Kinetic characterization of the double mutant R148A/E182S of glycogen phosphorylase kinase catalytic subunit: the role of the activation loop

Many protein kinases are activated by phosphorylation in a highly conserved region of their catalytic subunit, termed activation loop. Phosphorylase kinase is constitutively active without the requirement for phosphorylation of residues in the activation loop. The residue which plays an analogous role to the phosphorylatable residues in other protein kinases is Glu182, which makes contacts to a highly conserved Arg148. In turn, Arg148 adjacent to the catalytic Asp149, enabling information to be transmitted from the activation loop to the catalytic machinery. The double mutant R148A/E182S has been kinetically characterized. The mutation resulted in an approximate 16- to 22-fold decrease in the k _cat/K _m value of the enzyme. The kinetic data, discussed in the light of the structural data from previously determined complexes of the enzyme, lead to the suggestion that the activation loop has a major role in substrate binding but also in correct orientation of the groups participating in catalysis.     

Unequal cleavage in the early Tubifex embryo

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non-cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin-based, cell contact-dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4-cell stage.     

Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

Analysis of zinc binding sites in protein crystal structures

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.     

α-Bn-o-AMPA as a cis peptide bond mimic in somatostatin analogues

Summary In a previous communication we reported the racemic synthesis of the cis peptide bond mimic α-benzyl-o-aminomethylphenylacetic acid and its incorporation in the cyclic somatostatin analoguesc[α(R andS)Bn-o-AMPA-Phe⁷-d-Trp⁸-Lys⁹-Thr¹⁰]. Since the epimeric peptides exhibit different binding affinities, we completed the structure-activity study with an asymmetric synthesis. A model for the solution conformation ofc[α(R andS)Bn-o-AMPA-Phe⁷-d-Trp⁸-Lys⁹-Thr¹⁰] is proposed on the basis of a 2D NMR study in CD₃OH and restrained molecular dynamics.     

Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.     

海洋微生物裂解二甲基巯基丙酸内盐(DMSP)的酶促催化机制

二甲基巯基丙酸内盐(dimethylsulfoniopropionate,DMSP)是全球硫循环和碳循环的重要载体物质。海洋浮游植物、大型藻类和临海被子植物是DMSP的主要生产者。每年DMSP的产量可以达到1×10~9吨。在北大西洋表面的某些区域,DMSP的产量可以达到碳固定总量的10%。微生物介导的DMSP的裂解是全球硫循环和碳循环的重要步骤。目前,8种参与裂解DMSP的DMSP裂解酶已被报道。在已发现的8种DMSP裂解酶中,3种DMSP裂解酶的催化机制得到了研究和阐明。本文根据国内外研究成果,主要对DMSP裂解过程的酶促催化机制的研究进展进行综述,认为在今后工作中需要继续发现新的DMSP裂解酶,并进一步揭示海洋微生物裂解DMSP的分子机制。     

Characterization of conserved motifs in HIV-1 Vif required for APOBEC3G and APOBEC3F interaction

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, or A3G) and related cytidine deaminases such as apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F) are potent inhibitors of retroviruses. Formation of infectious human immunodeficiency virus (HIV)-1 requires suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through a common mechanism by recruiting Cullin5, ElonginB, and ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. Domains in Vif that mediate APOBEC3 recognition have not been fully characterized. In the present study, we identified a VxIPLx_4-5LxΦx₂YWxL motif in HIV-1 Vif, which is required for efficient interaction between Vif and A3G, Vif-mediated A3G degradation and virion exclusion, and functional suppression of the A3G antiviral activity. Amino acids 52 to 72 of HIV-1 Vif (including the VxIPLx_4-5LxΦx₂YWxL motif) alone could mediate interaction with A3G, and this interaction was abolished by mutations of two hydrophobic amino acids in this region. We have also observed that a Vif mutant was ineffective against A3G, yet it retained the ability to interact with Cullin5-E3 ubiquitin complex and A3G, suggesting that interaction with A3G is necessary but not sufficient to inhibit its antiviral function. Unlike the previously identified motif of HIV-1 Vif amino acids 40 to 44, which is only important for A3G suppression, the VxIPLx_4-5LxΦx₂YWxL motif is also required for efficient A3F interaction and suppression. On the other hand, another motif, TGERxW, of HIV-1 Vif amino acids 74 to 79 was found to be mainly important for A3F interaction and inhibition. Both the VxIPLx_4-5LxΦx₂YWxL and TGERxW motifs are highly conserved among HIV-1, HIV-2, and various simian immunodeficiency virus Vif proteins. Our data suggest that primate lentiviral Vif molecules recognize their autologous APOBEC3 proteins through conserved structural features that represent attractive targets for the development of novel inhibitors.     

Boosting Superior Lithium Storage Performance of Alloy‐Based Anode Materials via Ultraconformal Sb Coating–Derived Favorable Solid‐Electrolyte Interphase

Alloy materials such as Si and Ge are attractive as high‐capacity anodes for rechargeable batteries, but such anodes undergo severe capacity degradation during discharge–charge processes. Compared to the over‐emphasized efforts on the electrode structure design to mitigate the volume changes, understanding and engineering of the solid‐electrolyte interphase (SEI) are significantly lacking. This work demonstrates that modifying the surface of alloy‐based anode materials by building an ultraconformal layer of Sb can significantly enhance their structural and interfacial stability during cycling. Combined experimental and theoretical studies consistently reveal that the ultraconformal Sb layer is dynamically converted to Li₃Sb during cycling, which can selectively adsorb and catalytically decompose electrolyte additives to form a robust, thin, and dense LiF‐dominated SEI, and simultaneously restrain the decomposition of electrolyte solvents. Hence, the Sb‐coated porous Ge electrode delivers much higher initial Coulombic efficiency of 85% and higher reversible capacity of 1046 mAh g^?1 after 200 cycles at 500 mA g^?1, compared to only 72% and 170 mAh g^?1 for bare porous Ge. The present finding has indicated that tailoring surface structures of electrode materials is an appealing approach to construct a robust SEI and achieve long‐term cycling stability for alloy‐based anode materials.