bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

In 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.
The amino terminus of PKA catalytic subunit—A site for introduction of posttranslation heterogeneities by deamidation: D‐Asp2 and D‐isoAsp2 containing isozymes
Authors: Kinzel Volker , König Norbert , Bossemeyer Dirk , Pipkorn Rüdiger , Lehmann Wolf D.
Published: February 2009

Conserved deamidation of PKA catalytic subunit isozymes Cα and Cβ—more than 25% at Asn2 in vivo in both cases—has been shown to yield Asp2‐ and isoAsp2‐containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7: 457–469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the “repair” of isoAsp to Asp by the action of protein L‐isoaspartyl (D‐aspartyl) O‐methyl transferase (PIMT). L‐Succinimide is prone to racemization to D‐succinimide, which may hydrolyze to D‐isoAsp‐ and D‐Asp‐containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N‐terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high‐performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp‐ and isoAsp‐residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D‐isoAsp‐ and D‐Asp‐containing T1 fragments in addition to the L‐isomers. The small amount of the L‐isoAsp isomer, representing only part of the D‐isoAsp isomer, and the relatively large amounts of the L‐Asp and D‐Asp isomers argues for an effective action of PIMT present in cardiac tissue.
Thermodynamic and structural characterization of Asn and Ala residues in the disallowed II′ region of the Ramachandran plot
Authors: Vega M. Cristina , Serrano Luis , Martínez Jose C.
Published: February 2009

Residue Asn47 at position L1 of a type II′ β‐turn of the α‐spectrin SH3 domain is located in a disallowed region of the Ramachandran plot (ϕ = 56 ± 12, ± = −118 ± 17). Therefore, it is expected that replacement of Asn47 by Gly should result in a considerable stabilization of the protein. Thermodynamic analysis of the N47G and N47A mutants shows that the change in free energy is small (∼0.7 kcal/mol; ∼3 kJ/mol) and comparable to that found when mutating a Gly to Ala in a α‐helix or β‐sheet. X‐ray structural analysis of these mutants shows that the conformation of the β‐turn does not change upon mutation and, therefore, that there is no relaxation of the structure, nor is there any gain or loss of interactions that could explain the small energy change. Our results indicate that the energetic definition of II' region of the Ramachandran plot (ϕ = 60 ± 30, ± = −115 ± 15) should be revised for at least Ala and Asn in structure validation and protein design.
Functional prediction: Identification of protein orthologs and paralogs
Authors: Chen Ridong , Jeong Soon‐Seog
Published: February 2009

Orthologs typically retain the same function in the course of evolution. Using β‐decarboxylating dehydrogenase family as a model, we demonstrate that orthologs can be confidently identified. The strategy is based on our recent findings that substitutions of only a few amino acid residues in these enzymes are sufficient to exchange substrate and coenzyme specificities. Hence, the few major specificity determinants can serve as reliable markers for determining orthologous or paralogous relationships. The power of this approach has been demonstrated by correcting similarity‐based functional misassignment and discovering new genes and related pathways, and should be broadly applicable to other enzyme families.
Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR
Authors: Cave John W. , Wemmer David E. , Kremer Werner
Published: February 2009

Backbone dynamics of the basic/helix‐loop‐helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states. These secondary structures are compared to the co‐crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T, Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15:4689–4697). The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex. Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region. The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics. We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue. These results are discussed in relation to the mechanism of sequence specific recognition and binding.
Evolution of binding affinity in a WW domain probed by phage display
Authors: Dalby Paul A. , Hoess Ronald H. , Degrado William F.
Published: February 2009

The WW domain is an approximately 38 residue peptide‐binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one‐third of its three‐stranded antiparallel β‐sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high‐affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild‐type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.
Delineation of the calcineurin‐interacting region of cyclophilin B
Authors: Carpentier Mathieu , Allain Fabrice , Slomianny Marie‐Christine , Spik Geneviève , Haendler Bernard
Published: February 2009

The immunosuppressant drug cyclosporin A (CsA) inhibits T‐cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high‐affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin‐binding region. To delineate the calcineurin‐binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild‐type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA‐binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.
The effects of disulfide bonds on the denatured state of barnase
Authors: Clarke Jane , Hounslow Andrea M. , Fersht Alan R. , Bond Chris J. , Daggett Valerie
Published: February 2009

