Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C₁₄-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C₁₄-CoA and can also bind the far more abundant palmitoyl-CoA (C₁₆-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C_18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C₁₆-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C₁₆-CoA. These results indicate that ACBD6 can locally sequester C₁₆-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C₁₆-CoA.     

Mode of Binding of the Tuberculosis Prodrug Isoniazid to Heme Peroxidases: BINDING STUDIES AND CRYSTAL STRUCTURE OF BOVINE LACTOPEROXIDASE WITH ISONIAZID AT 2.7 ? RESOLUTION*

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe⁴²² O, Gln⁴²³ O^ϵ1, and Phe²⁵⁴ O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe³⁺, His¹⁰⁹ N^ϵ2, and Gln¹⁰⁵ N^ϵ2. The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C^β and C^γ atoms of Glu²⁵⁸, and C^γ and C^δ atoms of Arg²⁵⁵. The binding studies indicate that INH binds to LPO with a value of 0.9 × 10⁻⁶ m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H₂O₂ with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.     

Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5′ termini by cleaving tRNA precursors immediately 5′ of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G₊₁C₊₇₂ base pair at the end of the aminoacyl acceptor-stem whereas in tRNA^Gln G₊₁C₊₇₂ is replaced with U₊₁A₊₇₂. Here, we investigated RNase P RNA-mediated cleavage as a function of having G₊₁C₊₇₂ versus U₊₁A₊₇₂ in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA^Gln and pre-tRNA^TyrSu3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G₊₁C₊₇₂ with U₊₁A₊₇₂ influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA^Gln and pre-tRNA^TyrSu3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg²⁺ binding, most likely in its vicinity.     

State-dependent modulation of CFTR gating by pyrophosphate

Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR''s two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg²⁺-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (τ ∼1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (τ ∼30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C₁ and C₂) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C₂) that can be locked open by MgPPi. The stability of the C₂ state is enhanced when the channel is initially opened by N⁶-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C₂ state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C₂-equivalent state (C₂*), which exists before the channel in the C₁ state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C₂ state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.     

Topographic analysis of the S7 binding subsite of the tachykinin neurokinin-1 receptor

Conformationally and configurationally restricted rotameric probes of phenylalanine have been incorporated in the sequence of substance P (SP)—Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂—for analyzing the binding pockets of Phe⁷ (S₇) and Phe⁸ (S₈), in the neurokinin-1 receptor. These analogues of phenylalanine are (2S, 3R)- and (2S, 3S)-indanylglycines, E- and Z-α, β-dehydrophenylalanines, and 2(S)-α, β-cyclopropylphenylalanines [Δ^E Phe, Δ^ZPhe, ▿^E2(S)Phe, and ▿^Z2(S)Phe]. Binding data obtained with either conformationally (Ing diastereoisomers) or configurationally (Δ^EPhe, Δ^ZPhe) probes have unveiled large differences in the binding potencies of these rotameric probes. With the support of nmr data and energy calculations done on these SP-substituted analogues, we attempt to answer questions inherent to such study. First, none of these six probes prevents the formation of bioactive conformation(s) of the backbone of SP. Second, both diastereoisomers (S, S) and (S, R) of indanylglycine preferentially adopt, in the sequence of SP, the gauche (−) and trans side-chain orientations, respectively, as previously postulated from energy calculations with model peptides. However, in solution, the difference in energy between these rotamers included in the sequence of SP, compared to model peptides, is smaller since the other rotamer can be detected in [(2S, 3R) Ing⁷]SP. Finally, from this study we can hypothesize that the large variations observed in the affinities of Phe⁷ substituted analogues of SP must come from steric hindrance in the S₇ binding site, which drastically restricts the space filling around the C_α (SINGLE BOND) C_β bond of residue 7. © 1996 John Wiley & Sons, Inc.     

Cooperative binding of 3'-fragments of transfer ribonucleic acid to the peptidyltransferase center of Escherichia coli ribosomes.

