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PubMed1970

Cooperative effects in models of steady-state transport across membranes. II. Oscillating phase transition.

A simple model for steady-state transport of a neutral molecule across a membrane is investigated in a preliminary way. In this model, there is a possible conformation change in each membrane unit which alters the access of the binding site for the transported molecule from one bath to the other. Thus, transport cannot be accomplished without a conformation change. Furthermore, we assume a cooperative interaction between nearest-neighbor membrane units in the same conformation. Then, with suitable rate constants and bath concentrations, and if the interaction energy is large enough, the membrane will oscillate back and forth between the two conformational phases, producing a surge of flux in each cycle. The period of the cycle depends on the times necessary to nucleate the two phase transitions.

Biological Transport↗
PubMed1993

Atomic resolution (0.83 A) crystal structure of the hydrophobic protein crambin at 130 K.

To enhance the already high quality of diffraction data for crystals of the hydrophobic protein crambin, X-ray data were collected at 130 K by the method of H. Hope to 0.83 A resolution. Refinement with PROLSQ yields a model with an R value of 10.5%. The final model had three parameter anisotropic vibration factors for all atoms, which included 367 protein heavy atoms, 372 hydrogen atoms and 144 solvent atoms with one ethanol molecule. Dihedral angles and hydrogen-bonding distances generally agree with earlier studies of high-resolution protein structures, but some new patterns are noted. Solvent-related helix distortions are reminiscent of those described by others. Helix and beta-sheet regions show distinct patterns in their side-chain conformations. Despite crambin's hydrophobic nature, its accessible surface area in the crystal is surprisingly close to that of water-soluble proteins like myoglobin and carboxypeptidase A. More of crambin's hydrophobic surface is buried in the crystal, perhaps accounting for its high order of diffraction. A total of 24% of the 46 residues show discrete disorder at 130 K. This includes five side-chains at both 300 and 130 K, and six more side-chains and an ethanol molecule at 130 K. Disorder is associated with the sequence microheterogeneity at Pro/Ser22 and Leu/Ile25, with space filling or with solvent disorder. Correlated conformations extend over three to five residues. The patterns of disorder in this structure reveal important principles of protein structure and its dynamics. Finding disordered groups correlated over 5 to 8 A suggests that co-ordinated motion extends in groups rather than simply as uncorrelated movement around an atom center. Thermal diffuse scattering experiments on insulin and lysozyme are consistent with this interpretation. Nearly all of the protein-bound solvent has been located. Less than 1% of protein accessible surface area remains uncovered by solvent or crystal contacts. Preliminary analysis of the solvent network reveals two main networks in each of four solvent regions.

Crystallography↗
PubMed1997

Effect of chemical glycosylation of RNase A on the protein stability and surface histidines accessibility in immobilized metal ion affinity electrophoresis (IMAGE) system.

Immobilized metal ion affinity gel electrophoresis (IMAGE) has been applied to study the change of the surface histidines topography of RNase A when chemically glycosylated on exposed carboxylic groups with glucosamine using carbodiimide as cross-linker, under mild conditions. Two populations of glycosylated RNase A, one with a single glucosamine and another with two glucosamine attached, were obtained. These chemically glycosylated RNase A conserved about 80% of native enzymatic activity and their pls were increased in comparison to native RNase A. The chemically glycosylated RNase A showed dramatic enhancement for thermal stability, while proteolytic resistance was similar to that of native RNase A. Chemically glycosylated RNase A showed a slightly increased affinity to IDA-Cu(II) as compared to the native enzyme, which indicates that a conformational change and/or a decrease in steric hindrance around accessible surface histidines (His 12 or His 119 and His 105) has occured. IMAGE is a useful method to analyse subtle conformational changes in proteins which result in subtle changes in histidine accessibilities.

Animals↗
PubMed1999

Agonist-induced changes in substituted cysteine accessibility reveal dynamic extracellular structure of M3-M4 loop of glutamate receptor GluR6.

