Structure and interactions of the endogenous human Commander complex.

The Commander complex, a 16-protein assembly, plays multiple roles in cell homeostasis, cell cycle and immune response. It consists of copper-metabolism Murr1 domain proteins (COMMD1-10), coiled-coil domain-containing proteins (CCDC22 and CCDC93), DENND10 and the Retriever subcomplex (VPS26C, VPS29 and VPS35L), all expressed ubiquitously in the body and linked to various diseases. Here, we report the structure and key interactions of the endogenous human Commander complex by cryogenic-electron microscopy and mass spectrometry-based proteomics. The complex consists of a stable core of COMMD1-10 and an effector containing DENND10 and Retriever, scaffolded together by CCDC22 and CCDC93. We establish the composition of Commander and reveal major interaction interfaces. These findings clarify its roles in intracellular transport, and uncover a strong association with cilium assembly, and centrosome and centriole functions.

Exploring thienothiadiazine dioxides as isosteric analogues of benzo- and pyridothiadiazine dioxides in the search of new AMPA and kainate receptor positive allosteric modulators.

The synthesis and biological evaluation on AMPA and kainate receptors of new examples of 3,4-dihydro-2H-1,2,4-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxides is described. The introduction of a cyclopropyl chain instead of an ethyl chain at the 4-position of the thiadiazine ring was found to dramatically improve the potentiator activity on AMPA receptors, with compound 32 (BPAM395) expressing in vitro activity on AMPARs (EC2x = 0.24 µM) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344). Interestingly, the 4-allyl-substituted thienothiadiazine dioxide 27 (BPAM307) emerged as the most promising compound on kainate receptors being a more effective potentiator than the 4-cyclopropyl-substituted thienothiadiazine dioxide 32 and supporting the view that the 4-allyl substitution of the thiadiazine ring could be more favorable than the 4-cyclopropyl substitution to induce marked activity on kainate receptors versus AMPA receptors. The thieno-analogue 36 (BPAM279) of the clinically tested S18986 (11) was selected for in vivo evaluation in mice as a cognitive enhancer due to a safer profile than 32 after massive per os drug administration. Compound 36 was found to increase the cognition performance in mice at low doses (1 mg/kg) per os suggesting that the compound was well absorbed after oral administration and able to reach the central nervous system. Finally, compound 32 was selected for co-crystallization with the GluA2-LBD (L504Y,N775S) and glutamate to examine the binding mode of thienothiadiazine dioxides within the allosteric binding site of the AMPA receptor. At the allosteric site, this compound established similar interactions as the previously reported BTD-type AMPA receptor modulators.

Differences between the GluD1 and GluD2 receptors revealed by GluD1 X-ray crystallography, binding studies and molecular dynamics.

Ionotropic glutamate receptors are ligand-gated ion channels essential for fast excitatory neurotransmission in the brain. In contrast to most other members of the iGluR family, the subfamily of delta receptors, GluD1 and GluD2, does not bind glutamate but glycine/D-serine. GluD1 is widely expressed in the brain and the inner ear, where it is required for high-frequency hearing. Furthermore, it has been associated with schizophrenia, autism and depression. X-ray structures of the ligand-binding domain (LBD) of GluD2 have been published; however, no high-resolution structure is available for the ligand-binding domain of GluD1 (GluD1-LBD). Here, we report the X-ray crystal structure of the GluD1-LBD in its apo form at 2.57 Å resolution. Using isothermal titration calorimetry, we show that D-serine binds to the GluD1-LBD in an exothermic manner with a Kd of 160 µm, which is approximately five-fold greater than at GluD2. Furthermore, we identify Glu822 as a critical determinant of receptor activation in GluD1 A654T. In contrast to studies on the GluD2 lurcher mutant A654T, we did not observe any effect of 1 mm D-serine on the spontaneous currents at mouse GluD1 A654T by electrophysiological recordings of Xenopus laevis oocytes as previously also reported by others. These results point towards differences in the structure and dynamics between GluD1 and GluD2. Molecular dynamics simulations were employed to address this observation, suggesting that the apo structure of GluD1 is less flexible than the apo structure of GluD2 and that Pro725 in GluD1 may affect the interlobe closure of the ligand-binding domain of GluD1.

Commander Complex-A Multifaceted Operator in Intracellular Signaling and Cargo.

Commander complex is a 16-protein complex that plays multiple roles in various intracellular events in endosomal cargo and in the regulation of cell homeostasis, cell cycle and immune response. It consists of COMMD1-10, CCDC22, CCDC93, DENND10, VPS26C, VPS29, and VPS35L. These proteins are expressed ubiquitously in the human body, and they have been linked to diseases including Wilson's disease, atherosclerosis, and several types of cancer. In this review we describe the function of the commander complex in endosomal cargo and summarize the individual known roles of COMMD proteins in cell signaling and cancer. It becomes evident that commander complex might be a much more important player in intracellular regulation than we currently understand, and more systematic research on the role of commander complex is required.

Human myelin protein P2: from crystallography to time-lapse membrane imaging and neuropathy-associated variants.

Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double-membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays, and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.