The binding of substrates to the A-site half (A′) and the P-site half (P′) of the peptidyltransferase center was measured by means of equilibrium dialysis. The tRNA fragments C-A-C-C-A-Leu and C-A-C-C-A-(N-acetyl)Leu were used as A′-site and P′-site substrates, respectively. The A′- and P′-substrates bound well to 50 S in contrast to 30 S subunits; significant binding to 23 S and 16 S RNA was also found. The binding of the P′-site substrate to 23 S RNA and 50 S subunits was very similar at various Mg²⁺ and K⁺ concentrations, indicating that the 23 S RNA is probably directly involved in the binding of the 3′-end of the peptidyl-tRNA. Cooperative effects at the peptidyltransferase center were found using chloramphenicol and deacylated tRNA as competitors, which completely inhibited the substrate binding to one site whilst drastically stimulating binding to the other. Chloramphenicol inhibited the binding of the A′-site substrate C-A-C-C-A-Leu, whereas the binding of the corresponding P′-site substrate was stimulated. In contrast, deacylated tRNA blocked the binding of the P′-site substrate, but stimulated the corresponding A′-site binding. Similarly, the trinucleotide C_p,C_pA inhibited binding of the P′-site substrate (showing complete inhibition at 70 μm) whereas binding of the A′-site substrate was slightly stimulated at concentrations below 70 μm.     

Simulated annealing reveals the kinetic activity of SGLT1, a member of the LeuT structural family

The Na⁺/glucose cotransporter (SGLT1) is the archetype of membrane proteins that use the electrochemical Na⁺ gradient to drive uphill transport of a substrate. The crystal structure recently obtained for vSGLT strongly suggests that SGLT1 adopts the inverted repeat fold of the LeuT structural family for which several crystal structures are now available. What is largely missing is an accurate view of the rates at which SGLT1 transits between its different conformational states. In the present study, we used simulated annealing to analyze a large set of steady-state and pre–steady-state currents measured for human SGLT1 at different membrane potentials, and in the presence of different Na⁺ and α-methyl-d-glucose (αMG) concentrations. The simplest kinetic model that could accurately reproduce the time course of the measured currents (down to the 2 ms time range) is a seven-state model (C₁ to C₇) where the binding of the two Na⁺ ions (C₄→C₅) is highly cooperative. In the forward direction (Na⁺/glucose influx), the model is characterized by two slow, electroneutral conformational changes (59 and 100 s⁻¹) which represent reorientation of the free and of the fully loaded carrier between inside-facing and outside-facing conformations. From the inward-facing (C₁) to the outward-facing Na-bound configuration (C₅), 1.3 negative elementary charges are moved outward. Although extracellular glucose binding (C₅→C₆) is electroneutral, the next step (C₆→C₇) carries 0.7 positive charges inside the cell. Alignment of the seven-state model with a generalized model suggested by the structural data of the LeuT fold family suggests that electrogenic steps are associated with the movement of the so-called thin gates on each side of the substrate binding site. To our knowledge, this is the first model that can quantitatively describe the behavior of SGLT1 down to the 2 ms time domain. The model is highly symmetrical and in good agreement with the structural information obtained from the LeuT structural family.     

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (K_a ≈ 10⁵ M⁻¹). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.     

A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes

Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC₅₀ values of 5.8 (meta isomer) and 3.0 μM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K_I of 0.46 μM. Radiolabeled forms of both analogues selectively labeled the β-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.     

Oxygen ligand exchange at metal sites – implications for the O2 evolving mechanism of photosystem II

The mechanism for photosynthetic O₂ evolution by photosystem II is currently a topic of intense debate. Important questions remain as to what is the nature of the binding sites for the substrate water and how does the O–O bond form. Recent measurements of the ¹⁸O exchange between the solvent water and the photogenerated O₂ as a function of the S-state cycle have provided some surprising insights to these questions (W. Hillier, T. Wydrzynski, Biochemistry 39 (2000) 4399–4405). The results show that one substrate water molecule is bound at the beginning of the catalytic sequence, in the S₀ state, while the second substrate water molecule binds in the S₃ state or possibly earlier. It may be that the second substrate water molecule only enters the catalytic sequence following the formation of the S₃ state. Most importantly, comparison of the observed exchange rates with oxygen ligand exchange in various metal complexes reveal that the two substrate water molecules are most likely bound to separate Mn^III ions, which do not undergo metal-centered oxidations through to the S₃ state. The implication of this analysis is that in the S₁ state, all four Mn ions are in the +3 oxidation state. This minireview summarizes the arguments for this proposal.     

