bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of an in vivo HIV‐1 protease mutant in complex with saquinavir: Insights into the mechanisms of drug resistance
Authors: Hong Lin , Zhang Xuenjun C. , Hartsuck Jean A. , Tang Jordan 
Published: February 2009

AbstractSaquinavir is a widely used HIV‐1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV‐1 viral enzymes. Three HIV‐1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5‐, 3‐, and 419‐fold increase in Ki values, respectively, compared to the wild‐type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364‐12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV‐1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 Å resolution. This mutant complex is compared with that of the wild‐type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340‐3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main‐chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1′ substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild‐type enzyme and therefore may facilitate catalysis by the resistant mutant.
The PA domain: A protease‐associated domain
Authors: Mahon Piers , Bateman Alex 
Published: February 2009

AbstractWe have identified a similarity between the apical domain of the human transferrin receptor and several other protein families. This domain is found associated with two different families of peptidases. Therefore, we term it the PA domain for protease‐associated domain. The PA domain is found inserted within a loop of the peptidase domain of family M8/M33 zinc peptidases. The PA domain is also found in a vacuolar sorting receptor and a ring finger protein of unknown function that may be a cell surface receptor. The PA domain may mediate substrate determination of peptidases or form protein‐protein interactions.
New insight on β‐lactoglobulin binding sites by 1‐anilinonaphthalene‐8‐sulfonate fluorescence decay
Authors: Collini Maddalena , D'Alfonso Laura , Baldini Giancarlo 
Published: February 2009

AbstractThe fluorescence time decay parameters of the β‐lactoglobulin‐1‐anilinonaphthalene‐8‐sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a “surface” site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye–Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the “surface” site mainly responds to the presence of these local titrating charges while the “internal” site more closely follows the overall protein net charge.
Role of a solvent‐exposed aromatic cluster in the folding of Escherichia coli CspA
Authors: Rodriguez Hector M. , Vu Dung M. , Gregoret Lydia M. 
Published: February 2009

AbstractEscherichia coli CspA is a member of the cold shock protein family. All cold shock proteins studied to date fold rapidly by an apparent two‐state mechanism. CspA contains an unusual cluster of aromatic amino acids on its surface that is necessary for nucleic acid binding and also provides stability to CspA (Hillier et al., 1998). To elucidate the role this aromatic cluster plays in the determining the folding rate and pathway of CspA, we have studied the folding kinetics of mutants containing either leucine or serine substituted for Phe18, Phe20, and/or Phe31. The leucine substitutions are found to accelerate folding and the serine substitutions to decelerate folding. Because these residues exert effects on the free energy of the folding transition state, they may be necessary for nucleating folding. They are not responsible, however, for the very compact, native‐like transition state ensemble seen in the cold shock proteins, as the refolding rates of the mutants all show a similar, weak dependence of unfolding rate on denaturant concentration. Using mutant cycle analysis, we show that there is energetic coupling among the three residues between the unfolded and transition states, suggesting that the cooperative nature of these interactions helps to determine the unfolding rate. Overall, our results suggest that separate evolutionary pressures can act simultaneously on the same group of residues to maintain function, stability, and folding rate.
Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248
Authors: Derst Christian , Henseling Johannes , Röhm Klaus‐Heinrich 
Published: February 2009

AbstractThe use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of A sp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild‐type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol−1 for l‐glutamine compared to 4 kJ mol−1 for l‐aspartic β‐hydroxamate and 7 kJ mol−1 for l‐asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active‐site residues and catalytically relevant water molecules.
Dimer formation by a “monomeric” protein
Authors: Park Chiwook , Raines Ronald T. 
Published: February 2009

AbstractDimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S‐peptide and S‐protein) of the protein. The H12A and H119A variants have 105‐fold less ribonucleolytic activity than does the wild‐type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65°C results in a 103‐fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65°C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer–dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37°C. This value of Kd is only 20‐fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of “monomeric” proteins could be more prevalent than is usually appreciated.
For the record: A logical sequence search for S100B target proteins
Authors: Mcclintock Kimberly A. , Shaw Gary S. 
Published: February 2009

