Mechanism of Atg9 recruitment by Atg11 in the cytoplasm-to-vacuole targeting pathway.

Autophagy is a lysosomal degradation pathway for the removal of damaged and superfluous cytoplasmic material. This is achieved by the sequestration of this cargo material within double-membrane vesicles termed autophagosomes. Autophagosome formation is mediated by the conserved autophagy machinery. In selective autophagy, this machinery including the transmembrane protein Atg9 is recruited to specific cargo material via cargo receptors and the Atg11/FIP200 scaffold protein. The molecular details of the interaction between Atg11 and Atg9 are unclear, and it is still unknown how the recruitment of Atg9 is regulated. Here we employ NMR spectroscopy of the N-terminal disordered domain of Atg9 (Atg9-NTD) to map its interaction with Atg11 revealing that it involves two short peptides both containing a PLF motif. We show that the Atg9-NTD binds to Atg11 with an affinity of about 1 µM and that both PLF motifs contribute to the interaction. Mutation of the PLF motifs abolishes the interaction of the Atg9-NTD with Atg11, reduces the recruitment of Atg9 to the precursor aminopeptidase 1 (prApe1) cargo, and blocks prApe1 transport into the vacuole by the selective autophagy-like cytoplasm-to-vacuole (Cvt) targeting pathway while not affecting bulk autophagy. Our results provide mechanistic insights into the interaction of the Atg11 scaffold with the Atg9 transmembrane protein in selective autophagy and suggest a model where only clustered Atg11 when bound to the prApe1 cargo is able to efficiently recruit Atg9 vesicles.

Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation.

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum.

1H, 15N, 13C resonance assignment of human GAP-43.

GAP-43 is a 25 kDa neuronal intrinsically disordered protein, highly abundant in the neuronal growth cone during development and regeneration. The exact molecular function(s) of GAP-43 remains unclear but it appears to be involved in growth cone guidance and actin cytoskeleton organization. Therefore, GAP-43 seems to play an important role in neurotransmitter vesicle fusion and recycling, long-term potentiation, spatial memory formation and learning. Here we report the nearly complete assignment of recombinant human GAP-43.

N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions.

Incorporation of myristic acid onto the N terminus of a protein is a crucial modification that promotes membrane binding and correct localization of important components of signaling pathways. Recombinant expression of N-myristoylated proteins in Escherichia coli can be achieved by co-expressing yeast N-myristoyltransferase and supplementing the growth medium with myristic acid. However, undesired incorporation of the 12-carbon fatty acid lauric acid can also occur (leading to heterogeneous samples), especially when the available carbon sources are scarce, as it is the case in minimal medium for the expression of isotopically enriched samples. By applying this method to the brain acid soluble protein 1 and the 1-185 N-terminal region of c-Src, we show the significant, and protein-specific, differences in the membrane binding properties of lauroylated and myristoylated forms. We also present a robust strategy for obtaining lauryl-free samples of myristoylated proteins in both rich and minimal media.

Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17.

The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 µm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational flexibility and thus not amenable to conventional structural biology techniques. Given their inherent structural flexibility NMR spectroscopy offers unique opportunities for structural and dynamic studies of IDPs. The past two decades have witnessed significant development of NMR spectroscopy that couples advances in spin physics and chemistry with a broad range of applications. This chapter will summarize key advances in NMR methodology. Despite the availability of efficient (multi-dimensional) NMR experiments for signal assignment of IDPs it is discussed that NMR of larger and more complex IDPs demands spectral simplification strategies capitalizing on specific isotope-labeling strategies. Prototypical applications of isotope labeling-strategies are described. Since IDP-ligand association and dissociation processes frequently occur on time scales that are amenable to NMR spectroscopy we describe in detail the application of CPMG relaxation dispersion techniques to studies of IDP protein binding. Finally, we demonstrate that the complementary usage of NMR and EPR data provide a more comprehensive picture about the conformational states of IDPs and can be employed to analyze the conformational ensembles of IDPs.

Structural basis for the interaction of protein S1 with the Escherichia coli ribosome.

In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1-ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the 'ribosome puzzle', namely the detailed molecular insight into the topology of the S1-ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome.

Selective isotope labelling of leucine residues by using α-ketoacid precursor compounds.

You can have one without the other: A new metabolic precursor compound can selectively introduce (13)C and (2)H patterns at leucine residues in proteins in cell-based expression systems without interfering with valine metabolism. The protocol provides selectively labelled macromolecules well suited for probing structure and dynamics in high-molecular-weight proteins by NMR spectroscopy.

Toward rational fragment-based lead design without 3D structures.

Fragment-based lead discovery (FBLD) has become a prime component of the armamentarium of modern drug design programs. FBLD identifies low molecular weight ligands that weakly bind to important biological targets. Three-dimensional structural information about the binding mode is provided by X-ray crystallography or NMR spectroscopy and is subsequently used to improve the lead compounds. Despite tremendous success rates, FBLD relies on the availability of high-resolution structural information, still a bottleneck in drug discovery programs. To overcome these limitations, we recently demonstrated that the meta-structure approach provides an alternative route to rational lead identification in cases where no 3D structure information about the biological target is available. Combined with information-rich NMR data, this strategy provides valuable information for lead development programs. We demonstrate with several examples the feasibility of the combined NMR and meta-structure approach to devise a rational strategy for fragment evolution without resorting to highly resolved protein complex structures.