DNA binding and bending by dinuclear complexes comprising ruthenium polypyridyl centres linked by a bis(pyridylimine) ligand

The interaction of enantiomerically pure dinuclear complexes of the form [Ru₂(L-L)₄L¹]⁴⁺ (where L-L = 2,2^′-bipyridine (bpy) or 1,10-phenanthroline (phen) and L¹ = bis(pyridylimine) ligand ((C₅H₄N)CN(C₆H₄))₂CH₂)) with ct-DNA have been investigated by absorbance, circular dichroism, fluorescence displacement assays, thermal analysis, linear dichroism and gel electrophoresis. The complexes all bind more strongly to DNA than ethidium bromide, stabilise DNA and have a significant bending effect on DNA. The data for Δ,Δ-[Ru₂(bpy)₄L¹]⁴⁺ are consistent with it binding to DNA outside the grooves wrapping the DNA about it. By way of contrast the other complexes are groove-binders. The phen complexes provide a chemically and enantiomerically stable alternative to the DNA-coiling di-iron triple-helical cylinder previously studied. In contrast to the di-iron helicates, the phen complexes show DNA sequence effects with Δ,Δ-[Ru₂(phen)₄L¹]⁴⁺ binding preferentially to GC and Λ,Λ-[Ru₂(phen)₄L¹]⁴⁺ to AT.     

First structural evidence for the mode of diffusion of aromatic ligands and ligand-induced closure of the hydrophobic channel in heme peroxidases

The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole (amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules, including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H₂O₂ to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic channel as well as the binding of H₂O₂. This explains the amitrole toxicity. From binding studies, the dissociation constant (K _d) for amitrole with lactoperoxidase was found to be approximately 5.5 × 10⁻⁷ M, indicating high affinity.     

MHC Class I/peptide interactions: Binding specificity and kinetics

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (k_d) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured k_d values for antigenic peptide/class I interactions at 25°C are in the range of ca 10^?4–10^?6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.     

Reproduction rate,feeding process,and liebig limitations in cell populations—II

A biological system consisting of a population of cells suspended in a liquid substrate is considered. The general problem addressed in the paper is the derivation of the kinetic pattern of population growth as a statistical effect of a very large number of elementary interactions between a single cell and the molecules of nutrient in substrate. Solution of the problem is obtained in the form of equation expressing the population growth ratec as a function of substrate concentration,C _s. The analytical expression derived is applied to a real bacterial population (Escherichi coli) and kinetic patterns are theoretically computed. The major findings, expressed roughly, without nuances, are: (i) the concentration of nutrient at the cell membrane,C _c, can only be equal to either 0 (for theC _s below some threshold valueC ^*) orC _s (forC _s>C ^*); (ii) the Michaelis-Menten-Monod kinetics observed in experiments is an artifact: the pure (not contaminated by foreign factors) dependence ofc onC _s is actually such that the functionc=c(C _s) has practically linear increase whenC _s<C ^*, and is constant,c=c(C ^*)=const, whenC _s>C ^*; (iii) the Liebig principle is strictly fulfilled: up to a feasible accuracy of observation, under no circumstances can population growth be limited (controlled) by more than one substrate component—replacement of a limiting component for another one is an instant event rather than a gradual process.     