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended β‐sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild‐type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.
Chaperonin‐assisted folding of glutamine synthetase under nonpermissive conditions: Off‐pathway aggregation propensity does not determine the co‐chaperonin requirement
Authors: Voziyan Paul A. , Fisher Mark T.
Published: February 2009

One of the proposed roles of the GroEL‐GroES cavity is to provide an “infinite dilution” folding chamber where protein substrate can fold avoiding deleterious off‐pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N‐oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these “nonpermissive” folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High‐performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these “low Mg2+” conditions exist as long‐lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL‐GroES encapsulated environment is not required under all nonpermissive folding conditions.
Thermal stability of Clostridium pasteurianum rubredoxin: Deconvoluting the contributions of the metal site and the protein
Authors: Bonomi Francesco , Iametti Stefania , Mazzini Stefania , Fessas Dimitrios , Kurtz Donald M.
Published: February 2009

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 °C, and during progressive heating of the iron‐containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ « Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 °C for Fe2+Rd and 83 °C for Fe3+Rd. Temperature‐dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50‐70 °C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower‐temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M‐SCys bond strength. In the case of Fe3+Rd, stoichiometric Fe3+/cysteine‐ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.
Fibers of tau fragments, but not full length tau, exhibit a cross β‐structure: Implications for the formation of paired helical filaments
Authors: Giannetti A. M. , Lindwall G. , Kohlstaedt L. A. , Chau M.‐F. , Radeke M. J. , Feinstein S. C.
Published: February 2009

We have used X‐ray fiber diffraction to probe the structure of fibers of tau and tau fragments. Fibers of fragments from the microtubule binding domain had a cross β‐structure that closely resembles that reported both for neurofibrillary tangles found in Alzheimer's disease brain and for fibrous lesions from other protein folding diseases. In contrast, fibers of full‐length tau had a different, more complex structure. Despite major differences at the molecular level, all fiber types exhibited very similar morphology by electron microscopy. These results have a number of implications for understanding the etiology of Alzheimer's and other tauopathic diseases. The morphology of the peptide fibers suggests that the region in tau corresponding to the peptides plays a critical role in the nucleation of fiber assembly. The dramatically different structure of the full length tau fibers suggests that some region in tau has enough inherent structure to interfere with the formation of cross β‐fibers. Additionally, the similar appearance by electron microscopy of fibrils with varying molecular structure suggests that different molecular arrangements may exist in other samples of fibers formed from tau.
Characterization of the interface structure of enzyme‐inhibitor complex by using hydrogen‐deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry
Authors: Akashi Satoko , Takio Koji
Published: February 2009

We investigated the interaction between a thiol protease inhibitor, cystatin, and its target enzyme, papain, by hydrogen‐deuterium (H/D) exchange in conjunction with successive analysis by collision‐induced dissociation (CID) in an rf‐only hexapole ion guide with electrospray ionization‐Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole‐CID. In the absence of papain, amide hydrogens in short amino‐terminal fragments, such as b2+10 and b2+12, were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin‐papain complex in the hexapole‐CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1‐10 persisted for hours, suggesting that the flexible N‐terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that enzyme‐inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI‐FTICR MS rapidly and using only a small amount of sample.
For the record: Effect of deamidation on folding of ribonuclease A
Authors: Orrù Stefania , Vitagliano Luigi , Esposito Luciana , Mazzarella Lelio , Marino Gennaro , Ruoppolo Margherita
Published: February 2009

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65‐72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn → isoAsp where the local structure of the loop 65‐72 has been modified keeping intact the C65‐C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild‐type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65‐72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.
Characterization of an anti‐Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody‐bound antigen and mass spectrometricpeptide mapping
Authors: Legros Vèronique , Jolivet‐Reynaud Colette , Battail‐Poirot Nicole , Saint‐Pierre Christine , Forest Eric
Published: February 2009

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.
Nonpolar contributions to conformational specificity in assemblies of designed short helical peptides
Authors: Boon Chandra L. , Chakrabartty Avijit
Published: February 2009

