Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP^C) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.     

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified “Shell” that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.     

Refolding the Engrailed Homeodomain: Structural Basis for the Accumulation of a Folding Intermediate

The ultrafast folding pathway of the engrailed homeodomain has been exceptionally well characterized by experiment and simulation. Helices II and III of the three-helix bundle protein form the native helix-turn-helix motif as an on-pathway intermediate within a few microseconds. The slow step is then the proper docking of the helices in ∼15 μs. However, there is still the unexplained puzzle of why helix docking is relatively slow, which is part of the more general question as to why rearrangements of intermediates occur slowly. To address this problem, we performed 46 all-atom molecular dynamics refolding simulations in explicit water, for a total of 15 μs of simulation time. The simulations started from an intermediate state structure that was generated in an unfolding simulation at 498 K and was then quenched to folding-permissive temperatures. The protein refolded successfully in only one of the 46 simulations, and in that case the refolding pathway mirrored the unfolding pathway at high temperature. In the 45 simulations in which the protein did not fully fold, nonnative salt bridges trapped the protein, which explains why the protein folds relatively slowly from the intermediate state.     

芋螺毒素氧化折叠研究进展

芋螺毒素(conotoxin,CTX)是当今生物毒素研究的热点之一,已经成为研究药理学和神经科学的重要工具。因此,芋螺毒素如何在体内和体外进行氧化折叠形成天然构象,获得有生物活性的芋螺毒素,越来越为人们所关注。根据当前研究状况,概述了芋螺毒素氧化折叠的一般过程,并总结了二硫键、PDI(二硫键异构酶)、前体肽等在芋螺毒素氧化折叠中所发挥的作用。此外,还就芋螺毒素氧化重折叠的研究前景和方向进行了展望。     

Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1–108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an α-helical structure extending from residues 14–29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.The discovery of parathyroid hormone (PTH)6 -related protein (PTHrP) as the cause of hypercalcemia in many patients with cancer provided new insights into the pathogenesis of the skeletal complications of malignancy (1). It revealed PTHrP as a previously unrecognized hormone, related in evolution to the calcium-regulating PTH, but important in the pathogenesis of the humoral hypercalcemia of malignancy, a syndrome in which hypercalcemia occurs without evident bone metastases. Whereas PTH consists of 84 amino acids, human PTHrP has three alternative splice products of 139, 141, and 173 residues. Apart from 8 of the first 13 residues of PTH and PTHrP being identical, there is no significant identity between these peptides (2). PTHrP actively promotes bone resorption, doing so in a manner identical to that of PTH by acting upon the receptor (PTH1R) it shares with PTH. The PTH1R is located on cells of the osteoblast lineage, which program the formation and activation of osteoclasts, and on cells of the kidney tubule, through which both PTHrP and PTH promote cyclic AMP and phosphorus excretion but reduce calcium excretion. Other actions of PTHrP that reflect those of PTH include the ability to relax vascular and other smooth muscle. This response may reflect a physiological function of PTHrP rather than of PTH and is consistent with PTHrP production and local action on smooth muscles at various sites (3).The first 34 amino acids of each hormone contain the full biological activities of both PTH and of PTHrP to activate the PTH1R (4). The sequences of PTHrP and PTH between residues 14 and 34 are interesting in that, although they are not homologous, nevertheless they appear to be critical for binding of each to the seven transmembrane G protein-coupled receptor, PTH1R (4). Within the first 34 amino acids of PTH and PTHrP two functional regions have been revealed based on structural and cross-linking studies (5–8). These studies have indicated that the C-terminal half of the first 34 residues of each hormone comprises the high affinity binding domain, interacting with the N-terminal portion of the extracellular domain of the receptor. The N-terminal half of each hormone activates the receptor through contact points on the extracellular loops and juxtamembrane regions (9).Despite their equal ability to activate through the PTH1R, it was clear from the earliest work, even with antibodies against peptides within the first 14 residues of PTHrP, that highly specific antibodies could be generated that discriminate between PTH and PTHrP (10). Likewise, polyclonal antibodies against PTHrP-(1–34) that neutralized its effects completely in vitro in promotion of cyclic AMP production in response to PTHrP without any detectable neutralizing effect on PTH were used to prevent and to treat hypercalcemia in nude mice bearing xenografts of PTHrP-secreting human cancers (11, 12). Similar results were obtained with a neutralizing mouse monoclonal antibody against PTHrP (13). Subsequently, after the finding that breast cancer metastases to bone were enriched in PTHrP production (14), Guise and Mundy (15) used an experimental model in nude mice in which human breast cancer cells grow as lytic deposits in bone after intracardiac injection and showed that PTHrP production by the cancers contributed to the process of tumor establishment and growth in bone by promoting osteoclast formation and bone resorption. Furthermore, the tumor establishment and growth in bone could be prevented by treating the mice with a monoclonal antibody against PTHrP (16) or with a bisphosphonate (17) to inhibit bone resorption.The efficacy of anti-PTHrP antibodies in treating both humoral-mediated hypercalcemia in cancer and bone metastasis formation and growth in mouse models raises the prospect of humanized forms of these antibodies being used as therapeutic agents in these diseases in human subjects, and preclinical data have been obtained in support of that (18, 19). With that in mind, the present project was undertaken in which we have made use of a monoclonal antibody prepared against human PTHrP (residues 1–34), which neutralizes the actions of PTHrP through PTH1R without any action against PTH. The antibody has been complexed with recombinant human PTHrP (residues 1–108) to generate crystals that have been used to analyze the three-dimensional structure with the aim of discovering the structural basis of neutralization of PTHrP action by the antibody.     

