A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.     

Crystal structure of microsomal prostaglandin E2 synthase provides insight into diversity in the MAPEG superfamily

Prostaglandin E₂ (PGE₂) is a key mediator in inflammatory response. The main source of inducible PGE₂, microsomal PGE₂ synthase-1 (mPGES-1), has emerged as an interesting drug target for treatment of pain. To support inhibitor design, we have determined the crystal structure of human mPGES-1 to 1.2 Å resolution. The structure reveals three well-defined active site cavities within the membrane-spanning region in each monomer interface of the trimeric structure. An important determinant of the active site cavity is a small cytosolic domain inserted between transmembrane helices I and II. This extra domain is not observed in other structures of proteins within the MAPEG (Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily but is likely to be present also in microsomal GST-1 based on sequence similarity. An unexpected feature of the structure is a 16-Å-deep cone-shaped cavity extending from the cytosolic side into the membrane-spanning region. We suggest a potential role for this cavity in substrate access. Based on the structure of the active site, we propose a catalytic mechanism in which serine 127 plays a key role. We have also determined the structure of mPGES-1 in complex with a glutathione-based analog, providing insight into mPGES-1 flexibility and potential for structure-based drug design.     

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.     

Insights into the molecular architecture of the 26S proteasome

Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the “base” part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the “wobbling” model that was previously proposed to explain the role of the regulatory complex in opening the gate in the α-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.     

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved "arginine finger" of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras.RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling "off" to the signaling "on" state with a rate of 3 s(-1). The next step is the movement of the "arginine finger" into the active site of Ras with a rate of k(2) = 0.8 s(-1). Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound P(i) intermediate forms. The switch-I reversal to the "off" state, the release of P(i), and the movement of arginine back into an aqueous environment is observed simultaneously with k(3) = 0.1 s(-1), the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the "arginine finger" movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a P(i)-intermediate.     

Insights into the Transcriptional Regulation of Steroidogenic Enzymes and StAR

Reviews in Endocrine and Metabolic Disorders -     

Characterization of Metal-Bound Benzimidazole Derivatives,Effects on Tumor Cells of Lung Cancer

Four new ligands and four new copper (II) coordination compounds were prepared and characterized by chemical, elemental analysis, cytotoxicity, and FTIR spectroscopy (Fourier transform infrared spectroscopy). The nature of metal–ligand coordination was investigated. The thermal properties of complexes in the solid state were studied using TG-MS techniques (thermogravimetric analysis coupled with mass spectrometry) under dynamic flowing air atmosphere to analyze the principal volatile thermal decomposition and fragmentation products that evolved during thermolysis. The intermediate and final solid thermolysis products were also determined. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay was used to evaluate active metabolic cells as an IC₅₀ (half maximal inhibitory concentration). The relationship between antitumor activity and the position of nitrogen atoms in the organic ligand has been shown.     

Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library

A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantioselectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an E value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently.     

