Structure of the LDL receptor extracellular domain at endosomal pH

The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH Ͻ 6. In the crystal structure at pH ϭ 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B, ␤ propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the ␤ propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the ␤ propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release. The low-density lipoprotein receptor (LDL-R) regulates cholesterol homeostasis in mammalian cells. LDL-R removes cholesterolcarrying lipoproteins from plasma circulation in a process known as receptor-mediated endocytosis (1). Ligands bound extracellularly by LDL-R at neutral pH are internalized and then released in the endosomes ( pH Ͻ 6), leading to their subsequent lysosomal degradation. The receptor then recycles to the cell surface. Mutations in the LDL-R gene cause familial hypercholesterolemia (FH), one of the most common simply inherited genetic diseases (2). FH heterozygotes exhibit a reduced rate of receptor-mediated removal of plasma LDL by the liver, ultimately leading to early onset coronary heart disease and atherosclerosis. More than 920 mutations in LDL-R are known, some of which have been functionally characterized (2, 3). The extracellular domain of LDL-R is composed of a "ligand-binding domain" (with cysteine-rich repeats R1 to R7) and an "epidermal growth factor (EGF) precursor homology domain" (with the EGF-like repeats A, B, and C, as well as a ␤ propeller between B and C) (4, 5). LDL-R binds LDL via the single protein in LDL, the 550-kD apolipoprotein B (apoB) (6); deleting R3, R4, R5, R6, or R7 reduces LDL binding to Ͻ20% of that of the wild-type LDL-R (7). LDL-R also binds to very low density lipoprotein (VLDL), ␤-VLDL, intermediate density lipoprotein (IDL), and chylomicron remnants via the 33-kD apolipoprotein E (apoE) (8, 9); disrupting R5 decreases ␤-VLDL binding to 30 to 50% of that of the wild-type receptor, whereas disrupting R4 or R6 reduces binding only slightly (7). At neutral pH, negative charges on repeats R1 to R7 are thought to interact with positive charges on apoB and apoE. Indeed, LDL binding to LDL-R can be disrupted competitively with polycations or permanently by selective chemical modification of positively charged residues on apoE or apoB Dissociation of ligands is crucial for receptor recycling and hence proper receptor function; mutations in LDL-R that impair ligand release produce FH (2). Deletion mutagenesis studies in LDL-R and the related VLDL-R have indicated that, although the ligand-binding domain is sufficient for binding lipoprotein particles, the receptor requires the EGF precursor homology domain for ligand release (16-18). The structural basis for LDL-R's ability to recognize a diverse group of lipoprotein particles, all varying in size, and release them at acidic pH is unknown. High-resolution crystal structures of modules R5 (12) and ␤ propeller-C (5) are known, and solution NMR structures are known for single and tandem repeats, including R1, R2, R5, R6, A, and B Structure determination. The extracellular domain of human LDL-R (residues 1 to 699) was crystallized at pH ϭ 5.3, with the symmetry of space group P3 1 21 (28). Soaking crystals in sodium 12-tungstophosphate (Na 3 PW 12 O 40 ) improved their diffractive quality and incorporated large anomalous scatterers. The asymmetric unit contains a single protein molecule and two tungsten clusters as well as half of a tungsten cluster on a crystallographic twofold axis. Data collection, structure determination, and model statistics are given in Monomer description. There is clear electron density in the crystal structure for the modules R2, R3, R4, R5, R6, R7, A, B, ␤ propeller, and C In the crystal, each monomer forms major contacts with five neighboring symmetry-related molecules. Although the relative orien

Threonine 149 Phosphorylation Enhances ΔFosB Transcriptional Activity to Control Psychomotor Responses to Cocaine

Stable changes in neuronal gene expression have been studied as mediators of addicted states. Of particular interest is the transcription factor ΔFosB, a truncated and stable FosB gene product whose expression in nucleus accumbens (NAc), a key reward region, is induced by chronic exposure to virtually all drugs of abuse and regulates their psychomotor and rewarding effects. Phosphorylation at Ser[superscript 27] contributes to ΔFosB's stability and accumulation following repeated exposure to drugs, and our recent work demonstrates that the protein kinase CaMKIIα phosphorylates ΔFosB at Ser[superscript 27] and regulates its stability in vivo. Here, we identify two additional sites on ΔFosB that are phosphorylated in vitro by CaMKIIα, Thr[superscript 149] and Thr[superscript 180], and demonstrate their regulation in vivo by chronic cocaine. We show that phosphomimetic mutation of Thr[superscript 149] (T149D) dramatically increases AP-1 transcriptional activity while alanine mutation does not affect transcriptional activity when compared with wild-type (WT) ΔFosB. Using in vivo viral-mediated gene transfer of ΔFosB-T149D or ΔFosB-T149A in mouse NAc, we determined that overexpression of ΔFosB-T149D in NAc leads to greater locomotor activity in response to an initial low dose of cocaine than does WT ΔFosB, while overexpression of ΔFosB-T149A does not produce the psychomotor sensitization to chronic low-dose cocaine seen after overexpression of WT ΔFosB and abrogates the sensitization seen in control animals at higher cocaine doses. We further demonstrate that mutation of Thr[superscript 149] does not affect the stability of ΔFosB overexpressed in mouse NAc, suggesting that the behavioral effects of these mutations are driven by their altered transcriptional properties

Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, forming trans-complexes spanning the synaptic cleft or cis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affect their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics

Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, forming trans-complexes spanning the synaptic cleft or cis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affect their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics

Structural Plasticity of Neurexin 1α: Implications for its Role as Synaptic Organizer.

