with PPB identified common proteins among a diverse panel of mammalian cell lineages. Pathway and quantitative difference analysis revealed PPB-bound proteins related to iron and heme metabolism and suggested that these processes might be altered by heme/porphyrin synthesis. We show that increased heme/porphyrin synthesis in cells promotes ferroptosis that is pharmacologically distinct from canonical ferroptosis driven by erastin, an inhibitor of the cystine/glutamate antiporter. Proteomic data derived from PPB revealed an interactor, PRDX3, a mitochondrial peroxidase, that modulated heme/porphyrin biosynthesis driven ferroptosis. Consistent with a role in porphyrin-induced ferroptotic death targeted gene knockdown of PRDX3, but not peroxidases, PRDX1 or 2, enhanced porphyrin-induced ferroptotic death. The relationship between increased heme/porphyrin synthesis and ferroptosis was also found in a ferrochelatase-deficient T-lymphoblastoid leukemia cell line, suggesting potential strategy for treating certain cancers. We demonstrate that when the PPB probe is coupled with unbiased proteomics a previously unreported relationship between heme/porphyrin synthesis, and ferroptosis was discovered.

Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation., Graphical Abstract Graphical AbstracthnRNPA1 phase separation is highly salt sensitive. Phase separation of the low-complexity domain (LCD) of hnRNPA1 increases with NaCl. In contrast, phase separation of full-length hnRNPA1 is salt-sensitive. At low NaCl concentrations, electrostatic RRM–LCD interactions occur and can contribute positively to phase separation, but they are screened at high NaCl concentrations. The folded domains solubilize hnRNPA1 under these conditions and prevent phase separation.

Not too sticky There is increasing evidence for a role of liquid-liquid phase separation (LLPS) in many cellular processes. Many proteins that undergo LLPS include prionlike domains (PLDs), which are enriched in polar amino acids and often interspersed with aromatic residues. Combining experimental data with simulations, Martin et al. quantified concentrations of PLDs in coexisting dilute and dense phases as a function of temperature and show that the phase behavior is determined by the number of aromatic residues and their patterning, with uniform patterning of aromatic residues promoting LLPS and inhibiting aggregation. They developed a sticker-and-spacers model that can predict the phase behavior of PLDs on the basis of their sequence. Science , this issue p. 694

Fatty acid (FA) transfer proteins extract FA from membranes and sequester their ligand to facilitate its movement through the cytosol. While detailed views of soluble protein-FA complexes are available, how FA exchange occurs at the membrane has remained unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we determine the conformational change from this closed state to an open state that engages the phospholipid bilayer. Upon membrane binding, a dynamic loop in FakB1 that covers the FA binding site disengages and folds into an amphipathic helix. This helix inserts below the phosphate plane of the bilayer to create a diffusion channel for the FA to exchange between the protein and the membrane. The structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a general conceptual framework for how these proteins interact with the membrane to promote lipid transfer.

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein-FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a "closed" FakB1 state to an "open" state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

Lipidation of Atg8-family ubiquitin-like proteins (UBLs) plays important roles in macroautophagy/autophagy. This process is catalyzed by an E1-E2-E3 trienzyme cascade, in which an E1 enzyme, Atg7, directs Atg8 to its E2 enzyme, Atg3, forming a thioester bond-linked Atg3~ Atg8 intermediate; then the composite E3, Atg12–Atg5-Atg16, interacts with the Atg3~ Atg8 intermediate and promotes Atg8 transfer from the catalytic cysteine of Atg3 to the head group of phosphatidylethanolamine (PE) lipids. Despite progress that has been made toward understanding the Atg8 lipidation pathway, the molecular mechanism of Atg3 as it orchestrates between the E1 and E3 remains unclear. Here we summarize our recent work reporting an element in Atg3, termed the E1, E2, and E3-interacting region (E123IR), is an allosteric switch: in the absence of other binding partners, the E123IR restrains Atg3′s catalytic loop, while the E1 or E3 enzyme directly binds this region to remove this brace and thereby conformationally activate Atg3 to elicit Atg8 lipidation in vitro and in vivo.