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.Hydrogen bond networks are ubiquitous structural features within proteins, and they play key roles linking secondary and tertiary structural elements and spanning protein–protein interfaces. Such networks are especially common within enzyme active sites, where they position protein and substrate groups for catalysis, stabilize charge rearrangements during chemical transformations, and mediate proton transfers (1). Despite the prevalence and critical structural and functional roles of hydrogen bond networks, incisive dissection of their physical properties within the idiosyncratic interior of folded proteins remains difficult.Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures due to the low X-ray scattering power of hydrogen atoms (2). Thus, the presence of hydrogen bond networks is typically inferred from the proximity and orientation of hydrogen bond donor and acceptor groups within refined protein structural models. The inherent inability of most X-ray diffraction studies to monitor proton positions imposes additional challenges for dissecting the physical features that influence the equilibrium protonation states of specific residues along a hydrogen-bonded proton transfer network. Furthermore, it remains extremely challenging to study the electrostatic consequences of charge rearrangements that accompany hydrogen bond-mediated proton transfers. Few experimental methods exist to vary the ionization properties of discrete protein groups incrementally, and structural rearrangements within the protein matrix that typically accompany charge rearrangements complicate computational modeling and the straightforward interpretation of the electrostatic properties of protein active sites and interiors (3–5).Bacterial ketosteroid isomerase (KSI) from Pseudomonas putida KSI (pKSI) and Comamonas testosteroni KSI has been a powerful system with which to study the physical properties of hydrogen bonds within an enzyme active site (6–13). KSI uses a general base, D40 (pKSI numbering), to deprotonate steroid substrates and form a dienolate reaction intermediate that is stabilized by hydrogen bonds donated by Y16 and protonated D103. Y16 is further linked via hydrogen bonds to Y57 and Y32, forming an extended active site hydrogen bond network in pKSI (Fig. 1A). Phenolic ligands, such as single-ring phenols, two-ring naphthols, and four-ring steroids like equilenin or estradiol, can bind in the KSI active site as negatively charged oxyanions and accept hydrogen bonds from Y16 and D103, mimicking the oxyanion charge localization of the dienolate reaction intermediate and dienolate-like transition states (6–8, 14, 15) (Fig. 1B). A homologous series of bound phenols or naphthols bearing different electron-withdrawing substituents provides a deft experimental tool with which to incrementally vary the proton affinity and negative charge density of the phenolic oxygen (16). These changes tune the structure and strength of hydrogen bonds formed to phenolic ligands (17–19), and thus provide a systematic probe of the physical and energetic properties of the hydrogen bond network within the KSI oxyanion hole (6–9, 11, 13) and the response of the surrounding protein matrix to such changes (20, 21).Open in a separate windowFig. 1.KSI reaction and reaction intermediate analog. (A) KSI reaction mechanism for isomerization of 5-androstene-3,17-dione. (B) Schematic depiction of an ionized substituted phenol bound at the KSI D40N active site.Recent studies with the D40N pKSI mutant, which mimics the protonated D40 present in the KSI–dienolate intermediate complex (Fig. 1A), have provided evidence that ligands of increasing pK_a are bound as an increasing population of neutral, protonated phenol (11, 13, 22). These results suggest that an unspecified active site residue can ionize with increasing phenol pK_a, resulting in a net proton transfer to the bound ligand. We have used site-specific NMR and IR probes and KSI semisynthesis to determine that either of two different Tyrs within the extended hydrogen bond network can ionize, and we have systematically mapped the changes in their equilibrium ionization states as a function of the proton affinity and hydrogen bonding capability of the phenolic ligand. We further measured the electric field changes at discrete active site positions due to charge rearrangements within the hydrogen bond network. We demonstrate that a static continuum electrostatic model with a low dielectric can accurately describe these changes, suggesting a high degree of structural organization and the absence of substantial conformational rearrangement in response to charge rearrangement within the active site.     

