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See detailEnergetics of protein stability at extreme environmental temperatures in bacterial trigger factors
Struvay, Caroline ULg; Negro, Sonia; Matagne, André ULg et al

in Biochemistry (2013), 52

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See detailInhibition of dd-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 dd-Peptidase.
Dzhekieva, Liudmila; Adediran, S. A.; Herman, Raphael et al

in Biochemistry (2013)

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state ... [more ▼]

Inhibitors of bacterial dd-peptidases represent potential antibiotics. In the search for alternatives to beta-lactams, we have investigated a series of compounds designed to generate transition state analogue structures upon reaction with dd-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter includes a boronic acid, two alcohols, an aldehyde, and a trifluoroketone. The compounds were tested against two low-molecular mass class C dd-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but rather unexpectedly from precedent, the trifluoroketone [d-alpha-aminopimelyl(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, we found the trifluoroketone was the strongest inhibitor of the Actinomadura R39 dd-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates as a warhead that can be incorporated into new and effective dd-peptidase inhibitors and therefore, perhaps, antibiotics. [less ▲]

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See detailFree Energy Rhythms in Saccharomyces cerevisiae: A Dynamic Perspective with Implications for Ribosomal Biogenesis
Gross, A.; Li, Caroline M.; Remacle, Françoise ULg et al

in Biochemistry (2013), 52(9), 1641-1648

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See detailSolution Nuclear Magnetic Resonance Structure and Molecular Dynamics Simulations of a Murine 18.5 kDa Myelin Basic Protein Segment (S72-S107) in Association with Dodecylphosphocholine Micelles.
Ahmed, Mumdooh A. M.; De Avila, Miguel; Polverini, Eugenia et al

in Biochemistry (2012), 51(38), 7475-87

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of ... [more ▼]

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic alpha-helix followed by a proline-rich segment that represents a ligand for SH3 domain-containing proteins. Here, we have determined using solution nuclear magnetic resonance spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the alpha2-peptide) comprising these two structural motifs, in association with dodecylphosphocholine (DPC) micelles. The structure was calculated using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The experimental studies were complemented by molecular dynamics simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in association with a DPC micelle in silico. The experimental and theoretical results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane association via the amphipathic alpha-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-containing proteins such as Fyn and contribute to our understanding of myelin architecture at the molecular level. [less ▲]

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See detailSubstrate Specificity of Low-Molecular Mass Bacterial DD-Peptidases
Nemmara, Venkatesh V; Dzhekieva, Liudmila; Subarno Sakar, Kumar et al

in Biochemistry (2011), 50

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high ... [more ▼]

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and lowmolecular mass (LMM) enzymes. The latter group, which is subdivided into classes A−C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 DD-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown. [less ▲]

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See detailKinetics of Reactions of the Actinomadura R39 dd-Peptidase with Specific Substrates.
Adediran, S. A.; Kumar, Ish; Nagarajan et al

in Biochemistry (2011), 50(3), 376-387

The Actinomadura R39 dd-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are ... [more ▼]

The Actinomadura R39 dd-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(d-cysteinyl)propanoyl-d-alanyl-d-alanine and 3-(d-cysteinyl)propanoyl-d-alanyl-d-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed dd-carboxypeptidase, dd-transpeptidase, and dd-endopeptidase activities. These results confirm the specificity of the enzyme for a free d-amino acid at the N-terminus of good substrates and indicated a preference for extended d-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a dd-endopeptidase in vivo. pH−rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases. [less ▲]

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See detailRapid Collapse into a Molten Globule Is Followed by Simple Two-State Kinetics in the Folding of Lysozyme from Bacteriophage lambda
Di Paolo, Alexandre ULg; Balbeur, D.; De Pauw, Edwin ULg et al

in Biochemistry (2010), 49

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass ... [more ▼]