Lipocalin Q83 reveals a dual ligand binding mode with potential implications for the functions of siderocalins.

Siderocalins are particular lipocalins that participate in the innate immune response by interfering with bacterial siderophore-mediated iron uptake. Additionally, siderocalins are involved in several physiological and pathological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. Here we show that siderocalin Q83 displays an unexpected dual ligand binding mode as it can bind enterobactin and unsaturated fatty acids simultaneously. The solution structure of the siderocalin Q83 in complex with arachidonic acid and enterobactin reveals molecular details of this novel dual binding mode and the determinants of fatty acid binding specificity. Our results suggest that Q83 is a metabolic hub linking iron and fatty acid pathways. This unexpected coupling might contribute to the pleiotropic functions of siderocalins.

Siderocalin Q83 exhibits differential slow dynamics upon ligand binding.

Siderocalin Q83 is a small soluble protein that has the ability to bind two different ligands (enterobactin and arachidonic acid) simultaneously in two distinct binding sites. Here we report that Q83 exhibits an intriguing dynamic behavior. In its free form, the protein undergoes significant micro-to-millisecond dynamics. When binding arachidonic acid, the motions of the arachidonic acid binding site are quenched while the dynamics at the enterobactin binding site increases. Reciprocally, enterobactin binding to Q83 quenches the motions at the enterobactin binding site and increases the slow dynamics at the arachidonic acid binding site. Additionally, in the enterobactin-bound state, the excited state of the arachidonic acid binding site resembles the arachidonic acid-bound state. These observations strongly suggest an allosteric regulation where binding of one ligand enhances the affinity of Q83 for the other one. Additionally, our data strengthen the emerging view of proteins as dynamic ensembles interconverting between different sub-states with distinct functionalities.

The metastasis-associated extracellular matrix protein osteopontin forms transient structure in ligand interaction sites.

Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein that was first identified as a major sialoprotein in bones. It functions as a cell attachment protein displaying a RGD cell adhesion sequence and as a cytokine that signals through integrin and CD44 cell adhesion molecules. OPN is also implicated in human tumor progression and cell invasion. OPN has intrinsic transforming activity, and elevated OPN levels promote metastasis. OPN gene expression is also strongly activated in avian fibroblasts simultaneously transformed by the v-myc and v-mil(raf) oncogenes. Here we have investigated the solution structure of a 220-amino acid recombinant OPN protein by an integrated structural biology approach employing bioinformatic sequence analysis, multidimensional nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism spectroscopy, and small-angle X-ray scattering. These studies suggest that OPN is an intrinsically unstructured protein in solution. Although OPN does not fold into a single defined structure, its conformational flexibility significantly deviates from random coil-like behavior. OPN comprises distinct local secondary structure elements with reduced conformational flexibility and substantially populates a compact subspace displaying distinct tertiary contacts. These compacted regions of OPN encompass the binding sites for α(V)ß(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.

The v-myc-induced Q83 lipocalin is a siderocalin.

Siderocalins are atypical lipocalins able to capture siderophores with high affinity. They contribute to the innate immune response by interfering with bacterial siderophore-mediated iron uptake but are also involved in numerous physiological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. The Q83 lipocalin was originally identified based on its overexpression in quail embryo fibroblasts transformed by the v-myc oncogene. We show here that Q83 is a siderocalin, binding the siderophore enterobactin with an affinity and mode of binding nearly identical to that of neutrophil gelatinase-associated lipocalin (NGAL), the prototypical siderocalin. This strengthens the role of siderocalins in cancer progression and inflammation. In addition, we also present the solution structure of Q83 in complex with intact enterobactin and a detailed analysis of the Q83 binding mode, including mutagenesis of the critical residues involved in enterobactin binding. These data provide a first insight into the molecular details of siderophore binding and delineate the common molecular properties defining the siderocalin protein family.

Pharmacophore mapping via cross-relaxation during adiabatic fast passage.

A novel NMR method is demonstrated for the investigation of protein ligand interactions. In this approach an adiabatic fast passage pulse, i.e. a long, weak pulse with a linear frequency sweep, is used to probe (1)H-(1)H NOEs. During the adiabatic fast passage the effective rotating-frame NOE is a weighted average of transverse and longitudinal cross-relaxation contributions that can be tuned by pulse power and frequency sweep rate. It is demonstrated that the occurrence of spin diffusion processes leads to sizable deviations from the theoretical relationship between effective relaxation rate and effective tilt angle in the spin lock frame and can be used to probe protein-ligand binding. This methodology comprises high sensitivity and ease of implementation. The feasibility of this technique is demonstrated with two protein complexes, vanillic acid bound to the quail lipocalin Q83 and NAD(+) and AMP binding to alcohol dehydrogenase (ADH).

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short-term synaptic plasticity.