Structural Basis of Potent and Broad HIV-1 Fusion Inhibitor CP32M

CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B', CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the (621)VEWNEMT(627) motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the (621)VEWNEMT(627) motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use.     

Structural Basis and Selectivity of Tankyrase Inhibition by a Wnt Signaling Inhibitor WIKI4

Recently a novel inhibitor of Wnt signaling was discovered. The compound, WIKI4, was found to act through tankyrase inhibition and regulate β-catenin levels in many cancer cell lines and human embryonic stem cells. Here we confirm that WIKI4 is a high potency tankyrase inhibitor and that it selectively inhibits tankyrases over other ARTD enzymes tested. The binding mode of the compound to tankyrase 2 was determined by protein X-ray crystallography to 2.4 Å resolution. The structure revealed a novel binding mode to the adenosine subsite of the donor NAD⁺ binding groove of the catalytic domain. Our results form a structural basis for further development of potent and selective tankyrase inhibitors based on the WIKI4 scaffold.     

Structural Basis for the Immunomodulatory Function of Cysteine Protease Inhibitor from Human Roundworm Ascaris lumbricoides

Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.     

Irreversible,Tight-Binding Inhibition of Adenosine Deaminase by Coformycins: Inhibitor Structural Features That Contribute to the Mode of Enzyme Inhibition

Abstract

Coformycin analogues 1–6 were synthesized and biochemically screened against adenosine deaminase in order to assess the relative contributions of N-4, N-6, and the N-3 sugar moiety to the mode of enzyme inhibition. Our results indicate that N-4 plays a relatively greater role than N-6 in enzyme tight-binding, and that a benzyl group can substitute for the sugar moiety at N-3. The absence of a sugar or benzyl group at N-3, however, leads to loss of activity. The hydroxyl group at C-8, while crucial for activity, does not alone confer the tight-binding characteristics to coformycins.     

Structural Basis of Drug Binding to CYP46A1, an Enzyme That Controls Cholesterol Turnover in the Brain

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.     

Heterogeneous Packing in the Folding/Unfolding Intermediate State of Bitter Gourd Trypsin Inhibitor

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2–3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.     

蛋白质磷酸化作用的结构基础

蛋白质可逆磷酸化涉及到几乎所有细胞活动的调节.着重探讨了影响蛋白激酶作用专一性的几个因素和磷酸化影响蛋白质功能的结构基础及作用机制.     

氨基糖苷类修饰酶引起的细菌耐药性机制的研究进展

氨基糖苷类抗生素是高效、广谱的杀菌药物。随着在临床的广泛应用,抗生素的抗药性日趋严重,这在很大程度上降低了其临床应用的潜力。其中,最主要的原因就是细菌产生了一系列修饰酶修饰抗生素的特定基团,使其失去药效。细菌产生的修饰酶种类众多,主要包括磷酸化、乙酰化和腺苷化修饰酶。研究发现,一种酶可以修饰多种抗生素,同时,一种抗生素也可以被多种修饰酶修饰。由于修饰酶底物的广谱性,使得细菌的耐药性难以克服。因此,本文就氨基糖苷类修饰酶和抗生素相互作用的热力学和动力学性质进行了详细的论述,试图找出不同修饰酶失活抗生素药物的共同作用机制。这将为设计新的抗生素药物及修饰酶抑制剂、克服细菌的耐药性,提供理论指导和技术支持。     

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1), represented by AZ3976, was identified in a high throughput screening campaign. AZ3976 displayed an IC₅₀ value of 26 μm in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC₅₀ of 16 μm. Surprisingly, AZ3976 did not bind to active PAI-1 but bound to latent PAI-1 with a K_D of 0.29 μm at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance direct binding experiments. The x-ray structure of AZ3976 in complex with latent PAI-1 was determined at 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of surface plasmon resonance experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Because AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like prelatent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting x-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.     

Reconstitution of the Mia40-Erv1 Oxidative Folding Pathway for the Small Tim Proteins

Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of −310 mV via Mia40 with an intermediate midpoint potential of −290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of −150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX₉C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.