A Quasielastic Neutron Scattering Investigation on the Molecular Self-Dynamics of Human Myelin Protein P2.

The human myelin protein P2 is a membrane binding protein believed to maintain correct lipid composition and organization in peripheral nerve myelin. Its function is related to its ability to stack membranes, and this function can be enhanced by the P38G mutation, whereby the overall protein structure does not change but the molecular dynamics increase. Mutations in P2 are linked to human peripheral neuropathy. Here, the dynamics of wild-type P2 and the P38G variant were studied using quasielastic neutron scattering on time scales from 10 ps to 1 ns at 300 K. The results suggest that the mutant protein dynamics are increased on both the fastest and the slowest measured time scales, by increasing the dynamics amplitude and/or the portion of atoms participating in the movement.

Sub-Atomic Resolution Crystal Structures Reveal Conserved Geometric Outliers at Functional Sites.

Myelin protein 2 (P2) is a peripheral membrane protein of the vertebrate nervous system myelin sheath, having possible roles in both lipid transport and 3D molecular organization of the multilayered myelin membrane. We extended our earlier crystallographic studies on human P2 and refined its crystal structure at an ultrahigh resolution of 0.72 Å in perdeuterated form and 0.86 Å in hydrogenated form. Characteristic differences in C-H…O hydrogen bond patterns were observed between extended ß strands, kinked or ending strands, and helices. Often, side-chain C-H groups engage in hydrogen bonding with backbone carbonyl moieties. The data highlight several amino acid residues with unconventional conformations, including both bent aromatic rings and twisted guanidinium groups on arginine side chains, as well as non-planar peptide bonds. In two locations, such non-ideal conformations cluster, providing proof of local functional strain. Other ultrahigh-resolution protein structures similarly contain chemical groups, which break planarity rules. For example, in Src homology 3 (SH3) domains, a conserved bent aromatic residue is observed near the ligand binding site. Fatty acid binding protein (FABP) 3, belonging to the same family as P2, has several side chains and peptide bonds bent exactly as those in P2. We provide a high-resolution snapshot on non-ideal conformations of amino acid residues under local strain, possibly relevant to biological function. Geometric outliers observed in ultrahigh-resolution protein structures are real and likely relevant for ligand binding and conformational changes. Furthermore, the deuteration of protein and/or solvent are promising variables in protein crystal optimization.

Crystal Structures of Potent Dimeric Positive Allosteric Modulators at the Ligand-Binding Domain of the GluA2 Receptor.

The ionotropic glutamate receptor GluA2 is considered to be an attractive target for positive allosteric modulation for the development of pharmacological tools or cognitive enhancers. Here, we report a detailed structural characterization of two recently reported dimeric positive allosteric modulators, TDPAM01 and TDPAM02, with nanomolar potency at GluA2. Using X-ray crystallography, TDPAM01 and TDPAM02 were crystallized in the ligand-binding domain of the GluA2 flop isoform as well as in the flip-like mutant N775S and the preformed dimer L504Y-N775S. In all structures, one modulator molecule binds at the dimer interface with two characteristic hydrogen bonds being formed from the modulator to Pro515. Whereas the GluA2 dimers and modulator binding mode are similar when crystallized in the presence of l-glutamate, the shape of the binding site differs when no l-glutamate is present. TDPAM02 has no effect on domain closure in both apo and l-glutamate bound GluA2 dimers compared to structures without modulator.

Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the ß barrel in fatty acid binding proteins.

BACKGROUND: Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress. RESULTS: We performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous ß barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2. CONCLUSIONS: Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.

Enhancing Action of Positive Allosteric Modulators through the Design of Dimeric Compounds.

The present study describes the identification of highly potent dimeric 1,2,4-benzothiadiazine 1,1-dioxide (BTD)-type positive allosteric modulators of the AMPA receptors (AMPApams) obtained by linking two monomeric BTD scaffolds through their respective 6-positions. Using previous X-ray data from monomeric BTDs cocrystallized with the GluA2 ligand-binding domain (LBD), a molecular modeling approach was performed to predict the preferred dimeric combinations. Two 6,6-ethylene-linked dimeric BTD compounds (16 and 22) were prepared and evaluated as AMPApams on HEK293 cells expressing GluA2o( Q) (calcium flux experiment). These compounds were found to be about 10,000 times more potent than their respective monomers, the most active dimeric compound being the bis-4-cyclopropyl-substituted compound 22 [6,6'-(ethane-1,2-diyl)bis(4-cyclopropyl-3,4-dihydro-2 H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC50 = 13 nM) was successfully cocrystallized with the GluA2o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

7-Phenoxy-Substituted 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides as Positive Allosteric Modulators of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors with Nanomolar Potency.