A series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity. The consensus sequence was designed to obtain short helices (17 residues) and to minimize the presence of interhelical polar interactions. Furthermore, the sequence contained a heptad repeat (abcdefg), where positions a and d were occupied by hydrophobic residues Leu, Ile, or Val, and positions e and g were occupied by Ala. The peptides were named according to the identities of the residues in the adeg positions, respectively. The peptides llaa, liaa, ilaa, iiaa, ivaa, viaa, lvaa, vlaa, and vvaa were synthesized, and their characterization revealed marked differences in specificity. An experimental methodology was developed to study the nine peptides and their pairwise mixtures. These peptides and their mixtures formed a vast array of structural states, which may be classified as follows: helical tetramers and pentamers, soluble and insoluble helical aggregates, insoluble unstructured aggregates, and soluble unstructured monomers. The peptide liaa formed stable helical pentamers, and iiaa and vlaa formed stable helical tetramers. Disulfide cross‐linking experiments indicated the presence of an antiparallel helix alignment in the helical pentamers and tetramers. Rates of amide proton exchange of the tetrameric form of vlaa were 10‐fold slower than the calculated exchange rate for unfolded vlaa. In other work, the control of specificity has been attributed to polar interactions, especially buried polar interactions; this work demonstrated that subtle changes in the configuration of nonpolar interactions resulted in a large variation in the extent of conformational specificity of assemblies of designed short helical peptides. Thus, nonpolar interactions can have a significant effect on the conformational specificity of oligomeric short helices.
For the record: Temperature‐sensitive suppressor mutations of the Escherichia coli DNA gyrase B protein
Authors: Blance Stephen J. , Williams Nicola L. , Preston ZOË A. , Bishara John , Smyth Michael S. , Maxwell Anthony
Published: February 2009

Escherichia coli strain LE316 contains a mutation in gyrB that results in the substitution of Val164 to Gly and confers both chlorobiocin resistance and temperature sensitivity. Selection for suppressors of the ts phenotype yielded second‐site mutations in GyrB at His38 and Thr157. The properties of proteins bearing these mutations have been characterized, and a mechanism of suppression is proposed based upon structural considerations.
NMR characterization of a pH‐dependent equilibrium between two folded solution conformations of the pheromone‐binding protein from Bombyx mori
Authors: Damberger Fred , Horst Reto , Wüthrich Kurt , Peng Guihong , Nikonova Larisa , Leal Walter Soares
Published: February 2009

NMR spectroscopic changes as a function of pH in solutions of the pheromone‐binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single “acid form” (A), and a homogeneous “basic form” (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a welldispersed NMR spectrum, indicating that it represents a more structured, “closed” conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant‐binding proteins, and it will be of interest to further investigate possible correlations with pH‐dependent regulation of ligand binding and release in the biological function of this class of proteins.
Removal of the N‐terminal hexapeptide from human β2‐microglobulin facilitates protein aggregation and fibril formation
Authors: Esposito G. , Michelutti R. , Verdone G. , Viglino P. , ÁNdez H. Hern , Robinson C. V. , Amoresano A. , Piaz F. Dal , Monti M. , Pucci P. , Mangione P. , Stoppini M. , Merlini G. , Ferri G. , Bellotti V.
Published: February 2009

The solution structure and stability of N‐terminally truncated β2‐microglobulin (δN6β2‐m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that δN6β2‐m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full‐length proteins at μM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that δN6β2‐m is significantly less protected than its wild‐type counterpart. Analysis of δN6β2‐m by NMR shows that this loss of protection occurs in β strands I, III, and part of II. At mM concentration gel filtration analysis shows that δN6β2‐m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of β2‐microglobulin was found to have a higher tendency to self‐associate than the intact molecule, and unlike wild‐type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that δN6β2‐m could be a key intermediate of a proteolytic pathway of β2‐microglobulin. Overall, the data suggest that removal of the six residues from the N‐terminus of β2‐microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between β‐strands far removed from this constrain is greatly perturbed.
Evidence for phosphorylation‐dependent conformational changes in methylesterase CheB
Authors: Anand Ganesh S. , Goudreau Paul N. , Lewis J. Kathleen , Stock Ann M.
Published: February 2009

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N‐terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation‐dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate residue within the N‐terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the regulatory domain of CheB facilitate a repositioning of the N‐ and C‐terminal domains, leading to stimulation of methylesterase activity.
Comparative model building of interleukin‐7 using interleukin‐4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation
Authors: Cosenza Larry , Murphy John R. , Smith Temple , Rosenbach Andrew , White James V.
Published: February 2009

