Nicolas Coudevylle

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

Ca2+ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca2+–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 Ca2+ signals. We solved the structure of Ca2+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/Ca2+2–CaM complex. The Ca2+ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca2+–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca2+]i, whereas the N‐module acts as a sensor at micromolar [Ca2+]i. This Ca2+/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca2+‐dependent modulation of short‐term synaptic plasticity.

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)β(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 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 Ca2+ and is characterized by a KD of 55 μM in the presence of a saturating concentration of Ca2+ (5 mM). Reciprocally, the binding of IP3 increases the apparent Ca2+‐binding affinity of the C2A domain in agreement with a Target‐Activated Messenger Affinity (TAMA) mechanism. The C2B domain binds IP3 in a Ca2+‐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.

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 μm) 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 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.

P160/SRC/NCoA coactivators form complexes via specific interaction of their PAS-B domain with the CID/AD1 domain.

Transcriptional activation involves the ordered recruitment of coactivators via direct interactions between distinct binding domains and recognition motifs. The p160/SRC/NCoA coactivator family comprises three members (NCoA-1, -2 and -3), which are organized in multiprotein coactivator complexes. We had identified the PAS-B domain of NCoA-1 as an LXXLL motif binding domain. Here we show that NCoA family members are able to interact with other full-length NCoA proteins via their PAS-B domain and they specifically interact with the CBP-interaction domain (CID/AD1) of NCoA-1. Peptide competition, binding experiments and mutagenesis of LXXLL motifs point at distinct binding motif specificities of the NCoA PAS-B domains. NMR studies of different NCoA-1-PAS-B/LXXLL peptide complexes revealed similar although not identical binding sites for the CID/AD1 and STAT6 transactivation domain LXXLL motifs. In mechanistic studies, we found that overexpression of the PAS-B domain is able to disturb the binding of NCoA-1 to CBP in cells and that a CID/AD1 peptide competes with STAT6 for NCoA-1 in vitro. Moreover, the expression of an endogenous androgen receptor target gene is affected by the overexpression of the NCoA-1 or NCoA-3 PAS-B domains. Our study discloses a new, complementary mechanism for the current model of coactivator recruitment to target gene promoters.

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.

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.

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).