Recent evidence suggests that the transmembrane topology of ionotropic glutamate receptors differs from other members of the ligand-gated ion channel superfamily. However, the structure of the segment linking membrane domains M3 and M4 (the M3-M4 loop) remains controversial. Although various data indicate that this loop is extracellular, other results suggest that serine residues in this segment are sites of phosphorylation and channel modulation by intracellular protein kinases. To reconcile these data, we hypothesized that the M3-M4 loop structure is dynamic and, more specifically, that the portion containing putative phosphorylation sites may be translocated across the membrane to the cytoplasmic side during agonist binding. To test this hypothesis, we mutated Ser 684, a putative cAMP-dependent protein kinase site in the kainate-type glutamate receptor GluR6, to Cys. Results of biochemical and electrophysiological experiments are consistent with Cys 684 being accessible, in the unliganded state, from the extracellular side to modification by a Cys-specific biotinylating reagent followed by streptavidin (SA). Interestingly, our data suggest that this residue becomes inaccessible to the extracellular biotinylating reagent during agonist binding. However, we find it unlikely that Cys 684 undergoes membrane translocation, because the addition of SA to Cys-biotinylated GluR6(S684C) has no effect on peak glutamate-evoked current and only a small effect on macroscopic desensitization. We conclude that residue 684 in GluR6 is extracellular in the receptor-channel's closed, unliganded state and does not cross the membrane after agonist binding. However, an agonist-induced conformational change in the receptor substantially alters accessibility of position 684 to the extracellular environment.

Biotinylation↗
PubMed2003

Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase.

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.

Binding Sites↗
PubMed1992

The disaccharide L-alpha-D-heptose1-->7-L-alpha-D-heptose1-->of the inner core domain of Salmonella lipopolysaccharide is accessible to antibody and is the epitope of a broadly reactive monoclonal antibody.

We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS.

Animals↗
PubMed1988

Conformational changes in the membrane-bound hydrogenase of Bradyrhizobium japonicum. Evidence that the redox state of the enzyme affects its accessibility to protease and membrane-impermeant reagents.

The sensitivity of the membrane-bound hydrogenase of Bradyrhizobium japonicum to inactivation by proteases and membrane-impermeant protein modification reagents was compared under hydrogen versus oxygen. In membrane vesicles, the half-life of enzyme inactivation by trypsin of the H2-reduced enzyme was approximately 10 min, whereas O2-oxidized enzyme was much less sensitive to trypsin inactivation (half-life of over 90 min). Diazobenzene sulfonate (DABS) affected the enzyme activity in a manner similar to proteases. With DABS, the enzyme had a half-life of 2-3 min under H2 versus over 30 min under O2. Experiments in which the gas phase (containing either H2 or O2) available to the membranes was changed prior to the protease or chemical modification treatments indicated that it is the redox state of the enzyme at the time of the treatment which determines the sensitivity of the enzyme to inactivation. The redox-dependent differences in the behavior of the membrane-bound enzyme were attributed to changes in the accessibility of the small (33 kDa) subunit. The kinetics of enzyme inactivation by trypsin, under H2, correlated very well with the degradation of the intact 33-kDa subunit, whereas the large subunit (65 kDa) was rather resistant to proteolytic degradation. DABS treatment was found to decrease the reactivity of the small subunit to its antibody concomitant with enzyme inactivation under H2, but without such an effect on the O2-oxidized enzyme. In contrast to the results with the membrane-bound enzyme, purified dehydrogenase was found to be equally susceptible to inactivation by proteolysis or chemical modification irrespective of whether the treatments were performed under H2 or O2. These results indicate that, in the membrane, hydrogenase undergoes a redox-linked conformational change, whereby the small subunit of the enzyme becomes more accessible to external reagents when the enzyme is in its reduced form.

Cell Membrane↗
PubMed1995

Conformational analysis of endothelin-1: effects of solvation free energy.

In order to investigate conformational preferences of the 21-residue peptide hormone endothelin-1 (ET-1), an extensive conformational search was carried out in vacuo using a combination of high temperature molecular dynamics/annealing and a Monte Carlo/minimization search in torsion angle space. Fully minimized conformations from the search were grouped into families using a clustering technique based on rms fitting over the Cartesian coordinates of the atoms of the peptide backbone of the ring region. A wide range of local energy mining were identified even though two disulfide bridges (Cys1-Cys15 and Cys3-Cys11) constrain the structure of the peptide. Low energy conformers of ET-1 as a nonionized species in vacuo are stabilized by intramolecular interaction of the ring region (residues 1-15) with the tail (residues 16-21). Strained conformations for individual residues are observed. Conformational similarity to protein loops is established by matching to protein crystal structures. In order to assess the influence of aqueous environment on conformational preference, the electrostatic contribution to the solvation energy was calculated for ET-1 as a fully ionized species (Asp8, Lys9, Glu10, Asp18, N- and C-terminus) using a continuum electrostatics model (DelPhi) for each of the conformers generated in vacuo, and the total solvation free energy was estimated by adding a hydrophobic contribution proportional to solvent accessible surface area. Solvation dramatically alters the relative energetics of ET-1 conformers from that calculated in vacuo. Conformers of ET-1 favored by the electrostatic solvation energy in water include conformers with helical secondary structure in the region of residues 9-15. Perhaps of most importance, it was demonstrated that the contribution to solvation by an individual charge depends not only on its solvent accessibility but on the proximity of other charges, i.e., it is a cooperative effect. This was shown by the calculation of electrostatic solvation energy as a function of conformation with individual charges systematically turned "on" and "off". The cooperative effect of multiple charges on solvation demonstrated in this manner calls into question models that relate solvation energy simply to solvent accessibility by atom or residue alone.