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage lambda (lambda lysozyme) at pH 5.6, 20 degrees C. The first step in the folding of lambda lysozyme occurs very rapidly (tau < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure but only weak protection from (1)H/(2)H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with tau = 0.15 +/- 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (tau = 5 +/- 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that lambda lysozyme is a highly dynamic protein, with relatively low conformational stability (DeltaG degrees (N-U) = 25 +/- 2 kJ.mol(-1)). [less ▲]

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See detailCrystal structure of a complex between the Actinomadura R39 DD-peptidase and a peptidoglycan-mimetic boronate inhibitor: interpretation of a transition state analogue in terms of catalytic mechanism.
Dzhekieva, Liudmila; Rocaboy, Mathieu ULg; Kerff, Frédéric ULg et al

in Biochemistry (2010), 49(30), 6411-9

The Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small D-alanyl-D-alanine ... [more ▼]

The Actinomadura R39 DD-peptidase is a bacterial low molecular weight class C penicillin-binding protein. It has previously been shown to catalyze hydrolysis and aminolysis of small D-alanyl-D-alanine terminating peptides, especially those with a side chain that mimics the amino terminus of the stem peptide precursor to the bacterial cell wall. This paper describes the synthesis of (D-alpha-aminopimelylamino)-D-1-ethylboronic acid, designed to be a peptidoglycan-mimetic transition state analogue inhibitor of the R39 DD-peptidase. The boronate was found to be a potent inhibitor of the peptidase with a K(i) value of 32 +/- 6 nM. Since it binds some 30 times more strongly than the analogous peptide substrate, the boronate may well be a transition state analogue. A crystal structure of the inhibitory complex shows the boronate covalently bound to the nucleophilic active site Ser 49. The aminopimelyl side chain is bound into the site previously identified as specific for this moiety. One boronate oxygen is held in the oxyanion hole; the other, occupying the leaving group site of acylation or the nucleophile site of deacylation, appears to be hydrogen-bonded to the hydroxyl group of Ser 298. The Ser 49 oxygen appears to be hydrogen bonded to Lys 52. If it is assumed that this structure does resemble a high-energy tetrahedral intermediate in catalysis, it seems likely that Ser 298 participates as part of a proton transfer chain initiated by Lys 52 or Lys 410 as the primary proton donor/acceptor. The structure, therefore, supports a particular class of mechanism that employs this proton transfer device. [less ▲]

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See detailInsight into the cellular uptake mechanism of a secondary amphipathic cell penetrating peptide for siRNA delivery.
Konate, K.; Crombez, L.; Deshayes, S. et al

in Biochemistry (2010)

Delivery of siRNA remains a major limitation to their clinical application and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle ... [more ▼]

Delivery of siRNA remains a major limitation to their clinical application and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modelling, spectroscopy and membrane interaction approaches, in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an -helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids. [less ▲]

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See detailFolding of class A beta-lactamases is rate-limited by peptide bond isomerization and occurs via parallel pathways.
Vandenameele, Julie ULg; Lejeune, Annabelle ULg; Di Paolo, Alexandre ULg et al

in Biochemistry (2010), 49(19), 4264-75

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues ... [more ▼]

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding. [less ▲]

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See detailSecondary structure and solvent accessibility of a calmodulin-binding C-terminal segment of membrane-associated myelin basic protein.
Homchaudhuri, Lopamudra; De Avila, Miguel; Nilsson, Stina B. et al

in Biochemistry (2010), 49(41), 8955-66

Myelin basic protein (MBP), specifically the 18.5 kDa isoform, is a peripheral membrane protein and a major component of mammalian central nervous system myelin. It is an intrinsically disordered and ... [more ▼]