亚砷酸钠对HaCaT细胞MGMT基因启动子区甲基化CpG结合蛋白-2、DNA甲基转移酶1、组蛋白去乙酰化酶1结合的影响

目的 观察亚砷酸钠(NaAsO2)对人肤角质形成细胞株(HaCaT细胞)MGMT基因启动子区甲基化CpG结合蛋白-2(MeCP2)、DNA甲基转移酶1(DNMT1)及组蛋白去乙酰化酶1(HDAC1)结合情况的影响,为深化阐释砷毒作用机制提供依据.方法 分别以0.00(空白对照)、3.13、6.25、12.50、25.00 μmol/L NaAsO2重复间隔处理HaCaT细胞72 h(NaAsO2处理24h,隔天再次相同处理,重复3次),以人表皮鳞癌细胞株(A431)作为阳性对照,定量染色质免疫共沉淀技术(Q-ChIP)检测MGMT基因转录调控区ChIP1、ChIP2区域及MGMT基因编码区ChIP3区域MeCP2、DNMT1、HDAC1结合情况.结果 各组HaCaT细胞MGMT基因转录调控区ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为7.387、84.634、78.442和19.263、69.649、26.546,P均＜0.05);其中各NaAsO2处理组ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平[3.13 μmol/L NaAsO2处理组:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％和(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％;6.25 μmol/L NaAsO2处理组:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％和(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2处理组:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％和(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO处理组:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％和(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]均高于空白对照组[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％和(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P均＜0.05].各组HaCaT细胞MGMT基因编码区ChIP3区域MeCP2蛋白结合水平比较,差异无统计学意义(F=1.670,P＞0.05),而DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为4.404、9.863,P均＜0.05),其中25.00 μmol/L NaAsO2处理组DNMT1、HDAC1蛋白结合水平[(615.85±29.63)％、(306.09±59.40)％]与空白对照组[(99.70±12.02)％、(92.45±48.79)％]比较,差异有统计学意义(P均＜0.05).结论 MeCP2可结合于砷所致高甲基化MGMT基因转录调控区,通过招募DNMT1及HDAC1使组蛋白去乙酰化,同时DNMT1可结合于MGMT基因编码区,以非甲基化DNA结合蛋白(MBD)依赖的方式招募HDAC1,通过染色质重塑方式导致MGMT基因沉默,可能是砷毒性表现的早期分子事件.     

Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). An important step in the mechanism involves proton donation to the N5 atom of DHF. The inability to determine the protonation states of active site residues and substrate has led to a lack of consensus regarding the catalytic mechanism involved. To resolve this ambiguity, we conducted neutron and ultrahigh-resolution X-ray crystallographic studies of the pseudo-Michaelis ternary complex of Escherichia coli DHFR with folate and NADP⁺. The neutron data were collected to 2.0-Å resolution using a 3.6-mm³ crystal with the quasi-Laue technique. The structure reveals that the N3 atom of folate is protonated, whereas Asp27 is negatively charged. Previous mechanisms have proposed a keto-to-enol tautomerization of the substrate to facilitate protonation of the N5 atom. The structure supports the existence of the keto tautomer owing to protonation of the N3 atom, suggesting that tautomerization is unnecessary for catalysis. In the 1.05-Å resolution X-ray structure of the ternary complex, conformational disorder of the Met20 side chain is coupled to electron density for a partially occupied water within hydrogen-bonding distance of the N5 atom of folate; this suggests direct protonation of substrate by solvent. We propose a catalytic mechanism for DHFR that involves stabilization of the keto tautomer of the substrate, elevation of the pK_a value of the N5 atom of DHF by Asp27, and protonation of N5 by water that gains access to the active site through fluctuation of the Met20 side chain even though the Met20 loop is closed.Dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase) (DHFR) is a housekeeping enzyme that catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8,-tetrahydrofolate (THF). Various redox states of THF are used in several one-carbon transfer reactions to generate thymidine, methionine, glycine, serine, and other molecules (1–3). Given its role in biosynthesis, DHFR is a target for anticancer, antimicrobial, and rheumatoid arthritis drugs, such as methotrexate (MTX) and trimethoprim (4–7).Although the kinetics, structure, and biophysical properties of Escherichia coli DHFR (ecDHFR) have been well characterized, unresolved questions with respect to its catalytic mechanism remain (1, 3, 8–13), as evidenced by the recent controversy over whether millisecond time-scale structural fluctuations can directly affect the chemical step in catalysis (14, 15). Folate is a poor substrate for DHFR, whereas DHF is reduced more efficiently (2). In addition, unlike DHF, folate cannot be further oxidized in solution. Thus, the abortive DHFR-folate-NADP⁺ complex is an excellent mimic of the DHFR-DHF-NADPH Michaelis complex (3, 16), and its stability makes it well suited for structural studies.During catalysis, a proton is donated to the N5 atom of the DHF pterin ring and a hydride equivalent is transferred from NADPH to the C6 atom of the pterin. With folate as a substrate, proton donation occurs at the N8 atom (10). The five intermediates in the catalytic cycle are E-NADPH, E-NADPH-DHF, E-NADP⁺-THF, E-THF, and E-NADPH-THF (3), with product release as the rate-limiting step at neutral pH. THF is released on binding of a new NADPH molecule. The enzyme displays pH dependence with a characteristic pK_a value of 6.5 (8).Previous crystallographic and NMR studies of the DHFR binary and ternary complexes have revealed the locations of the folate and nicotinamide cofactor optimal for hydride transfer and the juxtaposition of the substrate with respect to the catalytic Asp27, which forms hydrogen bonds with the N3 and NA2 atoms of folate (3, 12). The DHFR-folate-NADP⁺ complex structure is considered the closest mimic of the Michaelis complex and has been used as a reference model in studies of the molecular details required for proton donation and hydride transfer (2, 3, 14, 17). Although it is clear from the structure that the nicotinamide ring is optimally positioned for hydride transfer to the C6 atom of the DHF substrate, how a proton can be donated to the N5 atom is unclear, especially considering that the conserved Asp27 is almost 5 Å distant from it. Disagreement abounds as to the protonation state of the Asp27 during catalysis (10, 18, 19). The mutation of the other residues contacting the substrate diminishes but does not abrogate activity, suggesting that the enzyme is flexible and has built-in redundancies (9, 20).Several catalytic mechanisms have been proposed based on X-ray and NMR structures, molecular dynamics, enzyme kinetic measurements, and Raman spectroscopy studies (1, 17, 18, 21). According to Maharaj et al. (21), the pK_a of the N5 atom of DHF is 2.6 in solution. When bound in a binary complex to DHFR, its N5 pK_a remains strongly acidic. However, the pK_a is elevated from <4 in the binary complex to 6.5 in the catalytic mimic complex, where NADP⁺ is bound as well (1, 21). This value matches the pK_a describing the hydride transfer step (8), suggesting that the kinetic pK_a describes the level of N5 protonated substrate available. The accompanying article by Liu et al. (22) further explores the kinetic pH profile for ecDHFR and its relationship to the hydride transfer step, as well as the