Many cell signaling events are coordinated by intrinsically disordered protein regions (IDRs) that undergo multisite Serine/Threonine phosphorylation. The conformational properties of these IDRs prior to and following multi-site phosphorylation are directly relevant to understanding their functions. Here, we present results from biophysical studies and molecular simulations that quantify the conformational properties of an 81-residue IDR from the S. cerevisiae transcription factor Ash1. We show that the unphosphorylated Ash1 IDR adopts coil-like conformations that are expanded and well-solvated. This result contradicts inferences regarding global compaction that are derived from heuristics based on amino acid compositions for IDRs with low proline contents. Upon phosphorylation at ten distinct sites, the global conformational properties of pAsh1 are indistinguishable from those of unphosphorylated Ash1. This insensitivity derives from compensatory changes to the pattern of local and long-range intra-chain contacts. We show that the conformational properties of Ash1 and pAsh1 can be explained in terms of the linear sequence patterning of proline and charged residues vis-à-vis all other residues. The sequence features of the Ash1 IDR are shared by many other IDRs that undergo multisite phosphorylation. Accordingly, we propose that our findings might be generalizable to other IDRs involved in cell signaling.

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub’s C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at “exosites.” However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

Ubiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitin’s C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at “exosites”. However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.SIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12–Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3′s catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3′s E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy., Autophagy mediated by the conjugation pathway for ubiquitin-like proteins plays a key role in controlling homeostasis in eukaryotic cells. Here the authors provide a molecular basis for allosteric activation of the E2 ligase Atg3, uncovering the mechanism underlying Atg8 lipidation and a novel mechanism regulating E1-E2-E3-mediated ubiquitin-like protein conjugation.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli31-90) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3SPOP and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli31-90, the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli31-90 ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification.

Necroptosis is a cell death pathway regulated by the receptor interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) pseudokinase. How MLKL executes plasma membrane rupture upon phosphorylation by RIPK3 remains controversial. Here, we characterize the hierarchical transduction of structural changes in MLKL that culminate in necroptosis. The MLKL brace, proximal to the N-terminal helix bundle (NB), is involved in oligomerization to facilitate plasma membrane targeting through the low-affinity binding of NB to phosphorylated inositol polar head groups of phosphatidylinositol phosphate (PIP) phospholipids. At the membrane, the NB undergoes a “rolling over” mechanism to expose additional higher-affinity PIP-binding sites responsible for robust association to the membrane and displacement of the brace from the NB. PI(4,5)P2 is the preferred PIP-binding partner. We investigate the specific association of MLKL with PIPs and subsequent structural changes during necroptosis.

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

Necroptosis is an inflammatory form of programmed cell death executed through plasma membrane rupture by the pseudokinase mixed lineage kinase domain-like (MLKL). We previously showed that MLKL activation requires metabolites of the inositol phosphate (IP) pathway. Here we reveal that I(1,3,4,6)P(4), I(1,3,4,5,6)P(5), and IP(6) promote membrane permeabilization by MLKL through directly binding the N-terminal executioner domain (NED) and dissociating its auto-inhibitory region. We show that IP(6) and inositol pentakisphosphate 2-kinase (IPPK) are required for necroptosis as IPPK deletion ablated IP(6) production and inhibited necroptosis. The NED auto-inhibitory region is more extensive than originally described and single amino acid substitutions along this region induce spontaneous necroptosis by MLKL. Activating IPs bind three sites with affinity of 100–600 μM to destabilize contacts between the auto-inhibitory region and NED, thereby promoting MLKL activation. We therefore uncover MLKL’s activating switch in NED triggered by a select repertoire of IP metabolites.

The BCL-2-family protein BAK is responsible for mitochondrial outer-membrane permeabilization (MOMP), which leads to apoptosis. The BCL-2 homology 3 (BH3)-only protein BID activates BAK to perform this function. We report the NMR solution structure of the human BID BH3-BAK complex, which identified the activation site at the canonical BH3-binding groove of BAK. Mutating the BAK BH1 in the groove prevented activation and MOMP but not the binding of BID. BAK BH3 mutations allowed BID binding and activation but blunted function by blocking BAK oligomerization. BAK activation follows a 'hit-and-run' mechanism whereby BID dissociates from the trigger site, which allows BAK oligomerization at an overlapping interface. In contrast, the BH3-only proteins NOXA and BAD are predicted to clash with the trigger site and are not activators of BAK. These findings provide insights into the early stages of BAK activation.