Myelin basic protein (MBP), specifically the 18.5 kDa isoform, is a peripheral membrane protein and a major component of mammalian central nervous system myelin. It is an intrinsically disordered and multifunctional protein that binds cytoskeletal and other cytosolic proteins to a membrane surface and thereby acquires ordered structure. These associations are modulated by post-translational modifications of MBP, as well as by interactions of MBP with Ca(2+)-calmodulin (CaM). Enzymatic deimination of usually six arginine residues to citrulline results in a decrease in the net positive charge of the protein from 19 to </=13. This deiminated form is found in greater amounts in normal children and in adult patients with the demyelinating disease multiple sclerosis. In this paper, we examine the secondary structure of a calmodulin-binding domain, residues A141-L154, when associated with a lipid bilayer in recombinant murine 18.5 kDa forms rmC1 (unmodified) and rmC8 (pseudodeiminated). We demonstrate here by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy that the Y142-L154 segment in membrane-associated rmC1 forms an amphipathic alpha-helix, with high accessibility to O(2) and low accessibility to NiEDDA. In membrane-associated rmC8, this segment assumed a structure distorted from an alpha-helix. Spin-labeled residues in rmC1 in solution were more immobilized on binding Ca(2+)-CaM than those in rmC8. Furthermore, rmC8 was dissociated more readily from a lipid bilayer by Ca(2+)-CaM than was rmC1. These results confirm both a predicted induced ordering upon membrane association in a specific segment of 18.5 kDa MBP, and that this segment is a CaM-binding site, with both interactions weakened by deimination of residues outside of this segment. The deiminated form would be more susceptible to regulation of its membrane binding functions by Ca(2+)-CaM than the unmodified form. [less ▲]

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See detailCritical role of tryptophan 154 for the activity and stability of class D beta-lactamases.
Baurin, Stephane; Vercheval, Lionel ULg; Bouillenne, Fabrice ULg et al

in Biochemistry (2009), 48(47), 11252-63

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of ... [more ▼]

The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity. [less ▲]

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See detailTem-1 Beta-Lactamase Folds in a Nonhierarchical Manner with Transient Non-Native Interactions Involving the C-Terminal Region
Lejeune, Annabelle ULg; Pain, R. H.; Charlier, Paulette ULg et al

in Biochemistry (2008), 47(4), 1186-93

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and ... [more ▼]

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process. [less ▲]

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See detailLarge Changes In The Crac Segment Of Gp41 Of Hiv Do Not Destroy Fusion Activity If The Segment Interacts With Cholesterol
Vishwanathan, Sa.; Thomas, Annick ULg; Brasseur, Robert ULg et al

in Biochemistry (2008), 47(45),

The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears ... [more ▼]

The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G, or A substituting for W and I of the LWYIK sequence. The peptide having the greatest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild-type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, and did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol. [less ▲]

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See detailHydrophobic Substitutions In The First Residue Of The Crac Segment Of The Gp41 Protein Of Hiv
Vishwanathan, Sa.; Thomas, Annick ULg; Brasseur, Robert ULg et al

in Biochemistry (2008), 47(1), 124-30

We investigated the peptides N-acetyl-AWYIK-amide and N-acetyl-VWYIK-amide corresponding to single amino acid substitutions in LWYIK, a segment found in the gp41 protein of HIV and believed to play a role ... [more ▼]

We investigated the peptides N-acetyl-AWYIK-amide and N-acetyl-VWYIK-amide corresponding to single amino acid substitutions in LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. The effects of these peptides on the thermotropic phase transitions of 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and mixtures of SOPC and cholesterol were intermediate between that having the wild-type sequence (LWYIK) and another (IWYIK), the least active peptide previously studied. This correlated with results from studies of single mutations in the gp41 protein of HIV-1, in which L679 of the LWYIK segment is replaced with either A or V, measuring the capability of TZM-BL HeLa-based HIV-1 indicator cells to form syncytia. The peptides were also comparatively analyzed in silico. All together, the results suggest that the mode of interaction of this region of gp41 with the polar heads of membrane lipids contributes to its cholesterol selectivity and that this is somehow related to the biological activity of the viral glycoprotein. [less ▲]

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See detailEngineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils
Chan, Pak Ho; Pardon, Els; Menzer, Linda ULg et al

in Biochemistry (2008), 47

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic ... [more ▼]

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation. [less ▲]