sequential order of the mechanism. Outstanding questions remain, including, but not limited to, the following: (i) When a catalytically competent complex is present, how does the active site environment so drastically increase the N5 pK_a to promote protonation? (ii) What is the protonation state of Asp27 throughout catalysis? And (iii) what is the source and mechanism of proton donation to N5?An oft-proposed general mechanism based on several crystallography and Raman spectroscopy studies invokes a keto-enol tautomerization of the pterin substrate, initiating at the Asp27 and triggering a proton shuttle that ultimately results in a reduction of N5 (9, 10, 23). Two versions of this mechanism have been proposed, the major differences being the protonation state of Asp27 in the ground state and the ultimate proton source for reduction of N5 (SI Materials and Methods). The caveat regarding this mechanism is that keto-enol tautomerization as a critical step in the DHFR catalytic cycle remains a major point of ambiguity (24). Blakley et al. (25) have challenged the idea that substrate undergoes tautomerization during catalysis based on the NMR finding of a persistent substrate in an N3 imino-C4 keto tautomer across a pH range.Alternative catalytic mechanisms propose the direct involvement of water molecules in the proton transfer step. These mechanisms notably omit the necessity for a substrate tautomerization event and the requirement for protonation of Asp27 at some point in the catalytic cycle (17). Asp27 is mainly responsible for binding the substrate in a catalytically favorable conformation and maintaining a negative electrostatic field in the active site, which would be negated if its carboxylate were protonated even transiently. A recent study revealed that Met20 loop dynamics are critical for solvent access to N5, and proposed a mechanism involving direct solvent protonation of the substrate (22). There has been only one previous structural observation of a solvent molecule within hydrogen-bonding distance of the N5 atom, in a crystal structure of E. coli DHFR bound to folate and NADP⁺ (1RA2). It should be noted that the Met20 loop adopts an open conformation in this structure, likely because of crystal packing effects (3). A solvent molecule is typically modeled in this position in crystal structures with only substrate bound (2).A barrier to experimentally testing most proposed enzyme mechanisms is the inability to directly visualize the positions of important catalytic protons. The initial models used for most theoretical calculations are derived from X-ray structures, and determining the location of hydrogen atoms using X-rays is difficult even at atomic resolution (1.2 Å) (26). Neutron crystallography (NC) has a proven ability to determine the positions of hydrogen atoms or ions (protons) essential for catalysis (27–30). In fact, NC defines unique positions of hydrogen atoms within ordered water molecules (31), and H₃O⁺ molecules crucial for catalysis in xylose isomerase were recently identified (32). By virtue of the need to perform hydrogen/deuterium exchange (HDX) on crystals before data collection, NC can accurately identify hydrogen atom positions even at modest resolution. Deuterium coherently scatters neutrons with lengths similar to carbon and nitrogen, whereas hydrogen coherently scatters neutrons with negative lengths, rendering them invisible in positively contoured nuclear density maps. In the past, the determination of NC structures was hindered by the limited number of data collection facilities, low beam fluxes, and the requirement for extremely large crystals (>1 mm³ in volume). Recently, new spallation sources, enhanced deuterium labeling of samples, and improved detectors have allowed the collection of high-quality data from crystals of smaller volume, leading to a dramatic increase in the number of neutron structures deposited in the Protein Data Bank (PDB).In a previous NC study, we resolved a question pertaining to the protonation state of the classical antifolate inhibitor MTX and Asp27 when MTX binds DHFR (28). The DHFR-MTX neutron structure demonstrates that Asp27 is negatively charged, whereas the N1 atom of MTX is protonated and thus positively charged. After our initial success with the DHFR-MTX complex, we conducted NC studies of a DHFR pseudo-Michaelis complex to identify the protonation state of Asp27 in a catalytic mimic complex, the source of protonation for N5, as well as the presence (or absence) of a substrate keto-enol tautomerization event. Here we report the neutron diffraction structure of the DHFR-folate-NADP⁺ complex and complementary ultrahigh-resolution X-ray structures at three different temperatures. Refinement of the neutron structure allowed determination of the positions of crucial protons on the folate substrate and the ionization state of Asp27. Furthermore, our comprehensive map of backbone HDX sheds light on the dynamically driven changes in solvent accessibility of crystalline DHFR. The ultrahigh-resolution X-ray structures provide molecular details of Met20 loop fluctuations required for the entry of solvent, identifying a water molecule possibly involved in proton donation to N5.     