The anaphase-promoting complex/cyclosome (APC/C) is a similar to 1.5-MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell-cycle regulatory proteins. Inhibition of human APC/C-CDH1 during interphase by early mitotic inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy and enzymology, which reveal that EMI1's 143-residue C-terminal domain inhibits multiple APC/C-CDH1 functions. The intrinsically disordered D-box, linker and tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/C-CDH1 to synergistically both block the substrate-binding site and inhibit ubiquitin-chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down various functions of a 'molecular machine' nearly 100 times its size.

While the gene for p53 is mutated in many human cancers causing loss of function, many others maintain a wild-type gene but exhibit reduced p53 tumor suppressor activity through overexpression of the negative regulators, Mdm2 and/or MdmX. For the latter mechanism of loss of function, the activity of endogenous p53 can be restored through inhibition of Mdm2 or MdmX with small molecules. We previously reported a series of compounds based upon the Nutlin-3 chemical scaffold that bind to both MdmX and Mdm2 [Vara, B. A. et al. (2014) Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: A platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition. J. Org. Chem. 79, 6913-6938]. Here we present the first solution structures based on data from NMR spectroscopy for MdmX in complex with four of these compounds and compare them with the MdmX:p53 complex. A p53-derived peptide binds with high affinity (Kd value of 150nM) and causes the formation of an extensive network of hydrogen bonds within MdmX; this constitutes the induction of order within MdmX through ligand binding. In contrast, the compounds bind more weakly (Kd values from 600nM to 12μM) and induce an incomplete hydrogen bond network within MdmX. Despite relatively weak binding, the four compounds activated p53 and induced p21(Cip1) expression in retinoblastoma cell lines that overexpress MdmX, suggesting that they specifically target MdmX and/or Mdm2. Our results document structure-activity relationships for lead-like small molecules targeting MdmX and suggest a strategy for their further optimization in the future by using NMR spectroscopy to monitor small-molecule-induced protein order as manifested through hydrogen bond formation.

The corticotropin-releasing factor (CRF) peptide hormone family members coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play important roles within the cardiovascular, gastrointestinal, and central nervous systems, among others. The actions of the peptides are mediated by activation of two G-protein-coupled receptors of the B1 family, CRF receptors 1 and 2 (CRF-R1 and CRF-R2α,β). The recently reported three-dimensional structures of the first extracellular domain (ECD1) of both CRF-R1 and CRF-R2β (Pioszak, A. A., Parker, N. R., Suino-Powell, K., and Xu, H. E. (2008) J. Biol. Chem. 283, 32900-32912; Grace, C. R., Perrin, M. H., Gulyas, J., Digruccio, M. R., Cantle, J. P., Rivier, J. E., Vale, W. W., and Riek, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4858-4863) complexed with peptide antagonists provided a starting point in understanding the binding between CRF ligands and receptors at a molecular level. We now report the three-dimensional NMR structure of the ECD1 of human CRF-R1 complexed with a high affinity agonist, α-helical cyclic CRF. In the structure of the complex, the C-terminal residues (23-41) of α-helical cyclic CRF bind to the ECD1 of CRF-R1 in a helical conformation mainly along the hydrophobic face of the peptide in a manner similar to that of the antagonists in their corresponding ECD1 complex structures. Unique to this study is the observation that complex formation between an agonist and the ECD1-CRF-R1 promotes the helical conformation of the N terminus of the former, important for receptor activation (Gulyas, J., Rivier, C., Perrin, M., Koerber, S. C., Sutton, S., Corrigan, A., Lahrichi, S. L., Craig, A. G., Vale, W., and Rivier, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10575-10579).

In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53's WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1's "potentiating neddylation" domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12's active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1's interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.