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See detailUnusually strong binding to the DNA minor groove by a highly twisted benzimidazole diphenylether: induced fit and bound water.
Tanious, Farial A; Laine, William; Peixoto, Paul ULg et al

in Biochemistry (2007), 46(23), 6944-56

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the ... [more ▼]

RT29 is a dicationic diamidine derivative that does not obey the classical "rules" for shape and functional group placement that are expected to result in strong binding and specific recognition of the DNA minor groove. The compound contains a benzimidazole diphenyl ether core that is flanked by the amidine cations. The diphenyl ether is highly twisted and gives the entire compound too much curvature to fit well to the shape of the minor groove. DNase I footprinting, fluorescence intercalator displacement studies, and circular dichroism spectra, however, indicate that the compound is an AT specific minor groove binding agent. Even more surprisingly, quantitative biosensor-surface plasmon resonance and isothermal titration calorimetric results indicate that the compound binds with exceptional strength to certain AT sequences in DNA with a large negative enthalpy of binding. Crystallographic results for the DNA complex of RT29 compared to calculated results for the free compound show that the compound undergoes significant conformational changes to enhance its minor groove interactions. In addition, a water molecule is incorporated directly into the complex to complete the compound-DNA interface, and it forms an essential link between the compound and base pair edges at the floor of the minor groove. The calculated DeltaCp value for complex formation is substantially less than the experimentally observed value, which supports the idea of water being an intrinsic part of the complex with a major contribution to the DeltaCp value. Both the induced fit conformational changes of the compound and the bound water are essential for strong binding to DNA by RT29. [less ▲]

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See detailOligosaccharide binding in family 8 glycosidases: Crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product
De Vos, D.; Collins, T.; Nerinckx, W. et al

in Biochemistry (2006), 45(15), 4797-4807

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex ... [more ▼]

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 angstrom resolution) and product xylotriose (at 1.9 angstrom resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apoenzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position Occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking Studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum S-2(O) conformation. [less ▲]

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See detailGlycosyl transferase activity of the Escherichia coli penicillin-binding protein 1b: Specificity profile for the substrate
Fraipont, Claudine ULg; Sapunaric, Frédéric ULg; Zervosen, Astrid ULg et al

in Biochemistry (2006), 45(12), 4007-4013

The glycosyl transferase of the Escherichia coli bifunctional penicillin-binding protein (PBP) 1b catalyzes the assembly of lipid-transported N-acetylglucosaminyl-beta-1,4-N-acetylmuramoyl-L-Ala-gamma-D ... [more ▼]

The glycosyl transferase of the Escherichia coli bifunctional penicillin-binding protein (PBP) 1b catalyzes the assembly of lipid-transported N-acetylglucosaminyl-beta-1,4-N-acetylmuramoyl-L-Ala-gamma-D-Glu-meso-A(2)pm-D-Ala-D-Ala units (lipid II) into linear peptidoglycan chains. These units are linked, at C1 of N-acetylmuramic acid (MurNAc), to a C-55 undecaprenyl pyrophosphate. In an in vitro assay, lipid II functions both as a glycosyl donor and as a glycosyl acceptor substrate. Using substrate analogues, it is suggested that the specificity of the enzyme for the glycosyl donor substrate differs from that for the acceptor. The donor substrate requires the presence of both N-acetylglucosamine (GlcNAc) and MurNAc and a reactive group on C1 of the MurNAc and does not absolutely require the lipid chain which can be replaced by uridine. The enzyme appears to prefer an acceptor substrate containing a polyprenyl pyrophosphate on C1 of the MurNAc sugar. The problem of glycan chain elongation that presumably proceeds by the repetitive addition of disaccharide peptide units at their reducing end is discussed. [less ▲]

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See detailReactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?
Josephine, Helen R.; Charlier, Paulette ULg; Davies, Christopher et al

in Biochemistry (2006), 45(51), 15873-83

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of ... [more ▼]

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design. [less ▲]

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