Insights into pilus assembly and secretion from the structure and functional characterization of usher PapC

Ushers constitute a family of bacterial outer membrane proteins responsible for the assembly and secretion of surface organelles such as the pilus. The structure at 3.15-Å resolution of the usher pyelonephritis-associated pili C (PapC) translocation domain reveals a 24-stranded kidney-shaped β-barrel, occluded by an internal plug domain. The dimension of the pore allows tandem passage of individual folded pilus subunits in an upright pilus growth orientation, but is insufficient for accommodating donor strand exchange. The molecular packing revealed by the crystal structure shows that 2 PapC molecules in head-to-head orientation interact via exposed β-strand edges, which could be the preferred dimer interaction in solution. In vitro reconstitution of fiber assemblies suggest that PapC monomers may be sufficient for fiber assembly and secretion; both the plug domain and the C-terminal domain of PapC are required for filament assembly, whereas the N-terminal domain is mainly responsible for recruiting the chaperone–subunit complexes to the usher. The plug domain has a dual function: gating the β-pore and participating in pilus assembly.     

Structural Insights into the Chaperone-Assisted Assembly of a Simplified Tail Fiber of the Myocyanophage Pam3

At the first step of phage infection, the receptor-binding proteins (RBPs) such as tail fibers are responsible for recognizing specific host surface receptors. The proper folding and assembly of tail fibers usually requires a chaperone encoded by the phage genome. Despite extensive studies on phage structures, the molecular mechanism of phage tail fiber assembly remains largely unknown. Here, using a minimal myocyanophage, termed Pam3, isolated from Lake Chaohu, we demonstrate that the chaperone gp25 forms a stable complex with the tail fiber gp24 at a stoichiometry of 3:3. The 3.1-Å cryo-electron microscopy structure of this complex revealed an elongated structure with the gp25 trimer embracing the distal moieties of gp24 trimer at the center. Each gp24 subunit consists of three domains: the N-terminal α-helical domain required for docking to the baseplate, the tumor necrosis factor (TNF)-like and glycine-rich domains responsible for recognizing the host receptor. Each gp25 subunit consists of two domains: a non-conserved N-terminal β-sandwich domain that binds to the TNF-like and glycine-rich domains of the fiber, and a C-terminal α-helical domain that mediates trimerization/assembly of the fiber. Structural analysis enabled us to propose the assembly mechanism of phage tail fibers, in which the chaperone first protects the intertwined and repetitive distal moiety of each fiber subunit, further ensures the proper folding of these highly plastic structural elements, and eventually enables the formation of the trimeric fiber. These findings provide the structural basis for the design and engineering of phage fibers for biotechnological applications.     

Sequence variants of the sarco(endo)plasmic reticulum Ca2+-transport ATPase 3 gene (SERCA3) in Caucasian Type II diabetic patients (UK Prospective Diabetes Study 48)

Abstract Aims/hypothesis. Type II (non-insulin-dependent) diabetes mellitus is a common heterogeneous metabolic disorder of largely unknown genetic aetiology. The sarco(endo)plasmic reticulum Ca²⁺-transport ATPase (SERCA) plays an important part in the glucose-activated beta-cell Ca²⁺ signalling that regulates insulin secretion. Impaired function and expression of SERCA have been shown in islets of Langerhans from diabetic animal models and have also been associated with beta-cell apoptosis. Thus, the SERCA3 encoding gene is a plausible candidate for a primary pancreatic beta-cell defect. Methods. In this study, the entire coding and the promoter regions of SERCA3 gene were screened by single-strand conformation polymorphism analysis in white Caucasian Type II diabetic patients. Results. We found four rare missense mutations [Exon 4: Gln ₁₀₈→His (CAG→CAT), Exon 14: Val ₆₄₈→Met (GTG→ATG) and Arg ₆₇₄→Cys (CGC→TGC), and Exon 15: Ile ₇₅₃→Leu (ATC→CTC)]. The patients with Gln ₁₀₈→His, Val ₆₄₈→Met and Arg ₆₇₄→Cys mutations, which may affect the E1P-E2P transition of SERCA3 during its enzyme cycle, had normal body weight with marked hyperglycaemia and beta-cell dysfunction. That is an unusual phenotype only found in 6 % of the Type II diabetic patients recruited for the UK Prospective Diabetes Study. In addition, five silent polymorphisms, six intron variants and two polymorphisms in the 3' untranslated region of exon 22 were found with similar frequency in diabetic and control subjects. Conclusion/interpretation. Our result suggests that in white Caucasians, the SERCA3 locus possibly contributes to the genetic susceptibility to Type II diabetes [Diabetologia (1999) 42: 1240–1243]. Received: 9 March 1999 and in revised form: 26 April 1999     

