Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.

Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease1 and is also linked to its idiopathic form2. LRRK2 has been proposed to function in membrane trafficking3 and colocalizes with microtubules4. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure5. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

A structural explanation for ERalpha/ERbeta SERM discrimination.

Many NRs have multiple subtypes that possess distinct expression patterns and that regulate distinct target genes. Antagonists generated through the addition of bulky side chains to agonist scaffolds are limited to being antagonistic on one or more subtypes of a particular NR. The passive antagonism mechanism, as revealed in our studies through direct comparison of the two THC-ER LBD complexes, suggests a new approach to achieving NR antagonism. Compounds could be designed to selectively stabilize the inactive conformations of certain NR subtypes and the active conformations of others. Such ligands are likely to exert novel biological and therapeutic effects.

Hormone selectivity in thyroid hormone receptors.

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia. We previously determined the crystal structure of the rat (r) TRalpha ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTRalpha LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRbeta receptor LBD in a complex with either Triac or a TRbeta-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTRalpha and hTRbeta LBDs show close structural similarity. However, the hTRbeta structures extend into the DNA-binding domain and allow definition of a structural "hinge" region of only three amino acids. The two TR subtypes differ in the loop between helices 1 and 3, which could affect both ligand recognition and the effects of ligand in binding coactivators and corepressors. The two subtypes also differ in a single amino acid residue in the hormone-binding pocket, Asn (TRbeta) for Ser (TRalpha). Studies here with TRs in which the subtype-specific residue is exchanged suggest that most of the selectivity in binding derives from this amino acid difference. The flexibility of the polar region in the TRbeta receptor, combined with differential recognition of the chemical group at the 1-carbon position, seems to stabilize the complex with GC-1 and contribute to its beta-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.

Orphan nuclear receptors: from new ligand discovery technologies to novel signaling pathways.

Members of the nuclear receptor superfamily of ligand-regulated transcription factors play critical roles in multiple aspects of development, cellular differentiation and homeostasis. The ligand-dependent transcriptional effects of nuclear receptors are, in part, mediated by interactions with a group of proteins collectively known as transcriptional coactivators. Receptor agonists promote coactivator binding and receptor antagonists suppress coactivator binding. Recently, biochemical assays that detect ligand-binding based on coactivator recruitment have been developed for several 'orphan' nuclear receptors, i.e., receptors for which no bona fide endogenous ligands are known. We review how these assays have been used to identify naturally occurring and synthetic ligands for the liver X receptor, farnesoid X receptor and estrogen receptor-related receptor subfamilies of orphans, the use of these ligands in the discovery of novel biological signaling pathways and the potential clinical implications of these findings.

Estrogen receptor pathways to AP-1.

Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (alpha or beta) and on the ligand. We propose that estrogen-ERalpha complexes use their activation functions (AF-1 and AF-2) to bind to the p 160 component of the coactivator complex recruited by Jun/Fos and trigger the coactivator to a higher state of activity. We propose that selective estrogen receptor modulator (SERM) complexes with ERbeta and with truncated ERalpha derivatives use their DNA binding domain to titrate histone deacetylase (HDAC)-repressor complexes away from the Jun/Fos coactivator complex, thereby allowing unfettered activity of the coactivators. Finally, we consider the possible physiological significance of ER action at AP-1 sites.

Pro region C-terminus:protease active site interactions are critical in catalyzing the folding of alpha-lytic protease.

alpha-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L. , and Agard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The pro region also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the active site by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904]. Through mutagenesis and structure-function analyses we have begun to investigate the mechanism by which the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Of central interest has been mapping the interface between the pro region and the protease and identifying interactions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletions of the pro region were found to have drastic effects on the rate at which the pro region folds the protease but surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly support a model in which the detailed interactions between the pro region C-terminus and the protease are remarkably similar to those of known substrate/inhibitor complexes. Further, mutation of two protease residues near the active site have significant effects on stabilization of the folding transition state (kcat) or in binding to the folding intermediate (KM). Our results suggest a model for the alpha-lytic protease pro region-mediated folding reaction that may be generally applicable to other pro region-dependent folding reactions.

The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen.

Ligand-dependent activation of transcription by nuclear receptors (NRs) is mediated by interactions with coactivators. Receptor agonists promote coactivator binding, and antagonists block coactivator binding. Here we report the crystal structure of the human estrogen receptor alpha (hER alpha) ligand-binding domain (LBD) bound to both the agonist diethylstilbestrol (DES) and a peptide derived from the NR box II region of the coactivator GRIP1 and the crystal structure of the hER alpha LBD bound to the selective antagonist 4-hydroxytamoxifen (OHT). In the DES-LBD-peptide complex, the peptide binds as a short alpha helix to a hydrophobic groove on the surface of the LBD. In the OHT-LBD complex, helix 12 occludes the coactivator recognition groove by mimicking the interactions of the NR box peptide with the LBD. These structures reveal the two distinct mechanisms by which structural features of OHT promote this "autoinhibitory" helix 12 conformation.

