Recognition of Mesothelin by the Therapeutic Antibody MORAb-009: STRUCTURAL AND MECHANISTIC INSIGHTS*

Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.     

Streptococcus sanguinis Class Ib Ribonucleotide Reductase: HIGH ACTIVITY WITH BOTH IRON AND MANGANESE COFACTORS AND STRUCTURAL INSIGHTS*

Streptococcus sanguinis is a causative agent of infective endocarditis. Deletion of SsaB, a manganese transporter, drastically reduces S. sanguinis virulence. Many pathogenic organisms require class Ib ribonucleotide reductase (RNR) to catalyze the conversion of nucleotides to deoxynucleotides under aerobic conditions, and recent studies demonstrate that this enzyme uses a dimanganese-tyrosyl radical (Mn^III₂-Y^•) cofactor in vivo. The proteins required for S. sanguinis ribonucleotide reduction (NrdE and NrdF, α and β subunits of RNR; NrdH and TrxR, a glutaredoxin-like thioredoxin and a thioredoxin reductase; and NrdI, a flavodoxin essential for assembly of the RNR metallo-cofactor) have been identified and characterized. Apo-NrdF with Fe^II and O₂ can self-assemble a diferric-tyrosyl radical (Fe^III₂-Y^•) cofactor (1.2 Y^•/β₂) and with the help of NrdI can assemble a Mn^III₂-Y^• cofactor (0.9 Y^•/β₂). The activity of RNR with its endogenous reductants, NrdH and TrxR, is 5,000 and 1,500 units/mg for the Mn- and Fe-NrdFs (Fe-loaded NrdF), respectively. X-ray structures of S. sanguinis NrdI_ox and Mn^II₂-NrdF are reported and provide a possible rationale for the weak affinity (2.9 μm) between them. These streptococcal proteins form a structurally distinct subclass relative to other Ib proteins with unique features likely important in cluster assembly, including a long and negatively charged loop near the NrdI flavin and a bulky residue (Thr) at a constriction in the oxidant channel to the NrdI interface. These studies set the stage for identifying the active form of S. sanguinis class Ib RNR in an animal model for infective endocarditis and establishing whether the manganese requirement for pathogenesis is associated with RNR.     

Thiosulfate Dehydrogenase (TsdA) from Allochromatium vinosum: STRUCTURAL AND FUNCTIONAL INSIGHTS INTO THIOSULFATE OXIDATION*

Although the oxidative condensation of two thiosulfate anions to tetrathionate constitutes a well documented and significant part of the natural sulfur cycle, little is known about the enzymes catalyzing this reaction. In the purple sulfur bacterium Allochromatium vinosum, the reaction is catalyzed by the periplasmic diheme c-type cytochrome thiosulfate dehydrogenase (TsdA). Here, we report the crystal structure of the “as isolated” form of A. vinosum TsdA to 1.98 Å resolution and those of several redox states of the enzyme to different resolutions. The protein contains two typical class I c-type cytochrome domains wrapped around two hemes axially coordinated by His⁵³/Cys⁹⁶ and His¹⁶⁴/Lys²⁰⁸. These domains are very similar, suggesting a gene duplication event during evolution. A ligand switch from Lys²⁰⁸ to Met²⁰⁹ is observed upon reduction of the enzyme. Cys⁹⁶ is an essential residue for catalysis, with the specific activity of the enzyme being completely abolished in several TsdA-Cys⁹⁶ variants. TsdA-K208N, K208G, and M209G variants were catalytically active in thiosulfate oxidation as well as in tetrathionate reduction, pointing to heme 2 as the electron exit point. In this study, we provide spectroscopic and structural evidence that the TsdA reaction cycle involves the transient presence of heme 1 in the high-spin state caused by movement of the Sγ atom of Cys⁹⁶ out of the iron coordination sphere. Based on the presented data, we draw important conclusions about the enzyme and propose a possible reaction mechanism for TsdA.     