α-Neurexins are synaptic organizing molecules implicated in neuropsychiatric disorders. They bind and arrange an array of different partners in the synaptic cleft. The extracellular region of neurexin 1α (n1α) contains six LNS domains (L1-L6) interspersed by three Egf-like repeats. N1α must encode highly evolved structure-function relationships in order to fit into the narrow confines of the synaptic cleft, and also recruit its large, membrane-bound partners. Internal molecular flexibility could provide a solution; however, it is challenging to delineate because currently no structural methods permit high-resolution structure determination of large, flexible, multi-domain protein molecules. To investigate the structural plasticity of n1α, in particular the conformation of domains that carry validated binding sites for different protein partners, we used a panel of structural techniques. Individual particle electron tomography revealed that the N-terminally and C-terminally tethered domains, L1 and L6, have a surprisingly limited range of conformational freedom with respect to the linear central core containing L2 through L5. A 2.8-Å crystal structure revealed an unexpected arrangement of the L2 and L3 domains. Small-angle X-ray scattering and electron tomography indicated that incorporation of the alternative splice insert SS6 relieves the restricted conformational freedom between L5 and L6, suggesting that SS6 may work as a molecular toggle. The architecture of n1α thus encodes a combination of rigid and flexibly tethered domains that are uniquely poised to work together to promote its organizing function in the synaptic cleft, and may permit allosterically regulated and/or concerted protein partner binding

Structural Plasticity of Neurexin 1α: Implications for its Role as Synaptic Organizer.

α-Neurexins are synaptic organizing molecules implicated in neuropsychiatric disorders. They bind and arrange an array of different partners in the synaptic cleft. The extracellular region of neurexin 1α (n1α) contains six LNS domains (L1-L6) interspersed by three Egf-like repeats. N1α must encode highly evolved structure-function relationships in order to fit into the narrow confines of the synaptic cleft, and also recruit its large, membrane-bound partners. Internal molecular flexibility could provide a solution; however, it is challenging to delineate because currently no structural methods permit high-resolution structure determination of large, flexible, multi-domain protein molecules. To investigate the structural plasticity of n1α, in particular the conformation of domains that carry validated binding sites for different protein partners, we used a panel of structural techniques. Individual particle electron tomography revealed that the N-terminally and C-terminally tethered domains, L1 and L6, have a surprisingly limited range of conformational freedom with respect to the linear central core containing L2 through L5. A 2.8-Å crystal structure revealed an unexpected arrangement of the L2 and L3 domains. Small-angle X-ray scattering and electron tomography indicated that incorporation of the alternative splice insert SS6 relieves the restricted conformational freedom between L5 and L6, suggesting that SS6 may work as a molecular toggle. The architecture of n1α thus encodes a combination of rigid and flexibly tethered domains that are uniquely poised to work together to promote its organizing function in the synaptic cleft, and may permit allosterically regulated and/or concerted protein partner binding

Structural Plasticity of Neurexin 1α: Implications for its Role as Synaptic Organizer.

α-Neurexins are synaptic organizing molecules implicated in neuropsychiatric disorders. They bind and arrange an array of different partners in the synaptic cleft. The extracellular region of neurexin 1α (n1α) contains six LNS domains (L1-L6) interspersed by three Egf-like repeats. N1α must encode highly evolved structure-function relationships in order to fit into the narrow confines of the synaptic cleft, and also recruit its large, membrane-bound partners. Internal molecular flexibility could provide a solution; however, it is challenging to delineate because currently no structural methods permit high-resolution structure determination of large, flexible, multi-domain protein molecules. To investigate the structural plasticity of n1α, in particular the conformation of domains that carry validated binding sites for different protein partners, we used a panel of structural techniques. Individual particle electron tomography revealed that the N-terminally and C-terminally tethered domains, L1 and L6, have a surprisingly limited range of conformational freedom with respect to the linear central core containing L2 through L5. A 2.8-Å crystal structure revealed an unexpected arrangement of the L2 and L3 domains. Small-angle X-ray scattering and electron tomography indicated that incorporation of the alternative splice insert SS6 relieves the restricted conformational freedom between L5 and L6, suggesting that SS6 may work as a molecular toggle. The architecture of n1α thus encodes a combination of rigid and flexibly tethered domains that are uniquely poised to work together to promote its organizing function in the synaptic cleft, and may permit allosterically regulated and/or concerted protein partner binding