SUMMARY The effector B cell lymphoma-2 (BCL-2) protein BCL-2 ovarian killer (BOK) induces mitochondrial outer membrane permeabilization (MOMP) to initiate apoptosis upon inhibition of the proteasome. How BOK mediates MOMP is mechanistically unknown. The NMR structure of the BCL-2 core of human BOK reveals a conserved architecture with an atypical hydrophobic groove that undergoes conformational exchange. Remarkably, the BCL-2 core of BOK spontaneously associates with purified mitochondria to release cytochrome c in MOMP assays. Alanine substitution of a unique glycine in helix α1 stabilizes BOK, as shown by thermal shift and urea denaturation analyses, and significantly inhibits MOMP, liposome permeabilization, and cell death. Activated BID does not activate WT BOK or the stabilized alanine mutant to promote cell death. We propose that BOK-mediated membrane permeabilization is governed in part by its unique metastability of the hydrophobic groove and helix α1 and not through activation by BH3 ligands., In Brief Zheng et al. determined the structure of BOK, revealing intrinsic instability (metastability) in putative regulatory regions and supporting BOK’s independence of activators such as tBID. Structure-based stabilizing mutation in one of the metastable regulatory regions inhibits BOK-mediated liposome permeabilization, MOMP, and apoptosis, establishing a paradigm for apoptosis initiation., Graphical Abstract

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).

Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2 similar to Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2 similar to Ub catalytic modules such as APC11-UBCH10 similar to Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10 similar to Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2 similar to Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specifically with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.

The three-dimensional NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II’ β-turn reported in the literature for sst2/3/5-subtype-selective analogues. Correlating biological results and NMR studies led to the identification of the side chains of DPhe2, DTrp8 and Lys9 as the necessary components of the sst2 pharmacophore. This is the first study to show that the aromatic ring at position 7 (Phe7) is not critical for sst2 binding and that it plays an important role in sst3 and sst5 binding. This pharmacophore is therefore different from that proposed by others for sst2/3/5 analogues.

The corticotropin-releasing factor (CRF) ligand family has diverse effects on the CNS, including the modulation of the stress response. The ligands' effects are mediated by binding to CRF G protein-coupled receptors. We have determined the 3D NMR structure of the N-terminal extracellular domain (ECD 1 ) of the mouse CRF receptor 2β, which is the major ligand recognition domain, and identified its ligand binding site by chemical-shift perturbation experiments. The fold is identified as a short consensus repeat (SCR), a common protein interaction module. Mutagenesis reveals the integrity of the hormone-binding site in the full-length receptor. This study proposes that the ECD 1 captures the C-terminal segment of the ligand, whose N terminus then penetrates into the transmembrane region of the receptor to initiate signaling. Key residues of SCR in the ECD 1 are conserved in the G protein-coupled receptor subfamily, suggesting the SCR fold in all of the ECD 1 s of this subfamily.

An alternate technique for accurately monitoring the chemical shift in multidimensional NMR experiments using spin-state selective off-resonance decoupling is presented here. By applying off-resonance decoupling on spin S during acquisition of spin I, we scaled the scalar coupling J(I,S) between the spins, and the residual scalar coupling turns out to be a function of the chemical shift of spin S. Thus, the chemical shift information of spin S is indirectly retained, without an additional evolution period and the accompanying polarization transfer elements. The detection of the components of the doublet using spin-state selection enables an accurate measurement of the residual scalar coupling and a precise value for the chemical shift, concomitantly. The spin-state selection further yields two subspectra comprising either one of the two components of the doublet and thereby avoiding the overlap problems that arise from off-resonance decoupling. In general, spin-state selective off-resonance decoupling can be incorporated into any pulse sequence. Here, the concept of spin-state selective off-resonance decoupling is applied to 3D (13)C or (15)N-resolved [(1)H,(1)H]-NOESY experiments, adding the chemical shift of the heavy atom attached to the hydrogen ((13)C or (15)N nuclei) with high resolution resulting in a pseudo-4D. These pseudo-4D heavy-atom resolved [(1)H, (1)H]-NOESY experiments contain chemical shift information comparable to that of 4D (13)C or (15)N-resolved [(1)H,(1)H]-NOESY, but with an increase in chemical shift resolution by 1-2 orders of magnitude.

How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s.

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including Anaphase Promoting Complex/Cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2~ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain elongating E2 UBE2S in ways that differ completely from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal unexpectedly diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

The metabolic status of a cell is a fundamentally important factor in deciding whether or not a cell engages its apoptotic machinery. We have previously shown that active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII) mediated phosphorylation of caspase-2(1) but the link between metabolic activity and CaMKII is poorly understood. In this study(2) we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis, in a novel mechanism of CaMKII activation. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca2+ to activate CaMKII in a calmodulin-dependent manner; effectively lowering the activation threshold of CaMKII for Ca2+/calmodulin. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes, but also in Murine oocytes. These findings uncover a novel mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyt...