AbstractThe EF‐hand calcium‐binding protein S100B has been shown to interact in vitro in a calcium‐sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (α and β), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled‐coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of α and β tubulin found at the αβ tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.
Reversible denaturation of oligomeric human chaperonin 10: Denatured state depends on chemical denaturant
Authors: Guidry Jesse J. , Moczygemba Charmaine K. , Wittung‐Stafshede Pernilla , Steede N. Kalaya , Landry Samuel J. 
Published: February 2009

AbstractChaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far‐ultraviolet circular dichroism, and cross‐linking; all denaturation reactions were reversible. GuHCl‐induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea‐induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross‐linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy‐increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co‐chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.
Proline in α‐helical kink is required for folding kinetics but not for kinked structure, function, or stability of heat shock transcription factor
Authors: Hardy Jeanne A. , Nelson HILLARY C.M. 
Published: February 2009

AbstractThe DNA‐binding domain of the yeast heat shock transcription factor (HSF) contains a strictly conserved proline that is at the center of a kink. To define the role of this conserved proline‐centered kink, we replaced the proline with a number of other residues. These substitutions did not diminish the ability of the full‐length protein to support growth of yeast or to activate transcription, suggesting that the proline at the center of the kink is not conserved for function. The stability of the isolated mutant DNA‐binding domains was unaltered from the wild‐type, so the proline is not conserved to maintain the stability of the protein. The crystal structures of two of the mutant DNA‐binding domains revealed that the helices in the mutant proteins were still kinked after substitution of the proline, suggesting that the proline does not cause the α‐helical kink. So why are prolines conserved in this and the majority of other kinked α‐helices if not for structure, function, or stability? The mutant DNA‐binding domains are less soluble than wild‐type when overexpressed. In addition, the folding kinetics, as measured by stopped‐flow fluorescence, is faster for the mutant proteins. These two results support the premise that the presence of the proline is critical for the folding pathway of HSF's DNA‐binding domain. The finding may also be more general and explain why kinked helices maintain their prolines.
Homology modeling and molecular dynamics simulations of lymphotactin
Authors: Ma Buyong , Xiong Junjie , Lubkowski Jacek , Nussinov Ruth 
Published: February 2009

AbstractWe have modeled the structure of human lymphotactin (hLpnt), by homology modeling and molecular dynamics simulations. This chemokine is unique in having a single disulfide bond and a long C‐terminal tail. Because other structural classes of chemokines have two pairs of Cys residues, compared to one in Lpnt, and because it has been shown that both disulfide bonds are required for stability and function, the question arises how the Lpnt maintains its structural integrity. The initial structure of hLpnt was constructed by homology modeling. The first 63 residues in the monomer of hLpnt were modeled using the structure of the human CC chemokine, RANTES, whose sequence appeared most similar. The structure of the long C‐terminal tail, missing in RANTES, was taken from the human muscle fatty‐acid binding protein. In a Protein Data Bank search, this protein was found to contain a sequence that was most homologous to the long tail. Consequently, the modeled hLpnt C‐terminal tail consisted of both α‐helical and β‐motifs. The complete model of the hLpnt monomer consisted of two α‐helices located above the five‐stranded β‐sheet. Molecular dynamics simulations of the solvated initial model have indicated that the stability of the predicted fold is related to the geometry of Pro78. The five‐stranded β‐sheet appeared to be preserved only when Pro78 was modeled in the cis conformation. Simulations were also performed both for the C‐terminal truncated forms of the hLpnt that contained one or two (CC chemokine‐like) disulfide bonds, and for the chicken Lpnt (cLpnt). Our MD simulations indicated that the turn region (T30‐G34) in hLpnt is important for the interactions with the receptor, and that the long C‐terminal region stabilizes both the turn (T30‐G34) and the five‐stranded β‐sheet. The major conclusion from our theoretical studies is that the lack of one disulfide bond and the extension of the C‐terminus in hLptn are mutually complementary. It is very likely that removal of two Cys residues sufficiently destabilizes the structure of a chemokine molecule, particularly the core β‐sheet, to abolish its biological function. However, this situation is rectified by the long C‐terminal segment. The role of this long region is most likely to stabilize the first β‐turn region and α‐helix H1, explaining how this chemokine can function with a single disulfide bond.
The N‐terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two‐step binding by rapid‐kinetic studies of cystatin A labeled at the N‐terminus with a fluorescent reporter group
Authors: Estrada Sergio , Raub‐Segall Elke , Björk Ingemar , Olson Steven T. 
Published: February 2009