Amino Acid Sequence↗
PubMed2003

Crystal structure of S-ovalbumin as a non-loop-inserted thermostabilized serpin form.

Ovalbumin, a non-inhibitory member of serine proteinase inhibitors (serpin), is transformed into a heat-stabilized form, S-ovalbumin, under elevated pH conditions. The structural mechanism for the S-ovalbumin formation has long been a puzzling question in food science and serpin structural biology. On the basis of the commonly observed serpin thermostabilization by insertion of the reactive center loop into the proximal beta-sheet, the most widely accepted hypothetical model has included partial loop insertion. Here we demonstrate, for the first time, the crystal structure of S-ovalbumin at 1.9-A resolution. This structure unequivocally excludes the partial loop insertion mechanism; the overall structure, including the reactive center loop structure, is almost the same as that of native ovalbumin, except for the significant motion of the preceding loop of strand 1A away from strand 2A. The most striking finding is that Ser-164, Ser-236, and Ser-320 take the d-amino acid residue configuration. These chemical inversions can be directly related to the irreversible and stepwise nature of the transformation from native ovalbumin to S-ovalbumin. As conformational changes of the side chains, significant alternations are found in the values of the chi 1 of Phe-99 and the chi 3 of Met-241. The former conformational change leads to the decreased solvent accessibility of the hydrophobic core around Phe-99, which includes Phe-180 and Phe-378, the highly conserved residues in serpin. This may give a thermodynamic advantage to the structural stability of S-ovalbumin.

Amino Acid Sequence↗
PubMed2006

Transition from rolling to firm adhesion can be mimicked by extension of integrin alphaLbeta2 in an intermediate affinity state.

AlphaLbeta2 affinity for intercellular adhesion molecule-1 (ICAM-1) is regulated by the conformation of the alphaL I domain, which is in turn controlled by the conformation and orientation of other adjacent domains. Additionally, overall integrin conformation (bent versus straightened) influences the orientation of the I domain and access to its ligands, influencing adhesive efficiency. The open or high affinity I domain conformation supports strong adhesion, whereas the closed, low affinity conformation mediates weak interactions or rolling. We have previously suggested that alphaLbeta2 can also exist on the cell surface in an intermediate affinity state. Here we have studied the adhesive properties of integrin alphaLbeta2 containing mutant I domains with intermediate affinities for ICAM-1. In an overall bent conformation, the intermediate affinity state of alphaLbeta2 is hardly detected by conventional adhesion assays, but robust adhesion is seen when an extended conformation is induced by a small molecule alpha/beta I allosteric antagonist. Intermediate affinity alphaLbeta2 supports more stable rolling than wild-type alphaLbeta2 under shear conditions. Moreover, antagonist-induced extension transforms rolling adhesion into firm adhesion in a manner reminiscent of chemokine activation of integrin alphaLbeta2. These findings suggest the relevance of intermediate affinity states of alphaLbeta2 to the transition between inactive and active states and demonstrate the importance of both I domain affinity and overall integrin conformation for cell adhesion.

Allosteric Site↗
PubMed1998

A novel epitope on the C-terminus of SmD1 is recognized by the majority of sera from patients with systemic lupus erythematosus.

The SmD1 protein is a specific target for the autoantibody response in SLE. To further analyze this reactivity epitope, mapping was performed with cellulose-bound 13-mer peptides overlapping 10 amino acids (aa). In this initial approach, 4 out of 15 SLE sera recognized more than five overlapping peptides of the SmD1 C-terminus. Therefore, longer oligopeptides of up to 37 aa of this region were generated and probed for as antigens by ELISA. For the SmD1 aa 83-119 polypeptide, there was a striking increase of reactivity with 70.0% positive reactions out of 167 SLE sera. In contrast, 105 healthy control sera were negative, and only 8.3% of sera from patients with other inflammatory diseases (n = 267) exhibited a response, which was of low level only. The anti-SmD183-119 reactivity was significantly higher in anti-dsDNA antibody positive vs. negative sera (P < 0.001) and correlated with disease activity. Four of five human monoclonal anti-dsDNA antibodies also reacted with SmD183-119. The specificity for SmD1 was demonstrated by inhibition experiments and immunization of rabbits with SmD183-119 inducing SmD1-specific antibodies. In conclusion, the SmD183-119 peptide was demonstrated to be an important and highly specific target of the autoimmune response in SLE. The high sensitivity of this ELISA probably depends on a conformational epitope, which appears not to be accessible in the full-size SmD1 protein.