Significance Nucleophosmin (NPM1) is a multifunctional protein with critical roles in ribosome biogenesis, centrosome duplication, and tumor suppression. Despite the established importance of NPM1 as a tumor marker and potential drug target, little is currently known about the molecular mechanisms that govern its various functions. Our manuscript describes that the N-terminal domain of NPM1 (Npm-N) exhibits phosphorylation-dependent structural polymorphism along a broad conformational landscape between two extreme states: a stable, folded pentamer and a globally disordered monomer. We propose that phosphorylation-induced “regulated unfolding” of Npm-N provides a means to modulate NPM1 function and subcellular localization. Our findings will drive future structure-based studies on the roles of regulated unfolding in NPM1 biology and will provide a foundation for NPM1-targeted anticancer drug development.

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small-molecule modulators.

Summary Active metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca 2+ to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p, Author Summary We provide physical evidence, using NMR, FRET and SPR, that purine or pyrimidine-rich microRNAs can form triplexes with complementary purine-rich sequences of duplex DNA and provide an algorithm (Trident) to search genome-wide for potential microRNA double-stranded DNA triplex-forming sites. Using this algorithm we document enrichment of microRNA triplex binding sites in mammalian and non-mammalian genomes. We found in primary leukemia cells from patients a significant over-representation of positively correlated microRNA and mRNA expression for genes containing sequences favoring microRNA-duplex DNA triplex formation, suggesting this as a mechanism by which microRNA may enhance gene transcription.

Following DNA damage, nuclear p53 induces the expression of PUMA, a BH3-only protein that binds and inhibits the anti-apoptotic BCL-2 repertoire, including BCL-xL. PUMA, unique amongst BH3-only proteins, disrupts the interaction between cytosolic p53 and BCL-xL, allowing p53 to promote apoptosis via direct activation of the BCL-2 effector molecules, BAX and BAK. Structural investigations using nuclear magnetic resonance spectroscopy and X-ray crystallography revealed that PUMA binding induced partial unfolding of two α-helices within BCL-xL. Wild-type PUMA or a PUMA mutant incapable of causing binding-induced unfolding of BCL-xL equivalently inhibited the anti-apoptotic BCL-2 repertoire to sensitize for death receptor (DR)-activated apoptosis, but only wild-type PUMA promoted p53-dependent, DNA damage-induced apoptosis. Our data suggest that PUMA-induced partial unfolding of BCL-xL disrupts interactions between cytosolic p53 and BCL-xL, releasing the bound p53 to initiate apoptosis. We propose that regulated unfolding of BCL-xL provides a mechanism to promote PUMA-dependent signaling within the apoptotic pathways.

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined. The pharmacophore involves the side chains of Cpa2, DTrp/DAph(Cbm)8, and Lys9, with the backbone for most of the sst2-selective antagonists comprised a Type-II' beta-turn. Hence, the sst2-selective antagonist pharmacophore is very similar to the sst2-selective agonist pharmacophore previously described.

H-DPhe (2)-c[Cys (3)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Thr (15)-NH2 (1) (a somatostatin agonist, SRIF numbering) and H-Cpa (2)-c[DCys (3)-Tyr (7)-DTrp (8)-Lys (9)-Thr (10)-Cys (14)]-Nal (15)-NH2 (4) (a somatostatin antagonist) are based on the structure of octreotide that binds to three somatostatin receptor subtypes (sst 2/3/5) with significant binding affinity. Analogues of 1 and 4 were synthesized with norcysteine (Ncy), homocysteine (Hcy), or D-homocysteine (DHcy) at positions 3 and/or 14. Introducing Ncy at positions 3 and 14 constrained the backbone flexibility, resulting in loss of binding affinity at all sst s. The introduction of Hcy at positions 3 and 14 improved selectivity for sst 2 as a result of significant loss of binding affinity at the other sst s. Substitution by DHcy at position 3 in the antagonist scaffold (5), on the other hand, resulted in a significant loss of binding affinity at sst 2 and sst 3 as compared to the different affinities of the parent compound (4). The 3D NMR structures of the analogues in dimethylsulfoxide are consistent with the observed binding affinities.