Acetylene hydratase: a non-redox enzyme with tungsten and iron–sulfur centers at the active site

In living systems, tungsten is exclusively found in microbial enzymes coordinated by the pyranopterin cofactor, with additional metal coordination provided by oxygen and/or sulfur, and/or selenium atoms in diverse arrangements. Prominent examples are formate dehydrogenase, formylmethanofuran dehydrogenase, and aldehyde oxidoreductase all of which catalyze redox reactions. The bacterial enzyme acetylene hydratase (AH) stands out of its class as it catalyzes the conversion of acetylene to acetaldehyde, clearly a non-redox reaction and a reaction distinct from the reduction of acetylene to ethylene by nitrogenase. AH harbors two pyranopterins bound to W, and a [4Fe–4S] cluster. W is coordinated by four dithiolene sulfur atoms, one cysteine sulfur, and one oxygen ligand. AH activity requires a strong reductant suggesting W(IV) as the active oxidation state. Two different types of reaction pathways have been proposed. The 1.26 Å structure reveals a water molecule coordinated to W which could gain a partially positive net charge by the adjacent protonated Asp-13, enabling a direct attack of C₂H₂. To access the W–Asp site, a substrate channel was evolved distant from where it is found in other members of the DMSOR family. Computational studies of this second shell mechanism led to unrealistically high energy barriers, and alternative pathways were proposed where C₂H₂ binds directly to W. The architecture of the catalytic cavity, the specificity for C₂H₂ and the results from site-directed mutagenesis do not support this first shell mechanism. More investigations including structural information on the binding of C₂H₂ are needed to present a conclusive answer.     

Effect of Noradrenergic Lesions on Subtypes of α2-Adrenoceptors in Rat Brain

Abstract: The binding of [³H]rauwolscine to α_2A- (also referred to as α_2D-) and α_2C-adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for α_2A-adrenoceptors. Inhibition of [³H]rauwolscine binding by oxymetazoline was modeled best assuming binding to two sites (p < 0.001). Competition curves for oxymetazoline were shifted rightward by the addition of GTP (250 µM) but were still fit best by a two-site model (p < 0.001). A concentration of oxymetazoline was calculated that would optimally antagonize [³H]rauwolscine binding (with GTP present) to oxymetazoline-sensitive α_2A-adrenoceptors, minimally inhibiting binding to α_2C-adrenoceptors. Subsequently, [³H]rauwolscine binding to α_2A- and α_2C-adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to α_2C-adrenoceptors was increased significantly after treatment with 6-hydroxydopamine (6-OHDA) compared with vehicle-treated controls, whereas binding to α_2A-adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6-hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either α_2A- or α_2C-adrenoceptors. Thus, α_2C-adrenoceptors are regulated by changes in synaptic availability of norepinephrine. α_2A-Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post- and presynaptically so that up-regulation of postsynaptic α_2A-adrenoceptors is offset by a loss of presynaptic α_2A-adrenoceptors.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

Cation binding to beta-casein. A comparison of electrostatic models

The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I, as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [D]. Anticipated intrinsic binding constants are k_H,P^o = 3 × 10⁶, k_Ca,P^o = 120, k_Na,P^o = 1, k_H,C^o = 7 × 10⁴ and k_Ca,C^o = 5.6Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring k_H,P^o and k_Ca,C^o which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed.Discrete charge models accept anticipated k^o and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.     

Cooperative binding ensures the obligatory melibiose/Na+ cotransport in MelB

MelB catalyzes the obligatory cotransport of melibiose with Na⁺, Li⁺, or H⁺. Crystal structure determination of the Salmonella typhimurium MelB (MelB_St) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na⁺ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔC_p), and regulatory phosphotransferase EIIA^Glc binding with isothermal titration calorimetry (ITC). We found that MelB_St thermostability is increased by either substrate (Na⁺ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔC_p, suggesting MelB_St hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelB_St dehydration and cavity closure. Lastly, we observed that EIIA^Glc, which has been suggested to trap MelB_St at an outward-open state, readily binds to the MelB_St apo state at an affinity similar to MelB_St/Na⁺. However, it has a suboptimal binding to the ternary state, implying that MelB_St in the ternary complex may be conformationally distant from the EIIA^Glc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na⁺ or melibiose) favors MelB_St at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.     

Structural and biochemical characterization of a novel aldehyde dehydrogenase encoded by the benzoate oxidation pathway in Burkholderia xenovorans LB400

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH_C) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD ⁺ and NADP ⁺). Our data definitively show that ALDH_C is more active in the presence of NADP ⁺ and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDH_C in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP ⁺ and a threonine/lysine pair on ALDH_C. The catalytic preference of ALDH_C for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH_C induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH_C in benzoate degradation.