Dimeric Switch of Hakai-truncated Monomers during Substrate Recognition: INSIGHTS FROM SOLUTION STUDIES AND NMR STRUCTURE*

Hakai, an E3 ubiquitin ligase, disrupts cell-cell contacts in epithelial cells and is up-regulated in human colon and gastric adenocarcinomas. Hakai acts through its phosphotyrosine-binding (HYB) domain, which bears a dimeric fold that recognizes the phosphotyrosine motifs of E-cadherin, cortactin, DOK1, and other Src substrates. Unlike the monomeric nature of the SH2 and phosphotyrosine-binding domains, the architecture of the HYB domain consists of an atypical, zinc-coordinated tight homodimer. Here, we report a C-terminal truncation mutant of the HYB domain (HYB^ΔC), comprising amino acids 106–194, which exists as a monomer in solution. The NMR structure revealed that this deletion mutant undergoes a dramatic structural change caused by a rearrangement of the atypical zinc-coordinated unit in the C terminus of the HYB domain to a C₂H₂-like zinc finger in HYB^ΔC. Moreover, using isothermal titration calorimetry, we show that dimerization of HYB^ΔC can be induced using a phosphotyrosine substrate peptide. This ligand-induced dimerization of HYB^ΔC is further validated using analytical ultracentrifugation, size-exclusion chromatography, NMR relaxation studies, dynamic light scattering, and circular dichroism experiments. Overall, these observations suggest that the dimeric architecture of the HYB domain is essential for the phosphotyrosine-binding property of Hakai.     

Mechanism of Epac Activation: STRUCTURAL AND FUNCTIONAL ANALYSES OF Epac2 HINGE MUTANTS WITH CONSTITUTIVE AND REDUCED ACTIVITIES*

Epac2 is a member of the family of exchange proteins directly activated by cAMP (Epac). Our previous studies suggest a model of Epac activation in which cAMP binding to the enzyme induces a localized “hinge” motion that reorients the regulatory lobe relative to the catalytic lobe without inducing large conformational changes within individual lobes. In this study, we identified the location of the major hinge in Epac2 by normal mode motion correlation and structural alignment analyses. Targeted mutagenesis was then performed to test the functional importance of hinge bending for Epac activation. We show that substitution of the conserved residue phenylalanine 435 with glycine (F435G) facilitates the hinge bending and leads to a constitutively active Epac2 capable of stimulating nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan (F435W), impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters determined by small angle x-ray scattering further reveal that whereas the F435G mutant assumes a more extended conformation in the absence of cAMP, the F435W mutant is incapable of adopting the fully extended and active conformation in the presence of cAMP. These findings demonstrate the importance of hinge motion in Epac activation. Our study also suggests that phenylalanine at position 435 is the optimal size side chain to keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac extension and activation in the presence of cAMP.Exchange proteins directly activated by cAMP (Epac)² are a family of novel intracellular sensors for the second messenger cAMP (1, 2). Unlike the classic eukaryotic cAMP receptor, cAMP-dependent protein kinase (protein kinase A; PKA), Epac proteins do not function as protein kinases that phosphorylate downstream substrates. Instead, they act as guanine nucleotide exchange factors and exert their functions by activating small GTP-binding proteins, Rap1 and Rap2. At the cellular level, Epac proteins assume distinct subcellular localization (3), and depending upon the specific cellular environment, Epac and PKA may act independently, converge synergistically, or oppose each other in regulating a specific cellular function (4, 5).Both Epac and PKA share a common cyclic nucleotide binding domain (CBD), a compact and evolutionarily conserved structural motif found in a variety of proteins with diverse cellular functions (6). CBDs act as molecular switches for sensing intracellular second messenger cAMP levels and regulate the functionality of the domain(s) to which they are linked (6, 7). In depth structural and biophysical analyses of CBDs in several CBD-containing families, including cAMP receptor proteins, PKAs, and cyclic nucleotide-gated ion channels, have revealed a conserved structural core as well as functional motifs important for cyclic nucleotide-induced activation (8–11). The CBD is composed of an eight-strand β-barrel core that forms the base of the nucleotide binding pocket and a lateral α-helical bundle subdomain. Although the β-barrel core remains relatively constant between the ligand-free and nucleotide-bound states, binding of cAMP to a CBD leads to significant conformational changes in the overall arrangement of the α-helical bundle subdomain. A general mechanism of cyclic nucleotide-induced activation of CBD-containing proteins has been recently proposed (12). In this model, binding of cAMP leads to the retraction of the phosphate-binding cassette toward the cyclic nucleotide binding pocket and consequently releases the steric hindrance imposed on the α-helix C-terminal to the β-barrel, termed the CBD lid, by a conserved hydrophobic residue within the phosphate-binding cassette. These structural changes result in a hinge motion that allows the lid to move closer to the β-barrel core and to interact with the base of the cyclic nucleotide.The recently solved crystal structure of Epac2 reveals that, unlike other CBD-containing proteins, the corresponding lid region in Epac points away from the cAMP binding pocket as a two-strand β-sheet that forms the first part of the five-strand β-sheet “switchboard” structure (13). Although this major structural difference suggests that the detailed mechanisms of PKA and Epac activation by cAMP will most likely be different at the atomic level, it is not clear if the aforementioned general mechanism, namely the hinge motion, is conserved during Epac activation. To address this important question, we determined the effects of targeted hinge perturbations on Epac activation using site-directed mutagenesis that specifically targeted a key residue in the hinge region of Epac2.     

Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR*

Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.     

Serpins Flex Their Muscle: II. STRUCTURAL INSIGHTS INTO TARGET PEPTIDASE RECOGNITION,POLYMERIZATION, AND TRANSPORT FUNCTIONS*

Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.     

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL,FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES*

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro¹⁰³. A molecular dynamics simulation and mutational analyses revealed that Arg⁴⁰ in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.     

Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121: INSIGHTS FROM BIOCHEMICAL STUDIES AND CRYSTAL STRUCTURES*

Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn⁸⁵, Phe¹⁶⁸, and Trp¹⁸². The propensity of the cYY tyrosyl to point toward Arg³⁸⁶ was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.     

从新的古生物学及今生物学资料看羽毛的起源与早期演化(英文)

近年来关于羽毛和羽状皮肤衍生物的研究极大促进了我们对羽毛起源与早期演化的理解。结合最新的古生物学与今生物学资料,对一些保存了皮肤衍生物的非鸟恐龙标本进行观察研究,为这个重要的进化问题提供了新见解。推测羽毛的演化在鸟类起源之前就以下列顺序完成了5个主要的形态发生事件:1)丝状和管状结构的出现;2)羽囊及羽枝脊形成;3)羽轴的发生;4)羽平面的形成;5)羽状羽小支的产生。这些演化事件形成了多种曾存在于各类非鸟初龙类中的羽毛形态,但这些形态在鸟类演化过程中可能退化或丢失了;这些演化事件也产生了一些近似现代羽毛或者与现代羽毛完全相同的羽毛形态。非鸟恐龙身上的羽毛有一些现代羽毛具有的独特特征,但也有一些现生鸟羽没有的特征。尽管一些基于发育学资料建立的有关鸟类羽毛起源和早期演化的模型推测羽毛的起源是一个全新的演化事件,与爬行动物的鳞片无关,我们认为用来定义现代鸟羽的特征应该是逐步演化产生的,而不是突然出现。因此,对于羽毛演化而言,一个兼具逐步变化与完全创新的模型较为合理。从目前的证据推断,最早的羽毛既不是用来飞行也不是用来保暖,各种其他假说皆有可能,其中包括展示或者散热假说。展开整合性的研究有望为羽毛的起源问题提供更多思路。     

BILLENGSELLIDE AND ORTHIDE BRACHIOPODS: NEW INSIGHTS INTO EARLIEST ORDOVICIAN EVOLUTION AND BIOGEOGRAPHY FROM NORTHERN IRAN

Abstract:  The eastern Alborz Mountains of Iran comprise a significant peri-Gondwanan terrane relevant to the early evolution of late Cambrian – early Ordovician brachiopods incorporated into the emerging benthic biota of the Paleozoic Evolutionary Fauna. A low diversity brachiopod assemblage from the late Tremadocian unit of the Lashkarak Formation contains six new species including the polytoechioideans Polytoechia and Protambonites and the orthoideans Paralenorthis, Ranorthis, Tarfaya and Xianorthis . The fauna preserves the earliest records of Polytoechia , unknown previously outside Laurentia and the Uralian margin of Baltica, and of Paralenorthis and Ranorthis , which were widespread along Gondwanan margins and in Baltica from the Floian (Arenig), plus Xianorthis , known hitherto only from the Floian of South China. The enigmatic Tarfaya has an impunctate shell fabric and setigerous perforations along the posterior margin, indicating placement within the Orthoidea in a new Family Tarfayidae. New species of Polytoechia , Protambonites , Paralenorthis, Ranorthis , Tarfaya , Xianorthis are described.     