We report here the synthesis of 7-phenoxy-substituted 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides and their evaluation as AMPA receptor positive allosteric modulators (AMPApams). The impact of substitution on the phenoxy ring and on the nitrogen atom at the 4-position was examined. At GluA2(Q) expressed in HEK293 cells (calcium flux experiment), the most potent compound was 11m (4-cyclopropyl-7-(3-methoxyphenoxy)-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, EC50 = 2.0 nM). The Hill coefficient in the screening and the shape of the dimerization curve in small-angle X-ray scattering (SAXS) experiments using isolated GluA2 ligand-binding domain (GluA2-LBD) are consistent with binding of one molecule of 11m per dimer interface, contrary to most benzothiadiazine dioxides developed to date. This observation was confirmed by the X-ray structure of 11m bound to GluA2-LBD and by NMR. This is the first benzothiadiazine dioxide AMPApam to reach the nanomolar range.

Structure and Affinity of Two Bicyclic Glutamate Analogues at AMPA and Kainate Receptors.

Ionotropic glutamate receptors (iGluRs) are involved in most of the fast excitatory synaptic transmission in the central nervous system. These receptors are important for learning and memory formation, but are also involved in the development of diseases such as Alzheimer's disease, epilepsy and depression. To understand the function of different types of iGluRs, selective agonists are invaluable as pharmacological tool compounds. Here, we report binding affinities of two bicyclic, conformationally restricted analogues of glutamate (CIP-AS and LM-12b) at AMPA (GluA2 and GluA3) and kainate receptor subunits (GluK1-3 and GluK5). Both CIP-AS and LM-12b were found to be GluK3-preferring agonists, with Ki of 6 and 22 nM, respectively, at recombinant GluK3 receptors. The detailed binding mode of CIP-AS and LM-12b in the ligand-binding domains of the AMPA receptor subunit GluA2 (GluA2-LBD) and the kainate receptor subunits GluK1 (GluK1-LBD) and GluK3 (GluK3-LBD) was investigated by X-ray crystallography. CIP-AS stabilized all three receptor constructs in conformations similar to those with kainate. Remarkably, whereas LM-12b bound in a similar manner to CIP-AS in GluA2-LBD and GluK3-LBD, it introduced full closure of the ligand-binding domain in GluK1-LBD and formation of a D1-D2 interlobe hydrogen bond between Glu441 and Ser721, as also observed with glutamate. As the binding affinity of LM-12b at GluK1 is ∼8-fold better than that for CIP-AS (Ki of 85 and 656 nM, respectively), it shows that small changes in agonist structure can lead to prominent differences in structure and function.

Determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an enzyme highly abundant in the central nervous system myelin of terrestrial vertebrates. The catalytic domain of CNPase belongs to the 2H phosphoesterase superfamily and catalyzes the hydrolysis of nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-monophosphates. The detailed reaction mechanism and the essential catalytic amino acids involved have been described earlier, but the roles of many amino acids in the vicinity of the active site have remained unknown. Here, several CNPase catalytic domain mutants were studied using enzyme kinetics assays, thermal stability experiments, and X-ray crystallography. Additionally, the crystal structure of a perdeuterated CNPase catalytic domain was refined at atomic resolution to obtain a detailed view of the active site and the catalytic mechanism. The results specify determinants of ligand binding and novel essential residues required for CNPase catalysis. For example, the aromatic side chains of Phe235 and Tyr168 are crucial for substrate binding, and Arg307 may affect active site electrostatics and regulate loop dynamics. The ß5-α7 loop, unique for CNPase in the 2H phosphoesterase family, appears to have various functions in the CNPase reaction mechanism, from coordinating the nucleophilic water molecule to providing a binding pocket for the product and being involved in product release.

Production, crystallization and neutron diffraction of fully deuterated human myelin peripheral membrane protein P2.

The molecular details of the formation of the myelin sheath, a multilayered membrane in the nervous system, are to a large extent unknown. P2 is a peripheral membrane protein from peripheral nervous system myelin, which is believed to play a role in this process. X-ray crystallographic studies and complementary experiments have provided information on the structure-function relationships in P2. In this study, a fully deuterated sample of human P2 was produced. Crystals that were large enough for neutron diffraction were grown by a ten-month procedure of feeding, and neutron diffraction data were collected to a resolution of 2.4 Å from a crystal of 0.09 mm(3) in volume. The neutron crystal structure will allow the positions of H atoms in P2 and its fatty-acid ligand to be visualized, as well as shedding light on the fine details of the hydrogen-bonding networks within the P2 ligand-binding cavity.

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering--A Comparison between Wild-Type Protein and a Hinge Mutant.

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the ß barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.

Atomic resolution view into the structure-function relationships of the human myelin peripheral membrane protein P2.

P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Šallows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.

Production and crystallization of a panel of structure-based mutants of the human myelin peripheral membrane protein P2.

The myelin sheath is a multilayered membrane that surrounds and insulates axons in the nervous system. One of the proteins specific to the peripheral nerve myelin is P2, a protein that is able to stack lipid bilayers. With the goal of obtaining detailed information on the structure-function relationship of P2, 14 structure-based mutated variants of human P2 were generated and produced. The mutants were designed to potentially affect the binding of lipid bilayers by P2. All mutated variants were also crystallized and preliminary crystallographic data are presented. The structural data from the mutants will be combined with diverse functional assays in order to elucidate the fine details of P2 function at the molecular level.