AbstractThe three‐dimensional structures of cystatins, and other evidence, suggest that the flexible N‐terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two‐step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N‐terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS‐ and AEDANS‐labeled cystatin A to papain resulted in ∼4‐fold and 1.2‐fold increases of probe fluorescence, respectively, reflecting the interaction of the N‐terminal region with the enzyme. Observed pseudo‐first‐order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS‐cystatin A increased linearly with the concentration of the latter. In contrast, pseudo‐first‐order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N‐terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two‐step manner. Such independent binding of the N‐terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin–proteinase reactions.
Phosphoryl transfer by a concerted reaction mechanism in UMP/CMP‐kinase
Authors: Hutter Michael C. , Helms Volkhard 
Published: February 2009

AbstractThe reaction mechanism of phosphoryl transfer catalyzed by UMP/CMP‐kinase from Dictyostelium discoideum was investigated by semiempirical AM1 molecular orbital computations of an active site model system derived from crystal structures that contain a transition state analog or a bisubstrate inhibitor. The computational results suggest that the nucleoside monophosphate must be protonated for the forward reaction while it is unprotonated in the presence of aluminium fluoride, a popular transition state analog for phosphoryl transfer reactions. Furthermore, a compactification of the active site model system during the reaction and for the corresponding complex containing AlF3 was observed. For the active site residues that are part of the LID domain, conformational flexibility during the reaction proved to be crucial. On the basis of the calculations, a concerted phosphoryl transfer mechanism is suggested that involves the synchronous shift of a proton from the monophosphate to the transferred PO3‐group. The proposed mechanism is thus analogous to the phosphoryl transfer mechanism in cAMP‐dependent protein kinase that phosphorylates the hydroxyl groups of serine residues.
Trp42 rotamers report reduced flexibility when the inhibitor acetyl‐pepstatin is bound to HIV‐1 protease
Authors: Ullrich Beáta , Laberge Monique , TÖlgyesi Ferenc , Fidy Judit , Szeltner Zoltán , Polgár LÁszlö 
Published: February 2009

AbstractThe Q7K/L33I/L63I HIV‐1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate‐analog inhibitor, acetyl‐pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with kq = 6.85 × 109 M−1s−1 and kq2 = 1.88 × 109 M−1s−1, that merge into one in the complex with kq = 1.78 × 109 M−1s−1.500 ps trajectory analysis of Trp χ1/χ2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between χ1/χ2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole‐quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV‐1 protease to ground state conformational heterogeneity.
Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry
Authors: Lehmann Wolf D. , Schlosser Andreas , Erben Gerhard , Pipkorn Rüdiger , Bossemeyer Dirk , Kinzel Volker 
Published: February 2009

AbstractIn view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI‐MS/MS) for the possibility of a site‐specific detection of this posttranslational modification. For this purpose, synthetic L‐Asp/L‐isoAsp containing oligopeptide pairs were investigated by ESI‐MS/MS and low‐energy collision‐induced dissociation (CID). Replacement of L‐Asp by L‐isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L‐Asp/L‐isoAsp)‐X bond and of the X‐(L‐Asp/L‐isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b‐ion formation on both its N‐and C‐terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L‐Asp or L‐isoAsp, these identify one or the other form. Characterization and relative quantification of L‐Asp/L‐isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG‐D/isoD‐AAAAK (deamidated peptide 1–7 of protein kinase A catalytic subunit) and VQ‐D/isoD‐GLR (deamidated peptide 41–46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.