DETERMINANTS OF PRIMATE SOCIAL ORGANIZATION: COMPARATIVE EVIDENCE AND NEW INSIGHTS FROM MALAGASY LEMURS

The aim of this review is to summarize newly available information on lemur social systems, to contrast it with the social organization of other primates and to relate it to existing models of primate social evolution. Because of their evolutionary history, the primates of Madagascar constitute a natural experiment in social evolution. During millions of years of isolation, they converged with other primates only in the most fundamental way in the evolution of solitary, pair-living and group-living species, but deviate in several respects within these basic categories of social organization. Solitary lemurs remain poorly studied, but their social organization appears to be broadly similar to that of other solitary primates, even though the unexpected lack of sexual dimorphism may indicate that similar types of social organization can give rise to different mating systems. The determinants of a solitary lifestyle remain elusive. Pair-living lemurs show striking convergences with other monogamous primates in several behavioural traits, but also deviate in that the majority of species are at least partly nocturnal and do not exhibit direct paternal care of dependent young. Group-living lemurs have not evolved single-male groups, male-bonded and multi-level societies, and polyandrous groups may also be lacking. Female philopatry is common, but female bonds are generally weakly developed and eviction of females from natal groups is not unusual. Group-living lemurs also differ from anthropoids in that their groups have even adult sex ratios, smaller average size and may split up on a seasonal basis. Feeding competition, predation risk and reproductive competition can not fully explain these unusual aspects of lemur social organization. It has therefore been suggested that the social consequences of the risk of infanticide and of recent changes in activity may be ultimately responsible for these idiosyncracies of group-living lemurs, an explanation largely supported by the available evidence. Thus, social factors and fundamental life-history traits, in addition to ecological factors, contribute importantly to variation in social systems among lemurs, and possibly other primates. However, neither the diversity of lemur social systems, nor the evolutionary forces and mechanisms operating in these and other primates are yet fully understood.     

ALLOPOLYPLOID SPECIATION IN TRAGOPOGON: INSIGHTS FROM CHLOROPLAST DNA

Tragopogon mirus and T. miscellus are classic examples of recent allopolyploid speciation. Previous studies documented that the diploid parents of T. mirus are T. dubius and T. porrifolius and those of T. miscellus are T. dubius and T. pratensis. Restriction fragment analysis of chloroplast DNA (cpDNA) provided additional evolutionary information regarding the origin of the allotetraploids. We analyzed 39 populations of the three diploid and two allotetraploid species with 18 restriction endonucleases. Six restriction site mutations and three length mutations were identified; these unambiguously differentiated the parental diploids. Previous morphological, cytological, and electrophoretic analyses indicated that T. mirus arose independently at least three times. Chloroplast DNA data suggest that T. porrifolius has consistently been the maternal parent of T. mirus. Chloroplast DNA data also document a minimum of two independent origins of T. miscellus: 1) populations from Pullman, Washington, have T. dubius as the maternal parent; 2) all other populations have T. pratensis as the maternal parent. Two restriction site mutations implicate certain populations of T. dubius in the formation of the Pullman populations of T. miscellus. The two rare diploid species, T. porrifolius and T. pratensis, typically appear as maternal parents of the allotetraploids; the widespread and common T. dubius is the maternal parent only for two populations of T. miscellus. These data suggest that pollen load may be an important factor in determining the male and female parents of allopolyploid angiosperms.     