Using a combination of theoretical sequence structure recognition predictions and experimental disulfide bond assignments, a three‐dimensional (3D) model of human interleukin‐7 (hIL‐7) was constructed that predicts atypical surface chemistry in helix D that is important for receptor activation. A 3D model of hIL‐7 was built using the X‐ray crystal structure of interleukin‐4 (IL‐4) as a template (Walter MR et al., 1992, J Mol Biol. 224: 1075‐1085; Walter MR et al., 1992, J Biol Chem 267:20371–20376). Core secondary structures were constructed from sequences of hIL‐7 predicted to form helices. The model was constructed by superimposing IL‐7 helices onto the IL‐4 template and connecting them together in an up‐up down‐down topology. The model was finished by incorporating the disulfide bond assignments (Cys3, Cys142), (Cys35, Cys130), and (Cys48, Cys93), which were determined by MALDI mass spectroscopy and site‐directed mutagenesis (Cosenza L, Sweeney E, Murphy JR, 1997, J Biol Chem 272:32995–33000). Quality analysis of the hIL‐7 model identified poor structural features in the carboxyl terminus that, when further studied using hydrophobic moment analysis, detected an atypical structural property in helix D, which contains Cys130 and Cys142. This analysis demonstrated that helix D had a hydrophobic surface exposed to bulk solvent that accounted for the poor quality of the model, but was suggestive of a region in IL‐7 that may be important for protein interactions. Alanine (Ala) substitution scanning mutagenesis was performed to test if the predicted atypical surface chemistry of helix D in the hIL‐7 model is important for receptor activation. This analysis resulted in the construction, purification, and characterization of four hIL‐7 variants, hIL‐7 (K121A), hIL‐7(L136A), hIL‐7(K140A), and hIL‐7(W143A), that displayed reduced or abrogated ability to stimulate a murine IL‐7 dependent pre‐B cell proliferation. The mutant hIL‐7(W143A), which is biologically inactive and displaces [125I]‐hIL‐7, is the first reported IL‐7R system antagonist.
The energetics of phosphate binding to a protein complex
Authors: Edgcomb Stephen P. , Baker Brian M. , Murphy Kenneth P.
Published: February 2009

The heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease‐inhibitor complex; thus, the elastase‐ovomucoid system provides a unique opportunity to study phosphate‐protein interactions. We have used isothermal titration calorimetry to investigate this binding, thereby providing one of the few complete thermodynamic characterizations of phosphate interacting with proteins. The binding is characterized by a small favorable ΔG°, a large unfavorable ΔH°, and a positive ΔCp, thermodynamics consistent with the release of water being linked to phosphate binding. These measurements provide insight into the binding of phosphotyrosine containing peptides to SH2 domains by suggesting the energetic consequences of binding phosphate free from other interactions.
Role of Lys335 in the metastability and function of inhibitory serpins
Authors: Im Hana , Yu Myeong‐Hee
Published: February 2009

The native form of inhibitory serpins (serine protease inhibitors) is not in the thermodynamically most stable state but in a metastable state, which is critical to inhibitory functions. To understand structural basis and functional roles of the native metastability of inhibitory serpins, we have been characterizing stabilizing mutations of human α1‐antitrypsin, a prototype inhibitory serpin. One of the sites that has been shown to be critical in stability and inhibitory activity of α1‐antitrypsin is Lys335. In the present study, detailed roles of this lysine were analyzed by assessing the effects of 13 different amino acid substitutions. Results suggest that size and architect of the side chains at the 335 site determine the metastability of α1‐antitrypsin. Moreover, factors such as polarity and flexibility of the side chain at this site, in addition to the metastability, seem to be critical for the inhibitory activity. Substitutions of the lysine at equivalent positions in two other inhibitory serpins, human α1‐antichymotrypsin and human antithrombin III, also increased stability and decreased inhibitory activity toward α‐chymotrypsin and thrombin, respectively. These results and characteristics of lysine side chain, such as flexibility, polarity, and the energetic cost upon burial, suggest that this lysine is one of the structural designs in regulating metastability and function of inhibitory serpins in general.
Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA
Authors: Ferguson Andrew D. , Coulton James W. , Diederichs Kay , Welte Wolfram , Braun Volkmar , Fiedler Hans‐Peter
Published: February 2009

One alternative method for drug delivery involves the use of siderophore‐antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron‐chelating siderophores, can be actively transported across the outer membrane of Gram‐negative bacteria. These “Trojan Horse” antibiotics may prove useful as an efficient means to combat multi‐drug–resistant bacterial infections. Here we present the crystallo‐graphic structures of the natural siderophore‐antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad‐host range antibiotics that consist of a hydroxamate‐type iron‐chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate‐type siderophores, the three‐dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron‐chelating siderophores and offers a structural platform for the rational design of hydroxamate‐type siderophore‐antibiotic conjugates.
Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa
Authors: Brocca Stefania , Alberghina Lilia , Lotti Marina , Persson Mattias , Wehtje Ernst , Adlercreutz Patrick
Published: February 2009