Amino Acid Sequence↗
PubMed2001

The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain.

Binding of the extracellular matrix molecule fibronectin to the integrin receptor alpha(5)beta(1) elicits downstream signaling pathways that modulate cell function. Fibronectin-alpha(5)beta(1) interaction occurs via the conserved RGD sequence in the tenth FIII (FIII10) domain of fibronectin. A synergistic site containing the sequence PHSRN in the adjacent FIII9 domain has also been identified. Here we investigate the function of the eighth FIII domain in integrin-mediated cell adhesion using a wide range of methods, including biochemical, biological, and biophysical assays of integrin binding, cell adhesion, and protein denaturation. Mutation of the FIII9 synergistic site (PHSRN to PHAAA) in FIII9-10 reduced the binding activity for integrin alpha(5)beta(1) to levels observed for FIII10 alone, but the corresponding mutant in FIII8-9-10 showed no loss of binding activity. Cell adhesion assays also demonstrated enhanced functional activity of constructs containing FIII8. Equilibrium chemical denaturation studies indicated that FIII8 confers conformational stability upon FIII9, but only if the exposed loops, PHSRN and VKNEED on FIII9 and FIII8, respectively, are intact. These results demonstrate that the loss of integrin binding activity, observed upon alteration of the PHSRN synergistic site of FIII9-10, results partly from a loss of conformational stability of FIII9. Our data suggest a mechanism for integrin alpha(5)beta(1)-fibronectin interaction, which in addition to the primary RGD binding event, involves a conformation-sensitive scanning by the integrin for accessible sites on the ligand, whereupon full activation of downstream signaling occurs.

Binding Sites↗
PubMed1999

Nucleotide and Mg2+ induced conformational changes in GroEL can be detected by sulfhydryl labeling.

The accessibility of fluorescein-5-maleimide to sulfhydryl groups in the molecular chaperone GroEL was used to follow structural rearrangements in the protein triggered by binding Mg2+ and/or adenine nucleotides. Three peptides, each containing one of the cysteines of GroEL (C138, C458 and C519) were identified. GroEL labeled in 50 mM TrisHCl, pH 7.8, incorporated approximately 0.3 labels each on C138 and C458. With 10 mM MgCl2, the labeling increased to approximately 0.8 labels each on C138 and C458. The increase was partially due to a conformational change which occurred upon Mg2+ binding as well as to an increase in ionic strength. When ADP, ATP, or AMP-PNP were added to a solution of GroEL and Mg2+, C138 incorporated approximately 0.8 labels, while C458 incorporated approximately 0.1 labels. These results suggest that the binding of adenine nucleotides changed the conformation of GroEL and made a previously highly exposed sulfhydryl group inaccessible. GroEL slowly dissociated into monomers when it was extensively labeled at C458. GroEL labeled with fluorescein-5-maleimide, under any of the conditions examined, was able to bind but not release active rhodanese. The observed variations in sulfhydryl accessibility are consistent with mechanisms that suggest binding of GroES to GroEL differs from the binding of substrate protein to GroEL, and that the binding of Mg2+ or Mg-adenine nucleotides results in conformational changes in GroEL.

Adenine↗
PubMed2004

mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure.