New insights into the molecular mechanisms of priming of insulin exocytosis

Exocytosis of insulin vesicles in the pancreatic β-cell involves a sequence of regulated events, whose normal function and efficient adaptation to increased demand are essential for the maintenance of glucose homeostasis. These exocytotic events comprise the trafficking and docking of vesicles to the plasma membrane, followed by fusion triggered by Ca²⁺. Recent studies have unravelled post-docking steps mediated by novel factors, which, by their interactions with soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)- and SNARE-associated proteins, confer the docked vesicles fusion competence. These priming steps define the releasable pool of insulin vesicles, which accounts for the first phase of insulin secretion, and controls the rate at which vesicles are replenished for the second phase of secretion. This article aims to summarize what is currently known about the mechanisms that underlie the priming activity of these proteins, focusing on Munc13, a topic to which we have made some recent contributions. Abnormal glucose homeostasis in type 2 diabetes is because of the failure of islet β-cells to augment insulin secretion sufficiently to compensate for reduced insulin sensitivity. A better understanding of the priming steps may help develop novel approaches to increase insulin secretory capacity and thereby prevent the progression to type 2 diabetes.     

Insights into the regulation of the human COP9 signalosome catalytic subunit,CSN5/Jab1

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.     

Relative contribution of different l-arginine sources to the substrate supply of endothelial nitric oxide synthase

In certain cases of endothelial dysfunction l-arginine becomes rate-limiting for NO synthesis in spite of sufficiently high plasma concentrations of the amino acid. To better understand this phenomenon, we investigated routes of substrate supply to endothelial nitric oxide synthase (eNOS). Our previous data with human umbilical vein (HUVEC) and EA.hy.926 endothelial cells demonstrated that eNOS can obtain its substrate from the conversion of l-citrulline to l-arginine and from protein breakdown. In the present study, we determined the quantitative contribution of proteasomal and lysosomal protein degradation and investigated to what extent extracellular peptides and l-citrulline can provide substrate to eNOS. The RFL-6 reporter cell assay was used to measure eNOS activity in human EA.hy926 endothelial cells. Individual proteasome and lysosome inhibition reduced eNOS activity in EA.hy926 cells only slightly. However, the combined inhibition had a pronounced reducing effect. eNOS activity was fully restored by supplementing either l-citrulline or l-arginine-containing dipeptides. Histidine prevented the restoration of eNOS activity by the dipeptide, suggesting that a transporter accepting both, peptides and histidine, mediates the uptake of the extracellular peptide. In fact, the peptide and histidine transporter PHT1 was expressed in EA.hy926 cells and HUVECs (qRT/PCR). Our study thus demonstrates that l-citrulline and l-arginine-containing peptides derived from either intracellular protein breakdown or from the extracellular space seem to be good substrate sources for eNOS.     

Insights into the social context of living with a dual diagnosis of HIV and cancer: a qualitative,thematic analysis of popular discourse in London newspapers

ABSTRACT

As growing numbers of people living with HIV also develop cancer, a holistic understanding of their experiences is essential to the provision of patient centred care. Both conditions are linked to powerful beliefs in our society that may affect experiences. This study explored how HIV and cancer were represented in UK newspapers to gain insight into the social context of living with a dual diagnosis. We performed an initial content analysis of HIV articles and of cancer articles published in the free London newspapers, The Metro and The Evening Standard between 2012 and 2017, followed by qualitative thematic analysis and in-depth analysis of selected articles of exemplar cases. Both conditions were presented very differently. The underlying subtext was that cancer could happen to any of us. HIV was framed as a potentially dangerous, stigmatising phenomenon affecting “others”. Popular discourse about HIV within news media remains largely negative and stigmatising. People living with a dual diagnosis of HIV and cancer may choose to prioritise the sharing of the more socially acceptable condition, cancer, in order to access support. The negotiation of cancer healthcare services is likely to be adversely influenced by the social burden of HIV related stigma.     

Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomicroscopy

Yeast fatty acid synthase (FAS) is a 2.6-MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a three-dimensional map of yeast FAS at 5.9-Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution compared to the crystal structures, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fitted the substrate-binding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ∼18 Å from the ACP substrate-binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct structural insight into the substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.