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N‐linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild‐type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
The 1.9 Å crystal structure of Escherichia coli MurG, a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis
Authors: Ha Sha , Walker Deborah , Shi Yigong , Walker Suzanne
Published: February 2009

The 1.9 Å X‐ray structure of a membrane‐associated glycosyltransferase involved in peptidoglycan biosynthesis is reported. This enzyme, MurG, contains two α/β open sheet domains separated by a deep cleft. Structural analysis suggests that the C‐terminal domain contains the UDP‐GlcNAc binding site while the N‐terminal domain contains the acceptor binding site and likely membrane association site. Combined with sequence data from other MurG homologs, this structure provides insight into the residues that are important in substrate binding and catalysis. We have also noted that a conserved region found in many UDP‐sugar transferases maps to a β/α/β/α supersecondary structural motif in the donor binding region of MurG, an observation that may be helpful in glycosyltransferase structure prediction. The identification of a conserved structural motif involved in donor binding in different UDP‐sugar transferases also suggests that it may be possible to identify—and perhaps alter—the residues that help determine donor specificity.
Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: A quantitative model
Authors: Schulz Tanja , Pleiss Jürgen , Schmid Rolf D.
Published: February 2009

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast‐reacting enantiomer in a productive mode and the slow‐reacting enantiomer in a nonproductive mode. Nonproductive mode of fast‐reacting enantiomers was prohibited by repulsive interactions. For the slow‐reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(HNϵ – Oalc) between the histidine side chain and the alcohol oxygen increases. d(HNϵ – Oalc) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(HNϵ – Oalc) is>2.2 Å for slow‐reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(HNϵ – Oalc) is less than 2.0 Å, and slow‐ and fast‐reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E< 100), d(HNϵ – Oalc) is around 2.1 Å. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.
A new model for 20‐hydroxyecdysone and dibenzoylhydrazine binding: A homology modeling and docking approach
Authors: Wurtz Jean‐Marie , Guillot Benoit , Fagart Jérôme , Moras Dino , Tietjen Klaus , Schindler Michael
Published: February 2009

The ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand‐binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20‐hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N‐tert‐butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR–LBD protein models rationalize how 20E and dibenzoylhy‐drazines interact with the ligand‐binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides.
Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein
Authors: GrießLer Richard , D'Auria Sabato , Tanfani Fabio , Nidetzky Bernd
Published: February 2009

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5′‐phosphate as an active‐site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500‐fold stabilization against irreversible inactivation at elevated temperatures (≥50°C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate≈︁16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the α‐helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5′‐phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature‐dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to≈︁ 50°C, and (2) stabilizes the covalent protein cofactor linkage up to ≈︁ 70°C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50°C of ≈︁ 25 mM. The extra stability conferred by oxyanion‐ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a ≈︁ 20°C higher value on the temperature scale.
Cascaded multiple classifiers for secondary structure prediction
Authors: Ouali Mohammed , King Ross D.
Published: February 2009

We describe a new classifier for protein secondary structure prediction that is formed by cascading together different types of classifiers using neural networks and linear discrimination. The new classifier achieves an accuracy of 76.7% (assessed by a rigorous full Jack‐knife procedure) on a new nonredundant dataset of 496 nonhomologous sequences (obtained from G.J. Barton and JA. Cuff). This database was especially designed to train and test protein secondary structure prediction methods, and it uses a more stringent definition of homologous sequence than in previous studies. We show that it is possible to design classifiers that can highly discriminate the three classes (H, E, C) with an accuracy of up to 78% for β‐strands, using only a local window and resampling techniques. This indicates that the importance of long‐range interactions for the prediction of β‐strands has been probably previously overestimated.
The role of backbone conformational heat capacity in protein stability: Temperature dependent dynamics of the B1 domain of Streptococcal protein G
Authors: Seewald Michael J. , Pichumani Kumar , Stowell Cheri , Tibbals Benjamin V. , Regan Lynne , Stone Martin J.
Published: February 2009

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR‐derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross‐relaxation rates (ηxy), and steady state {1H}‐15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50°C for 89–100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model‐free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ∼9°C lower (78°C rather than 87°C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.