Inhibition of alpha-lytic protease by pro region C-terminal steric occlusion of the active site.

alpha-Lytic protease, a chymotrypsin-like serine protease, is synthesized with an N-terminal 166 amino acid pro region which is absolutely required for folding of the protease. The pro region is also the most potent inhibitor of the protease known with a Ki of approximately 10(-10) M. Compared to its role in the folding reaction, relatively little is known about the mechanism by which the pro region inhibits the mature protease. While proteinaceous protease inhibitors generally function by occluding the active sites of their respective targets [Bode, W., & Huber, R. (1992) Eur. J. Biochem. 204, 433-451], the pro region of alpha-lytic protease with its dual roles in folding and inhibition might be expected to show a novel mechanism of inhibition. However, experiments that probe both the structural and enzymatic consequences of pro region binding indicate that the pro region does not measurably distort the protease active site. Instead, the catalytic site is fully functional in the complex. Pro region inhibition of the protease is due to simple steric obstruction; the pro region C-terminus lies in the substrate binding sites of the protease. The implications of these results are discussed with regard to alpha-lytic protease maturation and folding. In addition, the proposed mechanism of alpha-lytic protease pro region inhibition is discussed with respect to data from other pro region families.

Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.

The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl. These results are entirely consistent with X-ray structures of Michaelis complexes that show a hydrogen bond positions the gamma-carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP.

P2U purinoceptors: cDNA cloning, signal transduction mechanisms and structure-function analysis.

The cloning of a P2U purinoceptor cDNA has made it possible to use molecular biological approaches to investigate P2U purinoceptor function. Expression of recombinant P2U purinoceptors in mammalian cells lacking endogenous P2U purinoceptors has enabled us to characterize the receptor protein and its downstream effectors, and has allowed a partial analysis of the role of certain amino acid residues in ligand binding. These approaches have placed the pharmacological classification of the P2U purinoceptor on a firm molecular footing and have generated model systems that can be used to investigate receptor-ligand binding, regulation and signal transduction.

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.

The role of pro regions in protein folding.

In vivo, many proteases are synthesized as larger precursors. During the maturation process, the catalytically active protease domain is released from the larger polypeptide or pro-enzyme by one or more proteolytic processing steps. In several well studied cases, amino-terminal pro regions have been shown to play a fundamental role in the folding of the associated protease domains. The mechanism by which pro regions facilitate folding appears to be significantly different from that used by the molecular chaperones. Recent results suggest that the pro region assisted folding mechanism may be used by a wide variety of proteases, and perhaps even by non-proteases.

Trypsin display on the surface of bacteriophage.

The gene III and VIII-encoded coat proteins (pIII and pVIII) from bacteriophage M13 have been fused to the C terminus of the serine protease, trypsin (Tsn). The genes encoding the fusions were then inserted directly into M13mp18 to create vectors which expressed both the Tsn-coat protein hybrids and the wild-type (wt) coat proteins. Immunoblot analysis confirmed that the bacteriophage express Tsn on their surface. Isolated fusion phage possess kinetic parameters which approximate those of the wt enzyme. An endogenous Escherichia coli protease inhibitor, ecotin, copurifies with the Tsn phage. Immobilized ecotin can be used to selectively bind bacteriophage which express Tsn::pIII fusion proteins.

Expression cloning of an ATP receptor from mouse neuroblastoma cells.

Extracellular ATP activates cell-surface metabotropic and ionotropic nucleotide (P2) receptors in vascular, neural, connective, and immune tissues. These P2 receptors mediate a wealth of physiological processes, including nitric oxide-dependent vasodilation of vascular smooth muscle and fast excitatory neurotransmission in sensory afferents. Although ATP is now recognized as a signaling molecule, the cellular and molecular mechanisms underlying its actions have been difficult to study due to the absence of selective P2 receptor antagonists and cloned receptor genes. Nonetheless, five mammalian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein-coupled peptide receptors. When expressed in Xenopus laevis oocytes, the cloned receptor resembles a metabotropic P2U receptor; activation by either ATP or UTP elicits the mobilization of intracellular calcium. mRNA encoding the P2U purinergic receptor is found in neural and nonneural tissues.