The C-terminally amidated CRF antagonist astressin binds to CRF-R1 or CRF-R2 receptors with low nanomolar affinity while the corresponding astressin-acid has >100 times less affinity. To understand the role of the amide group in binding, the conformations of astressin-amide and astressin-acid were studied in DMSO using NMR techniques. The 3D NMR structures show that the backbones of both analogs prefer an α-helical conformation, with a small kink around Gln26. However, astressin-amide has a well-defined helical structure from Leu27 to Ile41 and a conformation very similar to the bioactive conformation reported by our group (Grace et al., Proc Natl Acad Sci USA 2007, 104, 4858–4863). In contrast, astressin-acid has an irregular helical conformation from Arg35 onward, including a rearrangement of the side chains in that region. This structural difference highlights the crucial role of the C-terminal amidation for stabilization of astressin's bioactive conformation. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 196–205, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The corticotropin releasing factor (CRF) family of ligands and their receptors coordinate endocrine, behavioral, autonomic, and metabolic responses to stress and play additional roles within the cardiovascular, gastrointestinal, and other systems. The actions of CRF and the related urocortins are mediated by activation of two receptors, CRF-R1 and CRF-R2, belonging to the B1 family of G protein-coupled receptors. The short-consensus-repeat fold (SCR) within the first extracellular domain (ECD1) of the CRF receptor(s) comprises the major ligand binding site and serves to dock a peptide ligand via its C-terminal segment, thus positioning the N-terminal segment to interact with the receptor's juxtamembrane domains to activate the receptor. Here we present the 3D NMR structure of ECD1 of CRF-R2β in complex with astressin, a peptide antagonist. In the structure of the complex the C-terminal segment of astressin forms an amphipathic helix, whose entire hydrophobic face interacts with the short-consensus-repeat motif, covering a large intermolecular interface. In addition, the complex is characterized by intermolecular hydrogen bonds and a salt bridge. These interactions are quantitatively weighted by an analysis of the effects on the full-length receptor affinities using an Ala scan of CRF. These structural studies identify the major determinants for CRF ligand specificity and selectivity and support a two-step model for receptor activation. Furthermore, because of a proposed conservation of the fold for both the ECD1s and ligands, this structure can serve as a model for ligand recognition for the entire B1 receptor family.

Strategies for the design of peptide agonists and antagonists of gonadotropin releasing hormone (GnRH, one ligand and one receptor) and of receptor-selective agonists and antagonists of somatostatin (SRIF, three ligands and five receptors) and corticotropin releasing factor (CRF, three ligands and two receptors) are described. These strategies include the use of unusual amino acids, a versatile scaffold based on aminoglycine (betidamino acids) and side chain as well as backbone conformational constraints.

Spin-state selective off-resonance decoupling (SITAR) is applied to the amide proton-to-nitrogen-to-alpha-carbon correlation (HNCA) triple-resonance experiment by measuring the 15N chemical shift during the acquisition simultaneously with the 1H chemical shift. The simultaneous detection of both 1H and 15N chemical shifts in SITAR reduces the dimensionality of the HNCA-type experiment from three dimensions to two dimensions with a 15N chemical shift resolution of approximately 0.4 ppm. This enables the recording of triple-resonance experiments in several minutes. SITAR is furthermore applied to the amide proton-to-nitrogen-to-alpha-carbon-and-beta-carbon correlation (HNCACB) triple-resonance experiment and the 15N-resolved [1H,1H]-nuclear Overhauser enhancement spectroscopy (NOESY) experiment with similar success. The accompanied peak crowding and chemical shift degeneracy of the amide protons in the SITAR two-dimensional (2D) spectra, which are inherent properties of pseudo-dimensional experiments, are resolved by local correlation of the two sub-spectra. With this procedure a 13C--1H strip for each 15N--1H moiety is generated resulting in a three-dimensional (3D) strip list known from the conventional 3D spectra. The quality of the strip list in terms of peak crowding and chemical shift degeneracy is comparable to their corresponding 3D counterparts. An analysis-software within the CARA package is presented, which generates, visualizes and manages the SITAR spectra, the corresponding strip lists and the assignment process.