Ca(2+) signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca(2+)-CaM binds a conserved region in the priming proteins Munc13-1 and ubMunc13-2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca(2+) signals. We solved the structure of Ca(2+)(4)-CaM in complex with the CaM-binding domain of Munc13-1, which features a novel 1-5-8-26 CaM-binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13-2 isoform. The N-module can be dissociated with EGTA to form the half-loaded Munc13/Ca(2+)(2)-CaM complex. The Ca(2+) regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca(2+)-CaM interactions, where the C-module provides a high-affinity interaction activated at nanomolar [Ca(2+)](i), whereas the N-module acts as a sensor at micromolar [Ca(2+)](i). This Ca(2+)/CaM-binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca(2+)-dependent modulation of short-term synaptic plasticity.

1H, 13C and 15N resonance assignments of the Calmodulin-Munc13-1 peptide complex.

Ca(2+)-Calmodulin binding to the variable N-terminal region of the diacylglycerol/phorbol ester-binding UNC13/Munc13 family of proteins modulates the short-term synaptic plasticity characteristics in neurons. Here, we report the sequential backbone and side chain resonance assignment of the Ca(2+)-Calmodulin/Munc13-1(458-492) peptide complex at pH 6.8 and 35 degrees C (BMRB No. 15470).

Autocorrelation analysis of NOESY data provides residue compactness for folded and unfolded proteins.

A novel spectral entropy interpretation for protein NOESY data is presented for the investigation of the spatial distribution of residues in protein structures without the requirement of NOE cross peak assignments. In this approach individual traces S(i)(omega) from a 3D (15)N NOESY-HSQC taken at frequency positions corresponding to different amide groups (residue position i) are subjected to a self-convolution procedure thus leading to the autocorrelation function C(i)(omega) of the NOESY-trace for a particular backbone residue position. The characteristic spatial surrounding of a particular residue position is reflected in the corresponding autocorrelation function and can be quantified by taking the (spectral) entropy S(nu) as an information measure. The feasibility of this novel approach is demonstrated with applications to the proteins Cyclophilin D and Osteopontin and the protein complex between the lipocalin Q83 and the bacterial siderophore Enterobactin. Typically, large entropy values were found for residues located in structurally loosely defined regions, whereas small entropy values were found for residues in hydrophobic core regions of the protein with tightly interacting side chains and distinct chemical shift patterns. The applications to the unfolded Osteopontin and the Q83/Enterobactin protein complex indicated that both local compaction of the polypeptide chain due to transiently formed structural elements and subtle changes in side-chain packing can be efficiently probed by this novel approach.

Structural determinants for Ca2+ and phosphatidylinositol 4,5-bisphosphate binding by the C2A domain of rabphilin-3A.

Rabphilin-3A is a neuronal C2 domain tandem containing protein involved in vesicle trafficking. Both its C2 domains (C2A and C2B) are able to bind phosphatidylinositol 4,5-bisphosphate, a key player in the neurotransmitter release process. The rabphilin-3A C2A domain has previously been shown to bind inositol-1,4,5-trisphosphate (IP3; phosphatidylinositol 4,5-bisphosphate headgroup) in a Ca2+-dependent manner with a relatively high affinity (50 microm) in the presence of saturating concentrations of Ca2+. Moreover, IP3 and Ca2+ binding to the C2A domain mutually enhance each other. Here we present the Ca2+-bound solution structure of the C2A domain. Structural comparison with the previously published Ca2+-free crystal structure revealed that Ca2+ binding induces a conformational change of Ca2+ binding loop 3 (CBL3). Our IP3 binding studies as well as our IP3-C2A docking model show the active involvement of CBL3 in IP3 binding, suggesting that the conformational change on CBL3 upon Ca2+ binding enables the interaction with IP3 and vice versa, in line with a target-activated messenger affinity mechanism. Our data provide detailed structural insight into the functional properties of the rabphilin-3A C2A domain and reveal for the first time the structural determinants of a target-activated messenger affinity mechanism.

The PIP2 binding mode of the C2 domains of rabphilin-3A.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a key player in the neurotransmitter release process. Rabphilin-3A is a neuronal C2 domain tandem containing protein that is involved in this process. Both its C2 domains (C2A and C2B) are able to bind PIP2. The investigation of the interactions of the two C2 domains with the PIP2 headgroup IP3 (inositol-1,4,5-trisphosphate) by NMR showed that a well-defined binding site can be described on the concave surface of each domain. The binding modes of the two domains are different. The binding of IP3 to the C2A domain is strongly enhanced by Ca(2+) and is characterized by a K(D) of 55 microM in the presence of a saturating concentration of Ca(2+) (5 mM). Reciprocally, the binding of IP3 increases the apparent Ca(2+)-binding affinity of the C2A domain in agreement with a Target-Activated Messenger Affinity (TAMA) mechanism. The C2B domain binds IP3 in a Ca(2+)-independent fashion with low affinity. These different PIP2 headgroup recognition modes suggest that PIP2 is a target of the C2A domain of rabphilin-3A while this phospholipid is an effector of the C2B domain.