Approximately 3 000 genes are regulated in a time-, tissue-, and stimulus-dependent manner by degradation or stabilization of their mRNAs. The process is mediated by interaction of AU-rich elements (AREs) in the mRNA's 3'-untranslated regions with trans-acting factors. AU-rich element-controlled genes of fundamentally different functional relevance depend for their activation on one positive regulator, HuR. Here we present a methodology to exploit this central regulatory process for specific manipulation of AU-rich element-controlled gene expression at the mRNA level. With a combination of single-molecule spectroscopy, computational biology, and molecular and cellular biochemistry, we show that mRNA recognition by HuR is dependent on the presentation of the sequence motif NNUUNNUUU in single-stranded conformation. The presentation of the HuR binding site in the mRNA secondary structure appears to act analogously to a regulatory on/off switch that specifically controls HuR access to mRNAs in cis. Based on this finding we present a methodology for manipulating ARE mRNA levels by actuating this conformational switch specifically in a target mRNA. Computationally designed oligonucleotides (openers) enhance the NNUUNNUUU accessibility by rearranging the mRNA conformation. Thereby they increase in vitro and endogenous HuR-mRNA complex formation which leads to specific mRNA stabilization (as demonstrated for TNFalpha and IL-2, respectively). Induced HuR binding both inside and outside the AU-rich element promotes functional IL-2 mRNA stabilization. This opener-induced mRNA stabilization mimics the endogenous IL-2 response to CD28 stimulation in human primary T-cells. We therefore propose that controlled modulation of the AU-rich element conformation by mRNA openers or closers allows message stabilization or destabilization in cis to be specifically triggered. The described methodology might provide a means for studying distinct pathways in a complex cellular network at the node of mRNA stability control. It allows ARE gene expression to be potentially silenced or boosted. This will be of particular value for drug-target validation, allowing the diseased phenotype to ameliorate or deteriorate. Finally, the mRNA openers provide a rational starting point for target-specific mRNA stability assays to screen for low-molecular-weight compounds acting as inhibitors or activators of an mRNA structure rearrangement.

Antigens, Surface↗
PubMed1998

The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis.

BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols. RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction. CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.

Amino Acid Sequence↗
PubMed1998

Lidocaine accessibility to the open state of brain Na+ channels increases during development.

During postnatal brain development, the function of Na+ channels undergoes changes. We investigated at the single channel level whether the lidocaine sensitivity of the open state of brain Na+ channels changed during development and the underlying kinetic differences of the lidocaine-induced open channel block between brain and muscle Na+ channels. The lidocaine affinity for the open channel state was found to be about 30% higher in 15 day old than in newborn channels and reflected a higher binding rate constant in 15 day old channels. When compared with reported values from adult muscle Na+ channels, lidocaine blocked the open state of brain channels with about ten times higher potency and reflected a lower unbinding rate constant in brain channels. These results indicate that the conformations of the channel structures defining the lidocaine accessibility to its binding site must undergo changes during brain development and that the conformations of the channel structures interacting with lidocaine must be different in brain and muscle channels.

Animals↗
PubMed1993

1.8 A refined structure of the lipase from Geotrichum candidum.

A lipase from the fungus Geotrichum candidum is one of only three interfacially activated lipases whose structures have been reported to date. We have previously reported the partially refined 2.2 A structure of this enzyme. We have subsequently extended the resolution and here report the fully refined 1.8 A structure of this lipase. The structure observed in the crystal is apparently not the lipolytic conformation, as the active site is not accessible from the surface of the molecule. A single large cavity is found in the interior of the molecule and extends from the catalytic Ser to two surface helices, suggesting that this face may be the region that interacts with the lipid interface. The mobility of local segments on this face is indicated by temperature factors larger than elsewhere in the molecule and by the observation of several residues whose side-chains are discretely disordered. These observations strongly suggest that this portion of the molecule is involved in interfacial and substrate binding, but the exact nature of the conformational changes induced by binding to the lipid interface can not be determined.

Amino Acid Sequence↗
PubMed1988

Mechanism of inhibition of human renin by monoclonal antibodies.

The mechanism by which monoclonal antibodies directed against human renin (R3-36-16 and R3-47-10) inhibit renin activity was investigated using various substrates. Both antibodies acted as potent inhibitors of human renin activity when human angiotensinogen was used as a substrate. However, their effects differed clearly in the presence of synthetic tetradecapeptide. When low concentrations of tetradecapeptide were used as substrate, renin activity was only partially inhibited by R3-47-10, whereas it was stimulated by R3-36-16. At higher synthetic substrate concentrations, both antibodies stimulated angiotensin I production. This effect was independent of the pH. Both antibodies exerted their effects in the presence of CGP 29287, a peptidic transition-state competitive renin inhibitor, indicating that their binding sites differed from that of CGP 29287. In combination, the stimulatory effect of R3-36-16 was not blocked by R3-47-10, but the inhibition produced by R3-47-10 was reversed by R3-36-16. Both antibodies may prevent the large natural substrate angiotensinogen from entering the enzymatic cleft by steric hindrance. At a low substrate concentration, R3-47-10 may also partially hinder the access of synthetic tetradecapeptide into the active cleft by steric hindrance. In contrast, the stimulating effect of both antibodies may be due to a conformational change in the renin molecule, allowing an increased access of tetradecapeptide or a more rapid release of the product from the enzymatic cleft.

Angiotensinogen↗