中国种子植物区系统计分析

中国种子植物初步统计有３３７科,３２００属,２６２７６～２７２６８种,其中裸子植物有１０科,３６属,１９１～１９５种,单子叶植物有５７科,６７９属,４４９３～４６６１种。本文对我国种子植物分别就科、属、种分布区类型,大小顺序排列,特有性等方面进行区系统计分析,并在种级水平上对各区系地区或具体区系进行对比,为中国种子植物区系深入研究提供基本素材     

Allosteric Inhibition of Epac: COMPUTATIONAL MODELING AND EXPERIMENTAL VALIDATION TO IDENTIFY ALLOSTERIC SITES AND INHIBITORS*

Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is an effector of cAMP signaling and has been implicated to have roles in numerous diseases, including diabetes mellitus, heart failure, and cancer. We used a computational molecular modeling approach to predict potential binding sites for allosteric modulators of Epac and to identify molecules that might bind to these regions. This approach revealed that the conserved hinge region of the cyclic nucleotide-binding domain of Epac1 is a potentially druggable region of the protein. Using a bioluminescence resonance energy transfer-based assay (CAMYEL, cAMP sensor using YFP-Epac-Rluc), we assessed the predicted compounds for their ability to bind Epac and modulate its activity. We identified a thiobarbituric acid derivative, 5376753, that allosterically inhibits Epac activity and used Swiss 3T3 and HEK293 cells to test the ability of this compound to modulate the activity of Epac and PKA, as determined by Rap1 activity and vasodilator-stimulated phosphoprotein phosphorylation, respectively. Compound 5376753 selectively inhibited Epac in biochemical and cell migration studies. These results document the utility of a computational approach to identify a domain for allosteric regulation of Epac and a novel compound that prevents the activation of Epac1 by cAMP.     

CONSTRAINTS ON MAMMALIAN FORELIMB DEVELOPMENT: INSIGHTS FROM DEVELOPMENTAL DISPARITY

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark‐based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo‐wide constraint.     

Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2: INSIGHTS FROM INTACT CELL PHOSPHORYLATION AND RECRUITMENT ASSAYS*

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α_2A-adrenergic receptors (α_2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α_2AAR with EC₅₀ values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu⁴, Val⁷, Leu⁸, Val¹¹, and Ser¹², directly interacts with receptors, whereas residues such as Asp¹⁰, Tyr¹³, Ala¹⁶, Met¹⁷, Gly⁴⁷⁵, Val⁴⁷⁷, and Ile⁴⁸⁵ are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.     

拱状灵芝多糖的分离与鉴定

由拱状灵芝Ganoderma Fornicolum提取的水溶性多糖,经甲醇分级与DEAE-纤维素柱层析获拱状灵芝葡聚糖,平均分子量为50,000。该葡聚糖经酶试验、高碘酸氧化、红外光谱、Smith降解,乙酰化产物质谱分析,证明为多枝结构。由β(1→6)(1→2)-D-葡萄糖组成。     

Crystal Structure of the Protealysin Precursor: INSIGHTS INTO PROPEPTIDE FUNCTION*

Protealysin (PLN) belongs to the M4 family of peptidases that are commonly known as thermolysin-like proteases (TLPs). All TLPs are synthesized as precursors containing N-terminal propeptides. According to the primary structure of the N-terminal propeptides, the family is divided into two distinct groups. Representatives of the first group including thermolysin and all TLPs with known three-dimensional structures have long prosequences (∼200 amino acids). Enzymes of the second group, whose prototype is protealysin, have short (∼50 amino acids) propeptides. Here, we present the 1.8 Å crystal structure of PLN precursor (proPLN), which is the first three-dimensional structure of a TLP precursor. Whereas the structure of the catalytic domain of proPLN is similar overall to previously reported structures of mature TLPs, it has specific features, including the absence of calcium-binding sites, and different structures of the N-terminal region and substrate-binding site. PLN propeptide forms a separate domain in the precursor and likely acts as an inhibitor that blocks the substrate-binding site and fixes the “open” conformation of the active site, which is unfavorable for catalysis. Furthermore the conserved PPL motif identified in our previous studies directly interacts with the S′ subsites of the active center being a critical element of the propeptide-catalytic domain interface. Comparison of the primary structures of TLPs with short propeptides suggests that the specific features revealed in the proPLN crystal structure are typical for all protealysin-like enzymes. Thus, such proteins can be considered as a separate subfamily of TLPs.